US20030119104A1 - Chromosome-based platforms - Google Patents

Chromosome-based platforms Download PDF

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US20030119104A1
US20030119104A1 US10/161,403 US16140302A US2003119104A1 US 20030119104 A1 US20030119104 A1 US 20030119104A1 US 16140302 A US16140302 A US 16140302A US 2003119104 A1 US2003119104 A1 US 2003119104A1
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cell
aces
chromosome
dna
site
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Edward Perkins
Carl Perez
Michael Lindenbaum
Amy Greene
Josephine Leung
Elena Fleming
Sandra Stewart
Joan Shellard
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Glaxo Group Ltd
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Assigned to CHROMOS MOLECULAR SYSTEMS, INC. reassignment CHROMOS MOLECULAR SYSTEMS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FLEMING, ELENA, GREENE, AMY, LEUNG, JOSEPHINE, LINDENBAUM, MICHAEL, PEREZ, CARL, PERKINS, EDWARD, SHELLARD, JOAN, STEWART, SANDRA
Publication of US20030119104A1 publication Critical patent/US20030119104A1/en
Priority to US11/006,076 priority patent/US7521240B2/en
Priority to US11/082,154 priority patent/US20060024820A1/en
Priority to US11/480,175 priority patent/US20060246586A1/en
Assigned to PENDER NDI LIFE SCIENCES FUND (VCC) INC. reassignment PENDER NDI LIFE SCIENCES FUND (VCC) INC. SECURITY AGREEMENT Assignors: CELEXSYS, INC., CHROMOS CORPORATION, CHROMOS MOLECULAR SYSTEMS, INC.
Assigned to GLAXO GROUP LIMITED reassignment GLAXO GROUP LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHROMOS MOLECULAR SYSTEMS, INC.
Priority to US12/985,478 priority patent/US20120064578A1/en
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Definitions

  • U.S. Pat. Nos. 6,025,155 and 6,077,697 provide heterochromatic artificial chromosomes designated therein as satellite artificial chromosomes (SATACs) and now designated artificial chromosome expression systems (ACes). These chromosomes are prepared by introducing heterologous DNA into a selected plant or animal cell under conditions that result in integration into a region of the chromosome that leads to an amplification event resulting in production of a dicentric chromosome. Subsequent treatment and growth of cells with dicentric chromosomes, including further amplifications, ultimately results in the artificial chromosomes provided therein.
  • SATACs satellite artificial chromosomes
  • ACes artificial chromosome expression systems
  • the artificial chromosomes that permit tractable, efficient and rational engineering thereof.
  • the artificial chromosomes provided herein contain one or a plurality of loci (sites) for site-specific, recombination-directed integration of DNA.
  • sites loci
  • platform ACes platform artificial chromosome expression systems
  • the artificial chromosomes and ACes artificial chromosomes include plant and animal chromosomes. Any recombinase system that effects site-specific recombination is contemplated for use herein.
  • chromosomes including platform ACes, are provided that contain one or more lambda att sites designed for recombination-directed integration in the presence of lambda integrase, and that are mutated so that they do not require additional factors.
  • Methods for preparing such chromosomes, vectors for use in the methods, and uses of the resulting chromosomes are also provided.
  • bacteriophage lambda A integrase site-specific recombination system.
  • kits containing the combinations of vectors encoding a recombinase and integrase and primers for introduction of the site recognized thereby are also provided.
  • the kits optionally include instructions for performing site-directed integration or preparation of ACes containing such sites.
  • mammalian and plant cells comprising the artificial chromosomes and ACes described herein.
  • the cells can be nuclear donor cells, stem cells, such as a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell or an embryonic stem cell.
  • a lamba-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-integrase3.
  • transgenic animals and methods for producing a transgenic animal comprising introducing a ACes into an embryonic cell, such as a stem cell or embryo.
  • the ACes can comprise heterologous nucleic acid that encodes a therapeutic product.
  • the transgenic animal can be a fish, insect, reptile, amphibians, arachnid or mammal.
  • the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer.
  • the platform ACes including plant and animal ACes, such as MACs, provided herein can be introduced into cells, such as, but not limited to, animal cells, including mammalian cells, and into plant cells.
  • animal cells including mammalian cells
  • plant cells that contain platform MACs, animal cells that contain platform PACs and other combinations of cells and platform ACes are provided.
  • FIG. 1 provides a diagram depicting creation of an exemplary ACes artificial chromosome prepared using methods detailed in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183.
  • the nucleic acid is targeted to an acrocentric chromosome in an animal or plant, and the heterologous nucleic acid includes a sequence-specific recombination site and marker genes.
  • FIG. 2 provides a map of pWEPuro9K, which is a targeting vector derived from the vector pWE15 (GenBank Accession #X65279; SEQ ID No. 31). Plasmid pWE15 was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance encoding fragment with the PvuII/BamHI (Klenow filled) puromycin resistance-encoding fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc., Palo Alto, Calif.; GenBank Accession no. U07648; SEQ ID No. 30) resulting in plasmid pWEPuro.
  • FIG. 3 depicts construction of an ACes platform chromosome with a single recombination site, such as loxP sites or an attP or attB site.
  • This platform ACes chromosome is an exemplary artificial chromosome with a single recombination site.
  • FIG. 4 provides a map of plasmid pSV40-193attPsensePur.
  • FIG. 5 depicts a method for formation of a chromosome platform with multiple recombination integration sites, such as attP sites.
  • FIG. 6 sets forth the sequences of the core region of attP, attB, attL and attR (SEQ ID Nos. 33-36).
  • FIG. 7 depicts insertional recombination of a vector encoding a marker gene, DsRed and an attB site with an artificial chromosome containing an attP site.
  • FIG. 8 provides a map of plasmid pCXLamIntR (SEQ ID NO: 112), which includes the Lambda integrase (E174R)-encoding nucleic acid.
  • FIG. 9 diagrammatically summarizes the platform technology; marker 1 permits selection of the artificial chromosomes containing the integration site; marker 2, which is promoterless in the target gene expression vector, permits selection of recombinants. Upon recombination with the platform marker 2 is expressed under the control of a promoter resident on the platform.
  • FIG. 10 provides the vector map for the plasmid p18attBZEO-5′6XHS4eGFP (SEQ ID NO: 116).
  • FIG. 11 provides the vector map for the plasmid p18attBZEO-3′6XHS4eGFP (SEQ ID NO: 115).
  • FIG. 12 provides the vector map for the plasmid p18attBZEO-(6XHS4)2eGFP (SEQ ID NO: 110).
  • FIGS. 13 AND 14 depict the integration of a PCR product by site-specific recombination as set forth in Example 8.
  • FIG. 15 provides the vector map for the plasmid pPACrDNA as set forth in Example 9.A.
  • nucleic acid refers to single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. When referring to probes or primers, optionally labeled, with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated.
  • a detectable label such as a fluorescent or radiolabel
  • Such molecules are typically of a length such that they are statistically unique and of low copy number (typically less than 5, preferably less than 3) for probing or priming a library.
  • a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides long.
  • DNA is meant to include all types and sizes of DNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.
  • nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified-nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.
  • heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations and/or in amounts in a genome or cell that differ from that in which it occurs in nature.
  • Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Any DNA or RNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA.
  • Heterologous DNA and RNA may also encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes.
  • heterologous DNA examples include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced for purposes of modification of the endogenous genes or for production of an encoded protein.
  • a heterologous or foreign gene may be isolated from a different species than that of the host genome, or alternatively, may be isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene.
  • Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers traits including, but not limited to, herbicide, insect, or disease resistance; traits, including, but not limited to, oil quality or carbohydrate composition.
  • Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.
  • operative linkage or operative association, or grammatical variations thereof, of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides.
  • operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • a sequence complementary to at least a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, preferably under moderate or high stringency conditions, forming a stable duplex.
  • the ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. The longer the hybridizing nucleic acid, the more base mismatches it can contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • regulatory molecule refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a polypeptide that is capable of enhancing or inhibiting expression of a gene.
  • recognition sequences are particular sequences of nucleotides that a protein, DNA, or RNA molecule, or combinations thereof, (such as, but not limited to, a restriction endonuclease, a modification methylase and a recombinase) recognizes and binds.
  • a recognition sequence for Cre recombinase (see, e.g., SEQ ID NO:58) is a 34 base pair sequence containing two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core and designated loxP (see, e.g., Sauer (1994) Current Opinion in Biotechnology 5:521-527).
  • recognition sequences include, but are not limited to, attB and attP, attR and attL and others (see, e.g., SEQ ID Nos. 8, 41-56 and 72), that are recognized by the recombinase enzyme Integrase (see, SEQ ID Nos. 37 and 38 for the nucleotide and encoded amino acid sequences of an exemplary lambda phage integrase).
  • the recombination site designated attB is an approximately 33 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region;
  • attP (SEQ ID No. 72) is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins IHF, FIS, and Xis (see, e.g., Landy (1993) Current Opinion in Biotechnology 3:699-7071 see, e.g., SEQ ID Nos. 8 and 72).
  • a recombinase is an enzyme that catalyzes the exchange of DNA segments at specific recombination sites.
  • An integrase herein refers to a recombinase that is a member of the lambda ( ⁇ ) integrase family.
  • recombination proteins include excisive proteins, integrative proteins, enzymes, co-factors and associated proteins that are involved in recombination reactions using one or more recombination sites (see, Landy (1993) Current Opinion in Biotechnology 3:699-707).
  • the recombination proteins used herein can be delivered to a cell via an expression cassette on an appropriate vector, such as a plasmid, and the like.
  • the recombination proteins can be delivered to a cell in protein form in the same reaction mixture used to deliver the desired nucleic acid, such as a platform ACes, donor target vectors, and the like.
  • lox site means a sequence of nucleotides at which the gene product of the cre gene, referred to herein as Cre, can catalyze a site-specific recombination event.
  • a LoxP site is a 34 base pair nucleotide sequence from bacteriophage P1 (see, e.g., Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402).
  • the LoxP site contains two 13 base pair inverted repeats separated by an 8 base pair spacer region as follows: (SEQ ID NO. 57):
  • E. coli DH5 ⁇ lac and yeast strain BSY23 transformed with plasmid pBS44 carrying two loxP sites connected with a LEU2 gene are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53254 and ATCC 20773, respectively.
  • the lox sites can be isolated from plasmid pBS44 with restriction enzymes EcoRI and SalI, or XhoI and BamHI.
  • a preselected DNA segment can be inserted into pBS44 at either the SalI or BamHI restriction enzyme sites.
  • Other lox sites include, but are not limited to, LoxB, LoxL, LoxC2 and LoxR sites, which are nucleotide sequences isolated from E.
  • Lox sites can also be produced by a variety of synthetic techniques (see, e.g., Ito et al. (1982) Nuc. Acid Res. 10:1755 and Ogilvie et al. (1981) Science 270:270).
  • cre gene means a sequence of nucleotides that encodes a gene product that effects site-specific recombination of DNA in eukaryotic cells at lox sites.
  • One cre gene can be isolated from bacteriophage P1 (see, e.g., Abremski et al. (1983) Cell 32:1301-1311).
  • E. coli DH 1 and yeast strain BSY90 transformed with plasmid pBS39 carrying a cre gene isolated from bacteriophage P1 and a GALL regulatory nucleotide sequence are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53255 and ATCC 20772, respectively.
  • the cre gene can be isolated from plasmid pBS39 with restriction enzymes XhoI and SalI.
  • site-specific recombination refers to site-specific recombination that is effected between two specific sites on a single nucleic acid molecule or between two different molecules that requires the presence of an exogenous protein, such as an integrase or recombinase.
  • Cre-lox site-specific recombination can include the following three events:
  • DNA segment refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source.
  • DNA deoxyribonucleic acid
  • the lox site is an asymmetrical nucleotide sequence
  • two lox sites on the same DNA molecule can have the same or opposite orientations with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule.
  • the deleted DNA segment forms a circular molecule of DNA.
  • the original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites.
  • the Cre-lox system can be used to specifically delete, invert, or insert DNA.
  • the precise event is controlled by the orientation of lox DNA sequences, in cis the lox sequences direct the Cre recombinase to either delete (lox sequences in direct orientation) or invert (lox sequences in inverted orientation) DNA flanked by the sequences, while in trans the lox sequences can direct a homologous recombination event resulting in the insertion of a recombinant DNA.
  • a chromosome is a nucleic acid molecule, and associated proteins, that is capable of replication and segregation within a cell upon cell division.
  • a chromosome contains a centromeric region, replication origins, telomeric regions and a region of nucleic acid between the centromeric and telomeric regions.
  • centromere is any nucleic acid sequence that confers an ability to segregate to daughter cells through cell division.
  • a centromere may confer stable segregation of a nucleic acid sequence, including an artificial chromosome containing the centromere, through mitotic or meiotic divisions, including through both mitotic and meiotic divisions.
  • a particular centromere is not necessarily derived from the same species in which it is introduced, but has the ability to promote DNA segregation in cells of that species.
  • Euchromatin refers to chromatin that stains diffusely and that typically contains genes
  • heterochromatin refers to chromatin that remains unusually condensed and that has been thought to be transcriptionally inactive.
  • Highly repetitive DNA sequences are usually located in regions of the heterochromatin surrounding the centromere (pericentric or pericentromeric heterochromatin).
  • Constitutive heterochromatin refers to heterochromatin that contains the highly repetitive DNA which is constitutively condensed and genetically inactive.
  • an acrocentric chromosome refers to a chromosome with arms of unequal length.
  • endogenous chromosomes refer to genomic chromosomes as found in a cell prior to generation or introduction of an artificial chromosome.
  • artificial chromosomes are nucleic acid molecules, typically DNA, that stably replicate and segregate alongside endogenous chromosomes in cells and have the capacity to accommodate and express heterologous genes contained therein. It has the capacity to act as a gene delivery vehicle by accommodating and expressing foreign genes contained therein.
  • a mammalian artificial chromosome refers to chromosomes that have an active mammalian centromere(s).
  • Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include centromeres that function in plant, insect and avian cells, respectively.
  • a human artificial chromosome refers to chromosomes that include centromeres that function in human cells.
  • exemplary artificial chromosomes see, e.g., U.S. Pat. Nos. 6,025,155; 6,077,697; 5,288,625; 5,712,134; 5,695,967; 5,869,294; 5,891,691 and 5,721,118 and published International PCT application Nos, WO 97/40183 and WO 98/08964.
  • Artificial chromosomes include those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos.
  • minichromosomes that contain a de novo centromere (see, U.S. Pat. Nos. 5,712,134, 5,891,691 and 5,288,625), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of Vietnamese and heterochromatic DNA and in vitro assembled artificial chromosomes (see, copending U.S. provisional application Serial No. 60/294,687, filed on May 30, 2001).
  • SATAC tellite DNA-based artificial chromosome
  • ACes artificial chromosome expression system
  • ACes include those that are substantially all neutral non-coding sequences (heterochromatin) except for foreign heterologous, typically gene-encoding nucleic acid, that is interspersed within the heterochromatin for the expression therein (see U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183), or that is in a single locus as provided herein.
  • ACes that may include euchromatin and that result from the process described in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183 and outlined herein.
  • the delineating structural feature is the presence of repeating units, that are generally predominantly heterochromatin.
  • the precise structure of the ACes will depend upon the structure of the chromosome in which the initial amplification event occurs; all share the common feature of including a defined pattern of repeating units. Generally ACes have more heterochromatin than euchromatin.
  • Foreign nucleic acid molecules (heterologous genes) contained in these artificial chromosome expression systems can include any nucleic acid whose expression is of interest in a particular host cell.
  • Such foreign nucleic acid molecules include, but are not limited to, nucleic acid that encodes traceable marker proteins (reporter genes), such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively), other reporter genes, such as ⁇ -galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance.
  • traceable marker proteins reporter genes
  • reporter genes such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively
  • other reporter genes such as ⁇ -galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance.
  • heterologous nucleic acid molecules include, but are not limited to, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, DNA that encodes other types of proteins, such as antibodies, and DNA that encodes RNA molecules (such as antisense or siRNA molecules) that
  • an artificial chromosome platform also referred to herein as a “platform ACes” or “ACes platform”, refers to an artificial chromosome that has been engineered to include one or more sites for site-specific, recombination-directed integration.
  • ACes that are so-engineered are provided. Any sites, including but not limited to any described herein, that are suitable for such integration are contemplated.
  • Plant and animal platform ACes are provided. Among the ACes contemplated herein are those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos.
  • SATACs satellite artificial chromosomes
  • artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of Vietnamese and heterochromatic DNA resulting from an amplification event depicted in the referenced patent and herein.
  • ACes for use in generating platforms include artificial chromosomes that introduce and express heterologous nucleic acids in plants (see, copending U.S. provisional application Serial No. 60/294,687, filed on May 30, 2001). These include artificial chromosomes that have a centromere derived from a plant, and, also, artificial chromosomes that have centromeres that may be derived from other organisms but that function in plants.
  • reporter ACes refers to a an ACes that comprises one or a plurality of reporter constructs, where the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.
  • amplification is a process in which segments of DNA are duplicated to yield two or multiple copies of substantially similar or identical or nearly identical DNA segments that are typically joined as substantially tandem or successive repeats or inverted repeats.
  • amplification-based artificial chromosomes are artificial chromosomes derived from natural or endogenous chromosomes by virtue of an amplification event, such as one initiated by introduction of heterologous nucleic acid into rDNA in a chromosome. As a result of such an event, chromosomes and fragments thereof exhibiting segmented or repeating patterns arise. Artificial chromosomes can be formed from these chromosomes and fragments.
  • amplification-based artificial chromosomes refer to engineered chromosomes that exhibit an ordered segmentation that is not observed in naturally occurring chromosomes and that distinguishes them from naturally occurring chromosomes.
  • the segmentation which can be visualized using a variety of chromosome analysis techniques known to those of skill in the art, correlates with the structure of these artificial chromosomes.
  • the amplification-based artificial chromosomes throughout the region or regions of segmentation are predominantly made up of nucleic acid units also referred to as “amplicons”, that is (are) repeated in the region and that have a similar gross structure. Repeats of an amplicon tend to be of similar size and share some common nucleic acid sequences.
  • each repeat of an amplicon may contain a replication site involved in amplification of chromosome segments and/or some heterologous nucleic acid that was utilized in the initial production of the artificial chromosome.
  • the repeating units are substantially similar in nucleic acid composition and may be nearly identical.
  • the amplification-based artificial chromosomes differ depending on the chromosomal region that has undergone amplification in the process of artificial chromosome formation.
  • the structures of the resulting chromosomes can vary depending upon the initiating event and/or the conditions under which the heterologous nucleic acid is introduced, including modification to the endogenous chromosomes.
  • the region or regions of segmentation may be made up predominantly of heterochromatic DNA.
  • the region or regions of segmentation may be made up predominantly of Vietnamese amounts of heterochromatic and euchromatic DNA.
  • an amplicon is a repeated nucleic acid unit.
  • an amplicon may contain a set of inverted repeats of a megareplicon.
  • a megareplicon represents a higher order replication unit.
  • the megareplicon can contain a set of tandem DNA blocks (e.g., ⁇ 7.5 Mb DNA blocks) each containing satellite DNA flanked by non-satellite DNA or may be made up of substantially rDNA.
  • a primary replication site Contained within the megareplicon is a primary replication site, referred to as the megareplicator, which may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres.
  • the megareplicator may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres.
  • Within the megareplicon there may be smaller (e.g., 50-300 kb) secondary replicons.
  • each amplicon has the same gross structure but may contain sequence variations. Such variations will arise as a result of movement of mobile genetic elements, deletions or insertions or mutations that arise, particularly in culture. Such variation does not affect the use of the artificial chromosomes or their overall structure as described herein.
  • amplifiable when used in reference to a chromosome, particularly the method of generating artificial chromosomes provided herein, refers to a region of a chromosome that is prone to amplification. Amplification typically occurs during replication and other cellular events involving recombination (e.g., DNA repair). Such regions include regions of the chromosome that contain tandem repeats, such as satellite DNA, rDNA, and other such sequences.
  • a dicentric chromosome is a chromosome that contains two centromeres.
  • a multicentric chromosome contains more than two centromeres.
  • a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments is a replicable chromosome. If one of the chromosomes undergoes amplification of primarily euchromatic DNA to produce a fully functional chromosome that is predominantly (at least more than 50%) euchromatin, it is a minichromosome. The remaining chromosome is a formerly dicentric chromosome.
  • heterochromatin such as, for example, satellite DNA
  • a euchromatic portion such as, for example, an arm
  • a sausage chromosome A chromosome that is substantially all heterochromatin, except for portions of heterologous DNA, is called a predominantly heterochromatic artificial chromosome.
  • Predominantly heterochromatic artificial chromosomes can be produced from other partially heterochromatic artificial chromosomes by culturing the cell containing such chromosomes under conditions such as BrdU treatment that destabilize the chromosome and/or growth under selective conditions so that a predominantly heterochromatic artificial chromosome is produced.
  • the artificial chromosomes may not necessarily be produced in multiple steps, but may appear after the initial introduction of the heterologous DNA.
  • artificial chromosomes appear after about 5 to about 60, or about 5 to about 55, or about 10 to about 55 or about 25 to about 55 or about 35 to about 55 cell doublings after initiation of artificial chromosome generation, or they may appear after several cycles of growth under selective conditions and BrdU treatment.
  • an artificial chromosome that is predominantly heterochromatic i.e., containing more heterochromatin than euchromatin, typically more than about 50%, more than about 70%, or more than about 90% heterochromatin
  • cells such as, for example, animal or plant cells
  • Any nucleic acid may be introduced into cells in such methods of producing the artificial chromosomes.
  • the nucleic acid may contain a selectable marker and/or optionally a sequence that targets nucleic acid to the pericentric, heterochromatic region of a chromosome, such as in the short arm of acrocentric chromosomes and nucleolar organizing regions.
  • Targeting sequences include, but are not limited to, lambda phage DNA and rDNA for production of predominantly heterochromatic artificial chromosomes in eukaryotic cells.
  • a cell containing a predominantly heterochromatic artificial chromosome is selected.
  • Such cells may be identified using a variety of procedures. For example, repeating units of heterochromatic DNA of these chromosomes may be discerned by G-banding and/or fluorescence in situ hybridization (FISH) techniques.
  • FISH fluorescence in situ hybridization
  • the cells to be analyzed may be enriched with artificial chromosome-containing cells by sorting the cells on the basis of the presence of a selectable marker, such as a reporter protein, or by growing (culturing) the cells under selective conditions.
  • a “selectable marker” is a nucleic acid segment, generally DNA, that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds and compositions.
  • selectable markers include but are not limited to: (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be identified, such as phenotypic markers, including ⁇ -galactosidase, red, blue and/or green fluorescent proteins (FPs), and cell surface proteins; (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos.
  • phenotypic markers including ⁇ -galactosidase, red, blue and/or green fluorescent proteins (FPs), and cell surface proteins
  • FPs red, blue and/or green fluorescent proteins
  • nucleic acid segments that bind products that modify a substrate e.g. restriction endonucleases
  • nucleic acid segments that can be used to isolate a desired molecule e.g. specific protein binding sites
  • nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional, such as for PCR amplification of subpopulations of molecules e.g., antisense oligonucleotides or siRNA molecules for use in RNA interference
  • nucleic acid segments that bind products that modify a substrate e.g. restriction endonucleases
  • nucleic acid segments that can be used to isolate a desired molecule e.g. specific protein binding sites
  • nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional, such as for PCR amplification of subpopulations of molecules e.g., specific protein binding sites
  • selectable markers include nucleic acids encoding fluorescent proteins, such as green fluorescent proteins, ⁇ -galactosidase and other readily detectable proteins, such as chromogenic proteins or proteins capable of being bound by an antibody and FACs sorted. Selectable markers such as these, which are not required for cell survival and/or proliferation in the presence of a selection agent, are also referred to herein as reporter molecules.
  • Other selectable markers e.g., the neomycin phosphotransferase gene, provide for isolation and identification of cells containing them by conferring properties on the cells that make them resistant to an agent, e.g., a drug such as an antibiotic, that inhibits proliferation of cells that do not contain the marker.
  • RNA interference As another example, interference of gene expression by double stranded RNA has been shown in Caenorhabditis elegans, plants, Drosophila, protozoans and mammals. This method is known as RNA interference (RNAi) and utilizes short, double-stranded RNA molecules (siRNAs).
  • siRNAs are generally composed of a 19-22 bp double-stranded RNA stem, a loop region and a 1-4 bp overhang on the 3′ end.
  • the reduction of gene expression has been accomplished by direct introduction of the siRNAs into the cell (Harborth J et al., 2001, J Cell Sci 114(pt 24):4557-65) as well as the introduction of DNA encoding and expressing the siRNA molecule.
  • RNAi in mammalian cells may have advantages over other therapeutic methods. For example, producing siRNA molecules that block viral genetic activities in infected cells may reduce the effects of the virus.
  • Platform ACes provided herein encoding siRNA molecule(s) are an additional utilization of the platform ACes technology.
  • the platform ACes could be engineered to encode one or more siRNA molecules to create gene “knockdowns”.
  • a platform ACes can engineered to encode both the siRNA molecule and a replacement gene.
  • a mouse model or cell culture system could be generated using a platform ACes that has a knockdown of the endogenous mouse gene, by siRNA, and the human gene homolog expressing in place of the mouse gene.
  • the placement of siRNA encoding sequences under the regulation of a regulatable or inducible promoter would allow one to temporally and/or spatially control the knockdown effect of the corresponding gene.
  • a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Generally reporter genes are readily detectable. Examples of reporter genes include, but are not limited to nucleic acid encoding a fluorescent protein, CAT (chloramphenicol acetyl transferase) (Alton et al. (1979) Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984) Proc. Natl.
  • CAT chloramphenicol acetyl transferase
  • growth under selective conditions means growth of a cell under conditions that require expression of a selectable marker for survival.
  • an agent that destabilizes a chromosome is any agent known by those skilled in the art to enhance amplification events, and/or mutations. Such agents, which include BrdU, are well known to those skilled in the art.
  • nucleic acid in order to generate an artificial chromosome containing a particular heterologous nucleic acid of interest, it is possible to include the nucleic acid in the nucleic acid that is being introduced into cells to initiate production of the artificial chromosome.
  • a nucleic acid can be introduced into a cell along with nucleic acid encoding a selectable marker and/or a nucleic acid that targets to a heterochromatic region of a chromosome.
  • a heterologous nucleic acid into the cell it can be included in a fragment that includes a selectable marker or as part of a separate nucleic acid fragment and introduced into the cell with a selectable marker during the process of generating the artificial chromosomes.
  • heterologous nucleic acid can be introduced into an artificial chromosome at a later time after the initial generation of the artificial chromosome.
  • the minichromosome refers to a chromosome derived from a multicentric, typically dicentric, chromosome that contains more Vietnamese than heterochromatic DNA.
  • the minichromosome contains a de novo centromere (e.g., a neocentromere).
  • the minichromosome contains a centromere that replicates in animals, e.g., a mammalian centromere or in plants, e.g., a plant centromere.
  • in vitro assembled artificial chromosomes or synthetic chromosomes can be either more Vietnamese than heterochromatic or more heterochromatic than euchromatic and are produced by joining essential components of a chromosome in vitro. These components include at least a centromere, a megareplicator, a telomere and optionally secondary origins of replication.
  • in vitro assembled plant or animal artificial chromosomes are produced by joining essential components (at least the centromere, telomere(s), megareplicator and optional secondary origins of replication) that function in plants or animals.
  • the megareplicator contains sequences of rDNA, particularly plant or animal rDNA.
  • a plant is a eukaryotic organism that contains, in addition to a nucleus and mitochondria, chloroplasts capable of carrying out photosynthesis.
  • a plant can be unicellular or multicellular and can contain multiple tissues and/or organs. Plants can reproduce sexually or asexually and can be perennial or annual in growth. Plants can also be terrestrial or aquatic.
  • the term “plant” includes a whole plant, plant cell, plant protoplast, plant calli, plant seed, plant organ, plant tissue, and other parts of a whole plant.
  • stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95%, of the cells retain the chromosome. Stability is measured in the presence of a selective agent. Preferably these chromosomes are also maintained in the absence of a selective agent. Stable chromosomes also retain their structure during cell culturing, suffering no unintended intrachromosomal or interchromosomal rearrangements.
  • centromere As used herein, de novo with reference to a centromere, refers to generation of an excess centromere in a chromosome as a result of incorporation of a heterologous nucleic acid fragment using the methods herein.
  • BrdU refers to 5-bromodeoxyuridine, which during replication is inserted in place of thymidine. BrdU is used as a mutagen; it also inhibits condensation of metaphase chromosomes during cell division.
  • Ribosomal RNA is the specialized RNA that forms part of the structure of a ribosome and participates in the synthesis of proteins. Ribosomal RNA is produced by transcription of genes which, in eukaryotic cells, are present in multiple copies. In human cells, the approximately 250 copies of rRNA genes (i.e., genes which encode rRNA) per haploid genome are spread out in clusters on at least five different chromosomes (chromosomes 13, 14, 15, 21 and 22).
  • rDNA ribosomal DNA
  • rDNA DNA containing sequences that encode rRNA
  • rDNA In Arabidopsis thaliana the presence of rDNA has been verified on chromosomes 2 and 4 (18S, 5.8S, and 25S rDNA) and on chromosomes 3,4, and 5 (5S rDNA)(see The Arabidopsis Genome Initiative (2000) Nature 408:796-815).
  • the multiple copies of the highly conserved rRNA genes are located in a tandemly arranged series of rDNA units, which are generally about 40-45 kb in length and contain a transcribed region and a nontranscribed region known as spacer (i.e., intergenic spacer) DNA which can vary in length and sequence.
  • these tandem arrays of rDNA units are located adjacent to the pericentric satellite DNA sequences (heterochromatin).
  • the regions of these chromosomes in which the rDNA is located are referred to as nucleolar organizing regions (NOR) which loop into the nucleolus, the site of ribosome production within the cell nucleus.
  • a megachromosome refers to a chromosome that, except for introduced heterologous DNA, is substantially composed of heterochromatin. Megachromosomes are made up of an array of repeated amplicons that contain two inverted megareplicons bordered by introduced heterologous DNA (see, e.g., FIG. 3 of U.S. Pat. No. 6,077,697 for a schematic drawing of a megachromosome). For purposes herein, a megachromosome is about 50 to 400 Mb, generally about 250-400 Mb.
  • megachromosome Shorter variants are also referred to as truncated megachromosomes (about 90 to 120 or 150 Mb), dwarf megachromosomes ( ⁇ 150-200 Mb), and a micro-megachromosome ( ⁇ 50-90 Mb, typically 50-60 Mb).
  • megachromosome refers to the overall repeated structure based on an array of repeated chromosomal segments (amplicons) that contain two inverted megareplicons bordered by any inserted heterologous DNA. The size will be specified.
  • gene therapy involves the transfer or insertion of nucleic acid molecules into certain cells, which are also referred to as target cells, to produce specific products that are involved in preventing, curing, correcting, controlling or modulating diseases, disorders and deleterious conditions.
  • the nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced.
  • the nucleic acid may in some manner mediate expression of DNA that encodes a therapeutic product.
  • This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product.
  • nucleic acid encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
  • cells can be transfected in vitro, followed by introduction of the transfected cells into an organism. This is often referred to as ex vivo gene therapy. Alternatively, the cells can be transfected directly in vivo within an organism.
  • therapeutic agents include, but are not limited to, growth factors, antibodies, cytokines, such as tumor necrosis factors and interleukins, and cytotoxic agents and other agents disclosed herein and known to those of skill in the art.
  • agents include, but are not limited to, tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), pro-coagulants such as tissue factor and tissue factor variants, pro-apoptotic agents such FAS-ligand, fibroblast growth factors (FGF), nerve growth factor and other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin
  • a therapeutically effective product is a product that is encoded by heterologous DNA that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease.
  • transgenic plants and animals refer to plants and animals in which heterologous or foreign nucleic acid is expressed or in which the expression of a gene naturally present in the plant or animal has been altered by virtue of introduction of heterologous or foreign nucleic acid.
  • IRES internal ribosome entry site; see, e.g., SEQ ID No. 27 and nucleotides 2736-3308 SEQ ID No. 28
  • mRNA molecule a nucleic acid molecule
  • IRES refers to a region of a nucleic acid molecule, such as an mRNA molecule, that allows internal ribosome entry sufficient to initiate translation, which initiation can be detected in an assay for cap-independent translation (see, e.g., U.S. Pat. No. 6,171,821).
  • the presence of an IRES within an mRNA molecule allows cap-independent translation of a linked protein-encoding sequence that otherwise would not be translated.
  • IRES Internal ribosome entry site
  • a promoter refers to a sequence of DNA that contains a sequence of bases that signals RNA polymerase to associate with the DNA and initiate transcription of RNA (such as pol 11 for mRNA) from a template strand of the DNA.
  • a promoter thus generally regulates transcription of DNA into mRNA.
  • a particular promoter provided herein is the Ferritin heavy chain promoter (excluding the Iron Response Element, located in the 5′UTR), which was joined to the 37 bp Fer-1 enhancer element. This promoter is set forth as SEQ ID NO:128.
  • the endogenous Fer-1 enhancer element is located upstream of the Fer-1 promoter (e.g., a Fer-1 oligo was cloned proximal to the core promoter).
  • isolated, substantially pure nucleic acid such as, for example, DNA
  • nucleic acid fragments purified according to standard techniques employed by those skilled in the art, such as that found in Sambrook et al. ((2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 3rd edition).
  • expression refers to the transcription and/or translation of nucleic acid.
  • expression can be the transcription of a gene that may be transcribed into an RNA molecule, such as a messenger RNA (mRNA) molecule.
  • RNA messenger RNA
  • Expression may further include translation of an RNA molecule and translated into peptides, polypeptides, or proteins.
  • mRNA messenger RNA
  • expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
  • expression may refer to the transcription of the antisense DNA.
  • vector or plasmid refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors and plasmids are well within the level of skill of the art.
  • transformation/transfection refers to the process by which nucleic acid is introduced into cells.
  • transfection and transformation refer to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed.
  • transfection Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722; Potrykus et al. (1985) Mol. Gen. Genet. 199:169-177; Reich et al. (1986) Biotechnology 4:1001-1004; Klein et al.
  • PEG polyethylene glycol
  • lipid-mediated carrier systems see, e.g., Teifel et al. (1995) Biotechniques 19:79-80; Albrecht et al. (1996) Ann. Hematol. 72:73-79; Holmen et al. (1995) In Vitro Cell Dev. Biol. Anim. 31:347-351; Remy et al. (1994) Bioconjug. Chem. 5:647-654; Le Bolch et al. (1995) Tetrahedron Lett. 36:6681-6684; Loeffler et al. (1993) Meth. Enzymol. 217:599-618) or other suitable method.
  • Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell.
  • delivery refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.
  • injected refers to the microinjection, such as by use of a small syringe, needle, or pipette, for injection of nucleic acid into a cell.
  • substantially homologous DNA refers to DNA that includes a sequence of nucleotides that is sufficiently similar to another such sequence to form stable hybrids, with each other or a reference sequence, under specified conditions.
  • nucleic acid fragments with different sequences may, under the same conditions, hybridize detectably to the same “target” nucleic acid.
  • Two nucleic acid fragments hybridize detectably, under stringent conditions over a sufficiently long hybridization period, because one fragment contains a segment of at least about 10, 14 or 16 or more nucleotides in a sequence that is complementary (or nearly complementary) to a substantially contiguous sequence of at least one segment in the other nucleic acid fragment.
  • Two single-stranded nucleic acid segments have “substantially the same sequence”, if (a) both form a base-paired duplex with the same segment, and (b) the melting temperatures of the two duplexes in a solution of 0.5 ⁇ SSPE differ by less than 10° C. If the segments being compared have the same number of bases, then to have “substantially the same sequence”, they will typically differ in their sequences at fewer than 1 base in 10. Methods for determining melting temperatures of nucleic acid duplexes are well known (see, e.g., Meinkoth et al. (1984) Anal. Biochem. 138:267-284 and references cited therein).
  • a nucleic acid probe is a DNA or RNA fragment that includes a sufficient number of nucleotides to specifically hybridize to DNA or RNA that includes complementary or substantially complementary sequences of nucleotides.
  • a probe may contain any number of nucleotides, from as few as about 10 and as many as hundreds of thousands of nucleotides.
  • the conditions and protocols for such hybridization reactions are well known to those of skill in the art as are the effects of probe size, temperature, degree of mismatch, salt concentration and other parameters on the hybridization reaction. For example, the lower the temperature and higher the salt concentration at which the hybridization reaction is carried out, the greater the degree of mismatch that may be present in the hybrid molecules.
  • the nucleic acid is generally rendered detectable by labeling it with a detectable moiety or label, such as 32 P, 3 H and 14 C, or by other means, including chemical labeling, such as by nick-translation in the presence of deoxyuridylate biotinylated at the 5′-position of the uracil moiety.
  • the resulting probe includes the biotinylated uridylate in place of thymidylate residues and can be detected (via the biotin moieties) by any of a number of commercially available detection systems based on binding of streptavidin to the biotin.
  • detection systems can be obtained, for example, from Enzo Biochemicals, Inc.
  • any other label known to those of skill in the art, including non-radioactive labels, may be used as long as it renders the probes sufficiently detectable, which is a function of the sensitivity of the assay, the time available (for culturing cells, extracting DNA, and hybridization assays), the quantity of DNA or RNA available as a source of the probe, the particular label and the means used to detect the label.
  • sequences with a sufficiently high degree of homology to the probe are identified, they can readily be isolated by standard techniques (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press).
  • a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a polypeptide.
  • substantially homologous is meant having at least 75%, preferably 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.
  • the terms “homology” and “identity” are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program.
  • the GAP program utilizes the alignment method of Needleman and Wunsch ( J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman ( Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • sequence identity the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier.
  • Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Preferably the two molecules will hybridize under conditions of high stringency.
  • nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.
  • nucleic acid molecules have nucleotide sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.
  • sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988 ; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J.
  • identity represents a comparison between a test and a reference polypeptide or polynucleotide.
  • a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide.
  • the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides.
  • Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions.
  • conservative amino acid substitutions are those that do not eliminate biological activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. co., p.224).
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations.
  • nucleotides which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art.
  • a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.
  • a probe or primer based on a nucleotide sequence includes at least 10, 14, 16, 30 or 100 contiguous nucleotides from the reference nucleic acid molecule.
  • recombinant production by using recombinant DNA methods refers to the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA.
  • biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture.
  • Biological activity thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
  • Biological activities may be observed in in vitro systems designed to test or use such activities.
  • the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced.
  • substantially identical to a product means sufficiently similar so that the property is sufficiently unchanged so that the substantially identical product can be used in place of the product.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art.
  • a substantially chemically pure compound may, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound.
  • vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
  • the vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into a chromosome of the genome.
  • vectors that are artificial chromosomes such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art.
  • An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • protein-binding-sequence refers to a protein or peptide sequence that is capable of specific binding to other protein or peptide sequences generally, to a set of protein or peptide sequences or to a particular protein or peptide sequence.
  • composition refers to any mixture of two or more ingredients. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • a combination refers to any association between two or more items.
  • fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
  • a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell.
  • the term “subject” refers to animals, plants, insects, and birds and other phyla, genera and species into which nucleic acid molecules may be introduced. Included are higher organisms, such as mammals, fish, insects and birds, including humans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, hamsters, cats, dogs, horses, chicken and others.
  • flow cytometry refers to processes that use a laser based instrument capable of analyzing and sorting out cells and or chromosomes based on size and fluorescence.
  • Site-specific recombination systems typically contain three elements: a pair of DNA sequences (the site-specific recombination sequences) and a specific enzyme (the site-specific recombinase).
  • the site-specific recombinase catalyzes a recombination reaction between two site-specific recombination sequences.
  • a number of different site-specific recombinase systems are available and/or known to those of skill in the art, including, but not limited to: the Cre/lox recombination system using CRE recombinase (see, e.g., SEQ ID Nos. 58 and 59) from the Escherichia coli phage P1 (see, e.g., Sauer (1993) Methods in Enzymology 225:890-900; Sauer et al. (1990) The New Biologist 2:441-449), Sauer (1994) Current Opinion in Biotechnology 5:521-527; Odell et al. (1990) Mol Gen Genet. 223:369-378; Lasko et al.
  • the resolvases including Gin recombinase of phage Mu (Maeser et al. (1991) Mol Gen Genet. 230:170-176; Klippel, A. et al (1993) EMBO J. 12:1047-1057; see, e.g., SEQ ID Nos. 64-67), Cin, Hin, ⁇ Tn3; the Pin recombinase of E. coli (see, e.g., SEQ ID Nos. 68 and 69; Enomoto et al. (1983) J Bacteriol.
  • resolvase family such as ⁇ , Tn3 resolvase, Hin, Gin, and Cin are also available.
  • Members of this family of recombinases are typically constrained to intramolecular reactions (e.g., inversions and excisions) and can require host-encoded factors. Mutants have been isolated that relieve some of the requirements for host factors (Maeser et al. (1991) Mol. Gen. Genet. 230:170-176), as well as some of the constraints of intramolecular recombination (see, U.S. Pat. No. 6,171,861).
  • the bacteriophage P1 Cre/lox and the yeast FLP/FRT systems are particularly useful systems for site-specific integration, inversion or excision of heterologous nucleic acid into, and out of, chromosomes, particularly ACes as provided herein.
  • a recombinase (Cre or FLP) interacts specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences.
  • the sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT).
  • the FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells (U.S. Pat. No. 5,744,386), and, thus, can be used for producing plant artificial chromosome platforms.
  • short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites.
  • the system catalyzes intra- and intermolecular reactions, and, thus, can be used for DNA excision and integration reactions.
  • the recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation.
  • the site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.
  • Cre-lox In the Cre-lox system, discovered in bacteriophage P1, recombination between loxP sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Pat. No. 5,658,772).
  • This system can be used to insert, invert or excise nucleic acid located between two lox sites.
  • Cre can be expressed from a vector. Since the lox site is an asymmetrical nucleotide sequence, lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule.
  • the deleted DNA segment forms a circular molecule of DNA.
  • the original DNA molecule and the resulting circular molecule each contain a single lox site.
  • Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites.
  • reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region.
  • Any site-specific recombinase system known to those of skill in the art is contemplated for use herein. It is contemplated that one or a plurality of sites that direct the recombination by the recombinase are introduced into an artificial chromosome to produce platform ACes.
  • the resulting platform ACes are introduced into cells with nucleic acid encoding the cognate recombinase, typically on a vector, and nucleic acid encoding heterologous nucleic acid of interest linked to the appropriate recombination site for insertion into the platform ACes.
  • the recombinase-encoding-nucleic acid may be introduced into the cells on the same vector, or a different vector, encoding the heterologous nucleic acid.
  • E. coli phage lambda integrase system for ACes platform engineering and for artificial chromosome engineering is provided (Lorbach et al. (2000) J. Mol. Biol 296:1175-1181).
  • the phage lambda integrase (Landy, A. (1989) Annu. Rev. Biochem. 58:913-94) is adapted herein and the cognate att sites are provided.
  • Chromosomes, including ACes, engineered to contain one or a plurality of att sites are provided, as are vectors encoding a mutant integrase that functions in the absence other factors. Methods using the modified chromosomes and vectors for introduction of heterologous nucleic acid are also provided.
  • one or more of the sites (e.g., a single site or a pair of sites) required for recombination are introduced into an artificial chromosome, such as an ACes chromosome.
  • the enzyme for catalyzing site-directed recombination is introduced with the DNA of interest, or separately, or is engineered onto the artificial chromosome under the control of a regulatable promoter.
  • Att/Int system and vectors provided herein are not only intended for engineering ACes platforms, but may be used to engineer an Att/Int system into any chromosome.
  • Introduction of att sites into a chromosome will permit engineering of natural chromosomes, such as by permitting targeted integration genes or regulatory regions, and by controlled excision of selected regions.
  • genes encoding a particular trait may be added to a chromosome, such as plant chromosome engineered to contain one or plurality of att sites.
  • Such chromosomes may be used for screening DNA to identify genes. Large pieces of DNA can be introduced into cells and the cells screened phenotypically to select those having the desired trait.
  • platform artificial chromosomes containing single or multiple site-specific recombination sites.
  • Chromosome-based platform technology permits efficient and tractable engineering and subsequent expression of multiple gene targets.
  • Methods are provided that use DNA vectors and fragments to create platform artificial chromosomes, including animal, particularly mammalian, artificial chromosomes, and plant artificial chromosomes.
  • the artificial chromosomes contain either single or multiple sequence-specific recombination sites suitable for the placement of target gene expression vectors onto the platform chromosome.
  • the engineered chromosome-based platform ACes technology is applicable for methods, including cellular and transgenic protein production, transgenic plant and animal production and gene therapy.
  • the platform ACes are also useful for producing a library of ACes comprising random portions of a given genome (e.g., a mammalian, plant or prokaryotic genome) for genomic screening; as well as a library of cells comprising different and/or mutually exclusive ACes therein.
  • a given genome e.g., a mammalian, plant or prokaryotic genome
  • ACes artificial chromosome platforms are those based on ACes.
  • ACes artificial chromosomes are non-viral, self-replicating nucleic acid molecules that function as a natural chromosome, having all the elements required for normal chromosomal replication and maintenance within the cell nucleus.
  • ACes artificial chromosomes do not rely on integration into the genome of the cell to be effective, and they are not limited by DNA carrying capacity and as such the therapeutic gene(s) of interest, including regulatory sequences, can be engineered into the ACes.
  • ACes are stable in vitro and in vivo and can provide predictable long-term gene expression.
  • ACes work independently alongside host chromosomes, for ACes that are predominantly heterochromatin producing only the products (proteins) from the genes it carries.
  • ACes are modified by introduction of recombination site(s) to provide a platform for ready introduction of heterologous nucleic acid.
  • the ACes platforms can be used for production of transgenic animals and plants; as vectors for genetic therapy; for use as protein production systems; for animal models to identify and target new therapeutics; in cell culture for the development and production of therapeutic proteins; and for a variety of other applications.
  • ACes artificial chromosomes may be generated by any method known to those of skill in the art. Of particular interest herein are the ACes artificial chromosomes, which contain a repeated unit. Methods for production of ACes are described in detail in U.S. Pat. Nos. 6,025,155 and 6,077,697, which, as with all patents, applications, publications and other disclosure, are incorporated herein in their entirety.
  • ACes can be generated by cotransfecting exogenous DNA—such as a mammary tissue specific DNA cassette including the gene sequences for a therapeutic protein, with a rDNA fragment and a drug resistance marker gene into the desired eukaryotic cell, such as plant or animal cells, such as murine cells in vitro.
  • DNA with a selectable or detectable marker is introduced, and can be allowed to integrate randomly into pericentric heterochromatin or can be targeted to pericentric heterochromatin, such as that in rDNA gene arrays that reside on acrocentric chromosomes, such as the short arms of acrocentric chromosomes.
  • This integration event activates the “megareplicator” sequence and amplifies the pericentric heterochromatin and the exogenous DNA, and duplicates a centromere. Ensuing breakage of this “dicentric” chromosome can result in the production of daughter cells that contain the substantially-original chromosome and the new artificial chromosome.
  • the resulting ACes contain all the essential elements needed for stability and replication in dividing cells-centromere, origins of replications, and telomeres.
  • ACes have been produced that express marker genes (lacZ, green fluorescent protein, neomycin-resistance, puromycin-resistance, hygromycin-resistance) and genes of interest. Isolated ACes, for example, have been successfully transferred intact to rodent, human, and bovine cells by electroporation, sonoporation, microinjection, and transfection with lipids and dendrimers.
  • platform ACes can be produced that contain defined DNA sequences for enzyme-mediated homologous DNA recombination, such as by Cre or FLP recombinases (Bouhassira et al. (1996) Blood 88(supplement 1):190a; Bouhassira et al. (1997) Blood, 90:3332-3344; Siebler et al. (1997) Biochemistry: 36:1740-1747; Siebler et al. (1998) Biochemistry 37: 6229-6234; and Bethke et al. (1997) Nucl. Acids Res.
  • a lox site contains two 13 bp inverted repeats to which Cre-recombinase binds and an intervening 8 bp core region. Only pairs of sites having identity in the central 6 bp of the core region are proficient for recombination; sites having non-identical core sequences (heterospecific lox sites) do not efficiently recombine with each other (Hoess et al. (1986) Nucleic Acids Res. 14:2287-2300).
  • SATAC satellite DNA based artificial chromosome
  • Plant species may not have any acrocentric chromosomes with the same physical structure described, but “megareplicator” DNA sequences reside in the plant rDNA arrays, also known as the nucleolar organizing regions (NOR).
  • NOR nucleolar organizing regions
  • the Kan R cultivars are initially screened, such as by FISH, to identify two sets of candidate transgenic plants.
  • One set has one construct integrated in regions adjacent to the pericentric heterochromatin on the short arm of any chromosome.
  • the second set of candidate plants has the other construct integrated in the NOR region of appropriate chromosomes.
  • To obtain reciprocal translocation both sites must be in the same orientation. Therefore a series of crosses are required, Kan R plants generated, and FISH analyses performed to identify the appropriate “acrocentric” plant chromosome for de novo plant ACes formation.
  • An integral part of the platform technology includes a site-specific recombination system that allows the placement of selected gene targets or genomic fragments onto the platform chromosomes. Any such system may be used.
  • a method is provided for insertion of additional DNA fragments into the platform chromosome residing in the cell via sequence-specific recombination using the recombinase activity of the bacteriophage lambda integrase.
  • the lambda integrase system is exemplary of the recombination systems contemplated for ACes. Any known recombination system, including any described herein, particularly any that operates without the need for additional factors or that, by virtue of mutation, does not require additional factors, is contemplated.
  • the lambda integrase system provided herein can be used with natural chromosomes and artificial chromosomes in addition to ACes. Single or a plurality of recombination sites, which may be the same or different, are introduced into artificial chromosomes to produce artificial chromosome platforms.
  • the lambda phage-encoded integrase (designated Int) is a prototypical member of the integrase family. Int effects integration and excision of the phage in and out of the E. coli genome via recombination between pairs of attachment sites designated attB/attP and attL/attR. Each att site contains two inverted 9 base pair core Int binding sites and a 7 base pair overlap region that is identical in wild-type att sites. Each site, except for attB contains additional Int binding sites. In flanking regions, there are recognition sequences for accessory DNA binding proteins, such as integration host factor (IHF), factor for inversion stimulation (FIS) and the phage encoded excision protein (XIS). Except for attB, Int is a heterobivalent DNA-binding protein and, with assistance from the accessory proteins and negative DNA supercoiling, binds simultaneously to core and arm sites within the same att site.
  • IHF integration host factor
  • FIS factor for inversion stimulation
  • Int like Cre and FLP, executes an ordered sequential pair of strand exchanges during integrative and excisive recombination.
  • the natural pairs of target sequences for Int, attB and attP or attL and attR are located on the same or different DNA molecules resulting in intra or intermolecular recombination, respectively.
  • intramolecular recombination occurs between inversely oriented attB and attP, or between attL and attR sequences, respectively, leading to inversion of the intervening DNA segment.
  • Int Like the recombinase systems, such as Cre and FLP, Int directs site-specific recombination. Unlike the other systems, such Cre and FLP, Int generally requires additional protein factors for integrative and excisive recombination and negative supercoiling for integrative recombination. Hence, the Int system had not been used in eukaryotic targeting systems.
  • Mutant Int proteins designated Int-h (El 74K) and a derivative thereof Int-h/218(E174K/E218K) do not require accessory proteins to perform intramolecular integrative and excisive recombination in co-transfection assays in human cells (Lorbach et al. (2000) J Mol. Biol. 296:1175-1181); wild-type Int does not catalyze intramolecular recombination in human cells harboring target sites attB and attP.
  • mutant Int can catalyze factor-independent recombination events in human cells.
  • chromosomes including artificial chromosomes, such as but not limited to ACes that contain att sites (e.g., platform ACes), and the use of such chromosomes for targeted integration of heterologous DNA into such chromosomes in eukaryotic cells, including animal, such as rodent and human, and plant cells.
  • Mutant Int provided herein is shown to effect site-directed recombination between sites in artificial chromosomes and vectors containing cognate sites.
  • An additional component of the chromosome-based platform technology is the site-specific integration of target DNA sequences onto the platform.
  • the native bacteriophage lambda integrase has been modified to carry out this sequence specific DNA recombination event in eukaryotic cells.
  • the bacteriophage lambda integrase and its cognate DNA substrate att is a member of the site-specific recombinase family that also includes the bacteriophage P1 Cre/lox system as well as the Saccharomyces cerevisiae 2 micron based FLP/FRT system (see, e.g., Landy (1989) Ann. Rev. Biochem 58:913-949; Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402; Broach et al. (1982) Cell 29:227-234).
  • the engineered bacteriophage lambda integrase has been produced herein to carry out an intermolecular DNA recombination event between an incoming DNA molecule (primarily on a vector containing the bacterial attB site) and the chromosome-based platform carrying the lambda attP sequence independent of lambda bacteriophage or bacterial accessory proteins.
  • the engineered lambda recombination system derived for chromosome-based platform technology is advantageously unidirectional because accessory proteins, which are absent, are required for excision of integrated nucleic acid upon further exposure to the lambda Int recombinase.
  • artificial chromosomes are produced as depicted in FIG. 5 and Example 3.
  • artificial chromosomes can be produced using any suitable methodology, including those described in U.S. Pat. Nos. 5,288,625; 5,712,134; 5,891,691; 6,025,155.
  • nucleic acid is targeted into an amplifiable region of a chromosome, such as the pericentric region of a chromosome.
  • regions are the rDNA gene loci in acrocentric mammalian chromosomes.
  • targeting nucleic acid for integration into the rDNA region of mammalian acrocentric chromosomes can include the mouse rDNA fragments (for targeting into rodent cell lines) or large human rDNA regions on BAC/PAC vectors (or subclones thereof in standard vectors) for targeting into human acrocentric chromosomes, such as for human gene therapy applications.
  • the targeting nucleic acid generally includes a detectable or selectable marker, such as antibiotic resistance, such as puromycin and hygromycin, a recombination site (such as attP, attB, attL, attR or the like), and/or human selectable markers as required for gene therapy applications.
  • Cells are grown under conditions that result in amplification and ultimately production of ACes artificial chromosomes having multiple recombination (e.g., integration) sites therein. ACes having the desired size are selected for further engineering.
  • a mammalian platform artificial chromosome is generated containing a single sequence-specific recombination site.
  • this approach is demonstrated using a puromycin resistance marker for selection and a mouse rDNA fragment for targeting into the rDNA locus on mouse acrocentric chromosomes.
  • Other selection markers and targeting DNA sequences as desired and known to those of skill in the art can be used.
  • Additional resistance markers include genes conferring resistance to the antibiotics neomycin, blasticidin, hygromycin and zeocin.
  • host such as human, derived selectable markers or markers detectable with monoclonal antibodies (MAb) followed by fluorescent activated cell sorting (FACS) can be used.
  • FACS fluorescent activated cell sorting
  • examples in this class include, but are not limited to: human nerve growth factor receptor (detection with MAb); truncated human growth factor receptor (detection with MAb); mutant human dihydrofolate reductase (DHFR; detectable using a fluorescent methotrexate substrate); secreted alkaline phosphatase (SEAP; detectable with fluorescent substrate); thymidylate synthase (TS; confers resistance to fluorodeoxyuridine); human CAD gene (confers resistance to N-phosphonacetyl-L-aspartate (PALA)).
  • MAb monoclonal antibodies
  • FACS fluorescent activated cell sorting
  • an ACes artificial chromosome (or other artificial chromosome of interest) can be produced containing a selectable marker.
  • a single sequence specific recombination site is targeted onto ACes via homologous recombination.
  • DNA sequences containing the site-specific recombination sequence are flanked with DNA sequences homologous to a selected sequence in the chromosome.
  • a chromosome containing rDNA or satellite DNA such DNA can be used as homologous sequences to target the site-specific recombination sequence onto the chromosome.
  • a vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the chromosome, the DNA is transfected into cells harboring the chromosome. After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified, for example by screening single cell subclones via expression of resistance or a fluorescent marker and PCR analysis.
  • a platform artificial chromosome such as a platform ACes, that contains a single copy of the recombination site is selected. Examples 2B and 2D exemplify the process, and FIG. 3 provides a diagram depicting one method for the creation of a platform mammalian chromosome containing a single sequence-specific recombination site.
  • the third component of the chromosome-based platform technology involves the use of target gene expression vectors carrying, for example, genes for gene therapy, genes for transgenic animal or plant production, and those required for cellular protein production of interest.
  • target gene expression vectors carrying, for example, genes for gene therapy, genes for transgenic animal or plant production, and those required for cellular protein production of interest.
  • the target gene expression vectors are introduced onto the selected chromosome platform.
  • the use of target gene expression vector permits use of the de novo generated chromosome-based platforms for a wide range of gene targets.
  • chromosome platforms containing multiple attP sites provides the opportunity to incorporate multiple gene targets onto a single platform, thereby providing for expression of multiple gene targets, including the expression of cellular and genetic regulatory genes and the expression of all or parts of metabolic pathways.
  • the chromosome-based platform can be used for engineering and expressing large genomic fragments carrying target genes along with its endogenous genomic promoter sequences. This is of importance, for example, where the therapy requires precise cell specific expression and in instances where expression is best achieved from genomic clones rather than cDNA clones.
  • FIG. 9 provides a diagram summarizing one embodiment of the chromosome-based technology.
  • a feature of the target gene expression vector that is of interest to include is a promoterless marker gene, which as exemplified (see, FIG. 9) contains an upstream attB site (marker 2 on FIG. 9).
  • the nucleic acid encoding the marker is not expressed unless it is placed downstream from a promoter sequence.
  • a promoterless marker gene which as exemplified (see, FIG. 9) contains an upstream attB site (marker 2 on FIG. 9).
  • the nucleic acid encoding the marker is not expressed unless it is placed downstream from a promoter sequence.
  • site-specific recombination between the attB site on the vector and the promoter-attP site (in the “sense” orientation) on the chromosome-based platform results in the expression of marker 2 on the target gene expression vector, thereby providing a positive selection for the lambda INT mediated site-specific recombination event.
  • Site-specific recombination events on the chromosome-based platform versus random integrations next to a promoter in the genome can be quickly screened by designing primers to detect the correct event by PCR.
  • suitable marker 2 genes include, but are not limited to, genes that confer resistance to toxic compounds or antibiotics, fluorescence activated cell sorting (FACS) sortable cell surface markers and various fluorescent markers.
  • FACS fluorescence activated cell sorting
  • these genes include, but are not limited to, human L26a R (human homolog of Saccharomyces cerevisiae CYH 8 gene), neomycin, puromycin, blasticidin, CD24 (see, e.g., U.S. Pat. Nos. 5,804,177 and 6,074,836), truncated CD4, truncated low affinity nerve growth factor receptor (LNGFR), truncated LDL receptor, truncated human growth hormone receptor, GFP, RFP, BFP.
  • LNGFR low affinity nerve growth factor receptor
  • the target gene expression vectors contain a gene (target gene) for expression from the chromosome platform.
  • the target gene can be expressed using various constitutive or regulated promoter systems across various mammalian species.
  • the expression of the multiple targets can be coordinately regulated via viral-based or human internal ribosome entry site (IRES) elements (see, e.g., Jackson et al. (1990) Trends Biochem Sci. 15: 477-83; Oumard et al. (2000) Mol. Cell. Biol. 20: 2755-2759).
  • IRES internal ribosome entry site
  • the promoterless marker can be transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein.
  • the selectable marker and the heterologous nucleic acid can be transcriptionally linked by the presence of a IRES between them.
  • the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic or fluorescent. Expression from the target gene expression vector integrated onto the chromosome-based platform can be further enhanced using genomic insulator/boundary elements.
  • insulator sequences into the target gene expression vector helps define boundaries in chromatin structure and thus minimizes influence of chromatin position effects/gene silencing on the expression of the target gene (Bell et al. (1999) Current Opinion in Genetics and Development 9:191-198; Emery et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:9150-9155).
  • Examples of insulator elements that can be included onto target gene expression vector in order to optimize expression include, but are not limited to:
  • the copy number of the target gene can be controlled by sequentially adding multiple target gene expression vectors containing the target gene onto multiple integration sites on the chromosome platform.
  • the copy number of the target gene can be controlled within an individual target gene expression vector by the addition of DNA sequences that promote gene amplification.
  • gene amplification can be induced utilizing the dihydrofolate reductase (DHFR) minigene with subsequent selection with methotrexate (see, e.g., Schimke (1984) Cell 37:705-713) or amplification promoting sequences from the rDNA locus (see, e.g., Wegner et al. (1989) Nucl. Acids Res. 17: 9909-9932).
  • DHFR dihydrofolate reductase
  • a “double lox” targeting strategy mediated by Cre-recombinase (Bethke et al. (1997) Nucl. Acids Res. 25:2828-2834) can be used.
  • This strategy employs a pair of heterospecific lox sites-loxA and loxB, which differ by one nucleotide in the 8 bp spacer region. Both sites are engineered into the artificial chromosome and also onto the targeting DNA vector. This allows for a direct site-specific insertion of a commercially relevant gene or genes by a Cre-catalyzed double crossover event.
  • a platform ACes is engineered with a hygromycin-resistance gene flanked by the double lox sites generating lox-ACes, which is maintained in the thymidine kinase deficient cell, LMtk( ⁇ ).
  • the gene of interest for example, for testing purposes, the green fluorescence protein gene, GFP and a HSV thymidine kinase gene (tk) marker, are engineered between the appropriate lox sites of the targeting vector.
  • the vector DNA is cotransfected with plasmid pBS185 (Life Technologies) encoding the Cre recombinase gene into mammalian cells maintaining the dual-/ox artificial chromosome.
  • Transient expression of the Cre recombinase catalyzes the site-specific insertion of the gene and the tk-gene onto the artificial chromosome.
  • the transfected cells are grown in HAT medium that selects for only those cells that have integrated and expressed the thymidine kinase gene.
  • the HAT R colonies are screened by PCR analyses to identify artificial chromosomes with the desired insertion.
  • Lambda-Hyg R -lox DNA is transfected into the LMtk( ⁇ ) cell line harboring the precursor ACes. Hygromycin-resistant colonies are analyzed by FISH and Southern blotting for the presence of a single copy insert on the ACes.
  • cell lines containing candidate lox-ACes are cotransfected with pTK-GFP-lox and pBS185 (encoding the Cre recombinase gene) DNA.
  • pTK-GFP-lox encoding the Cre recombinase gene
  • transient expression of plasmid pBS185 will provide sufficient burst of Cre recombinase activity to catalyze DNA recombination at the lox sites.
  • a double crossover event between the ACes target and the exogenous targeting plasmid carrying the loxA and loxB permits the simple replacement of the hygromycin-resistance gene on the lox-ACes for the tk-GFP cassette from the targeting plasmid, with no integration of vector DNA.
  • Transfected cells are grown in HAT-media to select for tk-expression. Correct targeting will result in the generation of HAT R , hygromycin sensitive, and green fluorescent cells. The desired integration event is verified by Southern and PCR analyses. Specific PCR primer sets are used to amplify DNA sequences flanking the individual loxA and loxB sites on the lox-ACes before and after homologous recombination.
  • Platform ACes are applicable and tractable for different/optimized cell lines. Those that include a fluorescent marker, for example, can be purified and isolated using fluorescent activated cell sorting (FACS), and subsequently delivered to a target cell. Those with selectable markers provide for efficient selection and provide a growth advantage. Platform ACes allow multiple payload delivery of donor target vectors via a positive-selection site-specific, recombination system, and they allow for the inclusion of additional genetic factors that improve protein production and protein quality.
  • FACS fluorescent activated cell sorting
  • ACes can be produced from acrocentric chromosomes in rodent (mouse, hamster) cell lines via megareplicator induced amplification of heterochromatin/rDNA sequences. Such ACes are ideal for cellular protein production as well as other applications described herein and known to those of skill in the art. ACes platforms that contain a plurality of recombination sites are particularly suitable for engineering as cellular protein production systems.
  • CPP ACes involve a two-component system: the platform chromosome containing multiple engineering sites and the donor target vector containing a platform-specific recombination site with designed expression cassettes (see FIG. 9).
  • the platform ACes can be produced from any artificial chromosome, particularly the amplification-based artificial chromosomes. For exemplification, they are produced from rodent artificial chromosomes produced from acrocentric chromosomes using the technology of U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183, in which nucleic acid is targeted to the pericentric heterochromatic, and, particularly into rDNA to initiate the replication event(s).
  • the ACes can be produced directly in the chosen cellular protein production cell lines, such as, but not limited to, CHO cells, hybridomas, plant cells, plant tissues, plant protoplasts, stem cells and plant calli.
  • the initial de novo platform construction requires co-transfecting with excess targeting DNA, such as, rDNA or lambda DNA without an attP region, and an engineered selectable marker.
  • the engineered selectable marker should contain promoter, generally a constitutive promoter, such as human, viral, i.e., adenovirus or SV40 promoter, including the human ferritin heavy chain promoter (SEQ ID NO:128), SV40 and EF1 ⁇ promoters, to control expression of a marker gene that provides a selective growth advantage to the cell.
  • a constitutive promoter such as human, viral, i.e., adenovirus or SV40 promoter, including the human ferritin heavy chain promoter (SEQ ID NO:128), SV40 and EF1 ⁇ promoters
  • SEQ ID NO:128 human ferritin heavy chain promoter
  • SV40 and EF1 ⁇ promoters a marker gene that provides a selective growth advantage to the cell.
  • hisD typhimurium hisD a dominant marker selection system for mammalian cells previously described (see, Hartman et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8047-8051). Since histidine is an essential amino acid in mammals and a nutritional requirement in cell culture, the E. coli hisD gene can be used to select for histidine prototrophy in defined media. Furthermore more stringent selection can be placed on the cells by including histinol in the medium. Histidinol is itself permeable and toxic to cells. The hisD provides a means of detoxification.
  • bacteriophage lambda attP site Placed between the promoter and the marker gene is the bacteriophage lambda attP site to use the bacteriophage lambda integrase dependent site-specific recombination system (described herein).
  • the insertion of an attP site downstream of a promoter element provide forward selection of site-specific recombination events onto the platform ACes.
  • a second component of the CPP platform ACes system involves the construction of donor target vectors containing a gene product(s) of interest for the CPP platform ACes.
  • Individual donor target vectors can be designed for each gene product to be expressed thus enabling maximum usage of a de novo constructed platform ACes, so that one or a few CPP platform ACes will be required for many gene targets.
  • a key feature of the donor vector target is the promoterless marker gene containing an upstream attB site (marker 2 on FIG. 9). Normally the marker would not be expressed unless it is placed downstream of a promoter sequence.
  • AINT E174R on FIG. 8 and FIG. 9 site-specific recombination between the attB site on the vector and the promoter-attP site on the CPP platform ACes result in the expression of the donor target vector marker providing positive selection for the site-specific event.
  • Additional features of the donor target vector include gene target expression cassettes flanked by either chromatin insulator regions, matrix attachment regions (MAR) or scaffold attachment regions (SAR). The use of these regions will provide a more “open” chromatin environment for gene expression and help alleviate silencing.
  • MAR matrix attachment regions
  • SAR scaffold attachment regions
  • An example of such a cassette for expressing a monoclonal antibody is described.
  • a strong constitutive promoter e.g. chicken ⁇ -actin or RNA Poll, is used to drive the expression of the heavy and light chain open reading frames.
  • the heavy and light chain sequences flank a nonattenuated human IRES (IRES H ; from the 5′UTR of NRF1 gene; see Oumard et al., 2000 , Mol.
  • IRES HIV V modified ECMV internal ribosomal entry site (IRES)
  • IRES viral encoded IRES
  • hrGFP fluorescent marker gene from Renilla (Stratagene).
  • IRES viral encoded IRES
  • the heavy and light chains along with the hrGFP are monocistronic.
  • the identification of hrGFP fluorescing cells will provide a means to detect protein producing cells.
  • high producing cell lines can be identified and isolated by FACS thereby decreasing the time frame in finding high expressers. Functional monoclonal antibody will be confirmed by ELISA.
  • CPP ACes In addition to the aforementioned CPP ACes system, other genetic factors can be included to enhance the yield and quality of the expressed protein. Again to provide maximum flexibility, these additional factors can be inserted onto the CPP platform ACes by ⁇ INTE174R dependent site-specific recombination. Other factors that could be used with a CPP Platform ACes include for example, adenovirus E1a transactivation system which upregulates both cellular and viral promoters (see, e.g., Svensson and Akusjarvi (1984) EMBO 3:789-794; and U.S. Pat. Nos. 5,866,359; 4,775,630 and 4,920,211).
  • the host cell can be engineered to express these factors (see, below, targets for CHO-ACes engineering to enhance CHO cell growth and protein production/quality). Additional factors to consider including are addition of insulin or IGF-1 to sustain viabililty; human sialyltransferases or related factors to produce more human-like glycoproteins; expression of factors to decrease ammonium accumulation during cell growth; expression of factors to inhibit apoptosis; expression of factors to improve protein secretion and protein folding; and expression of factors to permit serum-free transfection and selection.
  • Stimulatory factors and/or their receptors are expressed to set up an autocrine loop, to improve cell growth, such as CHO cell growth.
  • Two exemplary candidates are insulin and IGF-1 (see, Biotechnol Prog 2000 Sep;16(5):693-7).
  • Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures.
  • Insulin and IGF-1 analog (LongR(3) serve as growth and viability factors for CHO cells.
  • CHO cells were modified to produce higher levels of essential nutrients and factors.
  • a serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44 cells) was prepared.
  • Chinese hamster ovary cells (DG44 cells) which are normally maintained in 10% serum medium, were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium (see, Kim et al. (199) In Vitro Cell Dev Biol Anim 35(4):178-82).
  • a SF medium (SF-DG44) was formulated by supplementing the basal medium with these components; basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Development of a SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.
  • CHO cells have been modified by increasing their ability to process protein via addition of complex carbohydrates. This has been achieved by overexpression of relevant processing enzymes, or in some cases, reducing expression of relevant enzymes (see, Bragonzi et al. (2000) Biochim Biophys Acta 1474(3):273-282; see, also Weikert et al. (1999) Nature biotech. 17:1116-11121; Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95).
  • a CHO cell line expressing alpha2,6-sialyltransferase was developed for the production of human-like sialylated recombinant glycoproteins.
  • the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA into the cells.
  • alpha2,6-ST alpha2,6-sialyltransferase
  • Glycoproteins produced by such CHO cells display alpha2,6-and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins.
  • a CHO cell line has been developed that constitutively expresses sialidase antisense RNA (see, Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95).
  • antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity.
  • Oligosaccharide biosynthesis pathways in mammalian cells have been engineered for generation of recombinant glycoproteins (see, e.g., Sburlati (1998) Biotechnol Prog 14(2):189-92), which describes a Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins. This cell line was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III) (see, also, Prati et al. (1998) Biotechnol Bioeng 59(4):445-50, which describes antisense strategies for glycosylation engineering of CHO cells).
  • GnT-III N-acetylglucosaminyltransferase III
  • the platform ACes provided herein are contemplated for use in mammalian gene therapy, particularly human gene therapy.
  • Human ACes can be derived from human acrocentric chromosomes from human host cells, in which the amplified sequences are heterochromatic and/or human rDNA. Different platform ACes applicable for different tissue cell types are provided.
  • the ACes for gene therapy can contain a single copy of a therapeutic gene inserted into a defined location on platform ACes.
  • Therapeutic genes include genomic clones, cDNA, hybrid genes and other combinations of sequences.
  • Preferred selectable markers are those from the mammalian host, such as human derived factors so that they are non-immunogenic, non-toxic and allow for efficient selection, such as by FACS and/or drug resistance.
  • Platform ACes useful for gene therapy and other applications, as noted herein, can be generated by megareplicator dependent amplification, such as by the methods in U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183.
  • human ACes are produced using human rDNA constructs that target rDNA arrays on human acrocentric chromosomes and induce the megareplicator in human cells, particularly in primary cell lines (with sufficient number of doublings to form the ACes) or stem cells (such as hematopoietic stem cells, mesenchymal stem cells, adult stem cells or embryonic stem cells) to avoid the introduction of potentially harmful rearranged DNA sequences present in many transformed cell lines.
  • Megareplicator induced ACes formation can result in multiple copies of targeting DNA/selectable markers in each amplification block on both chromosomal arms of the platform ACes.
  • human platform ACes for gene therapy applications employs a two component system analogous to the platform ACes designed for cellular protein production (CPP platform ACes).
  • the system includes a platform chromosome of entirely human DNA origin containing multiple engineering sites and a gene target vector carrying the therapeutic gene of interest.
  • the initial de novo construction of the platform chromosome employs the co-transfection of excess targeting DNA and a selectable marker.
  • the DNA is targeted to the rDNA arrays on the human acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22).
  • human acrocentric chromosomes chromosomes 13, 14, 15, 21 and 22.
  • two large human rDNA containing PAC clones 18714 and 18720 and the human PAC clone 558F8 are used for targeting (Genome Research (ML) now Incyte, BACPAC Resources, 747 52nd Street, Oakland Calif.).
  • the mouse rDNA clone pFK161 (SEQ ID NO: 118), which was used to make the human SATAC from the 94-3 hamster/human hybrid cell line (see, e.g., published International PCT application No. WO 97/40183 and Csonka, et al, Journal of Cell Science 113:3207-32161 and Example 1 for a description of pFK161) can also be used.
  • selectable markers should be non-immunogenic in the animal, such as a human, and include, but are not limited to: human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No.
  • a second consideration for the selection of the human marker is the resulting dose of the expressed marker after ACes formation.
  • High level of expression of certain markers may be detrimental to the cell and/or result in autoimmunity.
  • One method to decrease the dose of the marker protein is by shortening its half-life, such as via the fusion of the well-conserved human ubiquitin tag (a 76 amino acid sequence) thus leading to increased turnover of the selectable marker. This has been used successfully for a number of reporter systems including DHFR (see, e.g., Stack et al. (2000) Nature Biotechnology 18:1298-1302 and references cited therein).
  • a human selectable marker system analogous to the CPP ACes described herein is constructed. Briefly, a tagged selectable marker, such as for example one of those described herein, is cloned downstream of an attP site and expressed from a human promoter.
  • Exemplary promoters contemplated for use herein include, but are not limited to, the human ferritin heavy chain promoter (SEQ ID NO:128); RNA Poll; EF1 ⁇ ; TR; glyceraldehyde-3-phosphate dehydrogenase core promoter (GAP); a GAP core promoter including a proximal insulin inducible element the intervening GAP sequence; phosphofructokinase promoter; and phosphoglycerate kinase promoter.
  • GAP glyceraldehyde-3-phosphate dehydrogenase core promoter
  • aldolase A promoter H1 & H2 (representing closely spaced transcriptional start sites) along with the proximal H enhancer.
  • promoters e.g., transcriptional start sites
  • H most proximal 2 promoters are ubiquitously expressed off the H enhancer.
  • This resulting marker can then be co-transfected along with excess human rDNA targeting sequence into the host cells.
  • An important criteria for the selection of the recipient cells is sufficient number of cell doublings for the formation and detection of ACes. Accordingly, the co-transfections should be attempted in human primary cells that can be cultured for long periods of time, such as for example, stem cells (e.g., hematopoietic, mesenchymal, adult or embryonic stem cells), or the like.
  • Additional cell types include, but are not limited to: single gene transfected cells exhibiting increased life-span; over-expressing c-myc cells, e.g. MSU1.1 (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136); over-expressing telomerase lines, such as TERT cells; SV40 large T-antigen transfected lines; tumor cell lines, such as HT1080; and hybrid human cell lines, such as the 94-3 hamster/human hybrid cell line.
  • c-myc cells e.g. MSU1.1 (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136)
  • telomerase lines such as TERT cells
  • SV40 large T-antigen transfected lines such as TERT cells
  • tumor cell lines such as HT1080
  • hybrid human cell lines such as the 94-3 hamster/human hybrid cell line.
  • GT ACes The second component of the GT platform ACes (GT ACes) system involves the use of engineered target vectors carrying the therapeutic gene of interest. These are introduced onto the GT platform ACes via site-specific recombination. As with the CPP ACes, the use of engineered target vectors maximizes the use of the de novo generated GT platform ACes for most gene targets. Furthermore, using lambda integrase technology, GT platform ACes containing multiple attP sites permits the opportunity to incorporate multiple therapeutic targets onto a single platform. This could be of value in cases where a defined therapy requires multiple gene targets, a single therapeutic target requires an additional gene regulatory factor or a GT ACes requires a “kill” switch.
  • a feature of the gene target vector is the promoterless marker gene containing an upstream attB site (marker 2 on FIG. 9).
  • the marker in this case, a cell surface antigen that can be sorted by FACS would be ideal
  • the marker would not be expressed unless it is placed downstream of a promoter sequence.
  • ⁇ INT E174R on FIG. 9 site-specific recombination between the attB site on the vector and the promoter-attP site on the GT platform ACes results in the expression of marker#2 on the gene target vector, i.e. positive selection for the site-specific event.
  • Site-specific recombination events on the GT ACes versus random integrations next to a promoter in the genome can be quickly screened by designing primers to detect the correct event by PCR.
  • human specific promoters such as a ferritin heavy chain promoter (SEQ ID NO:128); EF1 ⁇ or RNA Poll, are used. These promoters are for high level expression of a cDNA encoded therapeutic protein.
  • the GT platform ACes are used for engineering and expressing large genomic fragments carrying therapeutic genes of interest expressed from native promoter sequences. This is of importance in situations where the therapy requires precise cell specific expression or in instances where expression is best achieved from genomic clones versus cDNA.
  • CTX cyclophosphamide
  • MTX methotrexate
  • generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors
  • a retroviral vector containing a human cytosolic ALDH-1-encoding DNA clone and a human doubly mutated DHFR-encoding clone (Phe22/Ser3l; termed F/S in the description of constructs) to generate increased resistance to CTX and MTX were constructed (Takebe et al. (2001) Mol Ther 3(1):88-96). This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression. ACes can be similarly constructed.
  • Rodent cells resistant to N-phosphonacetyl-L-aspartate invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells (Smith et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:1816-21).
  • amplification of CAD is very infrequent in several human tumor cell lines.
  • Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. Fantz et al. ((1998) Biochem Biophys Res Comm 243(1):6-12) have shown that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents.
  • TS thymidylate synthase
  • DHFR dihydrofolate reductase
  • Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold).
  • Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30to 140-fold).
  • a L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA that encodes TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents. ACes can be similarly constructed.
  • GSTA1 glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+cells and bone marrow was studied using RT-PCR and immunoblotting (Czerwinski M, Kiem et al. (1997) Gene Ther 4(3):268-70).
  • the GSTA1 protein conjugates glutathione to the stem cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body.
  • Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow.
  • baboon CD34+cells and dog bone marrow do not express GSTA1.
  • human GSTA1 is a chemoprotective selectable marker in human stem cell gene therapy and could be employed in ACes construction.
  • Pawliuk et al. ((1994) Blood 84(9):2868-2877) have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells.
  • the small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells.
  • MPSV myeloproliferative sarcoma virus
  • 5-FU 5-fluorouracil
  • CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen.
  • CD24 cell surface antigen as a retrovirally encoded marker permits rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and provides a tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells. ACes could be similarly constructed.
  • DeltahGHR a Biosafe Cell Surface-Labeling Molecule for Analysis and Selection of Genetically Transduced Human Cells
  • a selectable marker for retroviral transduction and selection of human and murine cells is known (see, Garcia-Ortiz et al. (2000) Hum Gene Ther 11(2):333-46).
  • the molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering.
  • the engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding.
  • the deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans.
  • MAbs monoclonal antibodies
  • the differential binding properties of several monoclonal antibodies (MAbs) are used in a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population is recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium. Such a system could be used to identify ACes containing cells.
  • artificial chromosomes derived from dicot plant species can be introduced into monocot plant species by transferring a dicot artificial chromosome.
  • the dicot artificial chromosome possessing a region of euchromatic DNA containing expressed genes.
  • the artificial chromosomes can be designed to allow the artificial chromosome to recombine with the naturally occurring plant DNA in such a fashion that a large region of naturally occurring plant DNA becomes incorporated into the artificial chromosome.
  • an artificial chromosome can be introduced into a wild relative of a crop plant under conditions whereby a portion of the DNA present in the chromosomes of the wild relative is transferred to the artificial chromosome. After isolation of the artificial chromosome, this naturally occurring region of DNA from the wild relative, now located on the artificial chromosome can be introduced into the domesticated crop species and the genes encoded within the transferred DNA expressed arid evaluated for utility. New traits and gene systems can be discovered in this fashion.
  • the artificial chromosome can be modified to contain sequences that promote homologous recombination within plant cells, or be modified to contain a genetic system that functions as a site-specific recombination system.
  • artificial chromosomes include the ability to transfer large regions of DNA from one plant species to another, such as DNA encoding potentially valuable traits such as altered oil, carbohydrate or protein composition, multiple genes encoding enzymes capable of producing valuable plant secondary metabolites, genetic systems encoding valuable agronomic traits such as disease and insect resistance, genes encoding functions that allow association with soil bacterium such as growth promoting bacteria or nitrogen fixing bacteria, or genes encoding traits that confer freezing, drought or other stress tolerances. In this fashion, artificial chromosomes can be used to discover regions of plant DNA that encode valuable traits.
  • the artificial chromosome can also be designed to allow the transfer and subsequent incorporation of these valuable traits now located on the artificial chromosome into the natural chromosomes of a plant species.
  • the artificial chromosomes can be used to transfer large regions of DNA encoding traits normally found in one plant species into another plant species. In this fashion, it is possible to derive a plant cell that no longer needs to carry an artificial chromosome to posses the novel trait.
  • the artificial chromosome would serve as the transfer mechanism to permit the formation of plants with greater degree of genetic diversity.
  • an artificial chromosome to accomplish the aforementioned purposes can include within the artificial chromosome the presence of specific DNA sequences capable of acting as sites for homologous recombination to take place.
  • DNA sequence of Arabidopsis is now known.
  • a sequence of Arabidopsis DNA normally located near a chromosomal location encoding genes of potential interest can be introduced into an artificial chromosome by methods provided herein.
  • the modified artificial chromosome containing the DNA sequences capable of homologous recombination region, can then be introduced into Arabidopsis cells and the homologous recombination event selected.
  • a marker gene it is convenient to include a marker gene to allow for the selection of a homologous recombination event.
  • the marker gene is preferably inactive unless activated by an appropriate homologous recombination event.
  • U.S. Pat. No. 5,272,071 describes a method where an inactive plant gene is activated by a recombination event such that desired homologous recombination events can be easily scored.
  • U.S. Pat. No. 5,501,967 describes a method for the selection of homologous recombination events by activation of a silent selection gene first introduced into the plant DNA, the gene being activated by an appropriate homologous recombination event.
  • both of these methods can be applied to enable a selective process to be included to select for recombination between an artificial chromosome and a plant chromosome.
  • the artificial chromosome Once the homologous recombination event is detected, the artificial chromosome, once selected, is isolated and introduced into a recipient cell, for example, tobacco, corn, wheat or rice, and the expression of the newly introduced DNA sequences evaluated.
  • Phenotypic changes in the recipient plant cells containing the artificial chromosome, or in regenerated plants containing the artificial chromosome allows for the evaluation of the nature of the traits encoded by the Arabidopsis DNA, under conditions naturally found in plant cells, including the naturally occurring arrangement of DNA sequences responsible for the developmental control of the traits in the normal chromosomal environment.
  • the large scale order and structure of the artificial chromosome provides a number of unique advantages in screening for new utilities or novel phenotypes within heterologous plant species.
  • the size of new DNA that can be carried by an artificial chromosome can be millions of base pairs of DNA, representing potentially numerous genes that may have novel utility in a heterologous plant cell.
  • the artificial chromosome is a “natural” environment for gene expression, the problems of variable gene expression and silencing seen for genes transferred by random insertion into a genome should not be observed. Similarly, there is no need to engineer the genes for expression, and the genes inserted would not need to be recombinant genes. Thus, one expects the expression from the transferred genes to be temporal and spatial, as observed in the species from where the genes were initially isolated.
  • a valuable feature for these utilities is the ability to isolate the artificial chromosomes and to further isolate, manipulate and introduce into other cells artificial chromosomes carrying unique genetic compositions.
  • artificial chromosomes In addition to the use of artificial chromosomes for the isolation and testing of large regions of naturally occurring DNA, methods for the use of artificial chromosomes and cloned DNA are also contemplated. Similar to that described above, artificial chromosomes can be used to carry large regions of cloned DNA, including that derived from other plant species.
  • the artificial chromosomes can be engineered as platforms to accept large regions of cloned DNA, such as that contained in Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs). It is further contemplated, that as a result of the typical structure of artificial chromosomes containing tandemly repeated DNA blocks, that sequences other than cloned DNA sequence can be introduced by recombination processes. In particular recombination within a predefined region of the tandemly repeated DNA within the artificial chromosome provides a mechanism to “stack” numerous regions of cloned DNA, including large regions of DNA contained within BACs or YACs clones.
  • BACs Bacterial Artificial Chromosomes
  • YACs Yeast Artificial Chromosomes
  • multiple combinations of genes can be introduced onto artificial chromosomes and these combinations tested for functionality.
  • multiple YACs or BACs can be stacked onto an artificial chromosomes, the BACs or YACs containing multiple genes of complex pathways or multiple genetic pathways.
  • the BACs or YACs are typically selected based on genetic information available within the public domain, for example from the Arabidopsis Information Management System (http://aims.cps.msu.edu/aims/index.html) or the information related to the plant DNA sequences available from the Institute for Genomic Research (http://www.tigr.org) and other sites known to those skilled in the art.
  • clones can be chosen at random and evaluated for functionality. It is contemplated that combinations providing a desired phenotype can be identified by isolation of the artificial chromosome containing the combination and analyzing the nature of the inserted cloned DNA.
  • site-specific recombination sequences can have considerable utility in developing artificial chromosomes containing DNA sequences recognized by recombinase enzymes and capable of accepting DNA sequences containing same.
  • site-specific recombination as a means to target an introduced DNA to a specific locus has been demonstrated in the art and such methods can be employed.
  • the recombinase systems can also be used to transfer the cloned DNA regions contained within the artificial chromosome to the naturally occurring plant or mammalian chromosomes.
  • the integration function of site-specific recombinases is contemplated as a means to assist in the derivation of genetic combinations on artificial chromosomes.
  • a first step of introducing site-specific recombinase sites into the genome of a plant cell in an essentially random manner is conducted, such that the plant cell has one or more site-specific recombinase recognition sequences on one or more of the plant chromosomes.
  • An artificial chromosome is then introduced into the plant cell, the artificial chromosome engineered to contain a recombinase recognition site (e.g., integration site) capable of being recognized by a site-specific recombinase.
  • a gene encoding a recombinase enzyme is also included, preferably under the control of an inducible promoter.
  • Expression of the site-specific recombinase enzyme in the plant cell either by induction of a inducible recombinase gene, or transient expression of a recombinase sequence, causes a site-specific recombination event to take place, leading to the insertion of a region of the plant chromosomal DNA (containing the recombinase recognition site) into the recombinase recognition site of the artificial chromosome, and forming an artificial chromosome containing plant chromosomal DNA.
  • the artificial chromosome can be isolated and introduced into a heterologous host, preferably a plant host, and expression of the newly introduced plant chromosomal DNA can be monitored and evaluated for desirable phenotypic changes. Accordingly, carrying out this recombination with a population of plant cells wherein the chromosomally located recombinase recognition site is randomly scattered throughout the chromosomes of the plant, can lead to the formation of a population of artificial chromosomes, each with a different region of plant chromosomal DNA, and each potentially representing a novel genetic combination.
  • Cells were re-transformed by electroporation with a plasmid that contained a promoter with a lox sequence and a transiently expressed Cre recombinase gene.
  • the expression of the Cre enzyme catalyzed the homologous recombination between the lox site in the chromosomally located promoter-less antibiotic resistance gene, and the lox site in the introduced promoter sequence, leading to the formation of a functional antibiotic resistance gene.
  • the authors demonstrated efficient and correct targeting of the introduced sequence, 54 of 56 lines analyzed corresponded to the predicted single copy insertion of the DNA due to Cre catalyzed site-specific homologous recombination between the lox sequences.
  • a lox sequence may be first added to a genome of a plant species capable of being transformed and regenerated to a whole plant to serve as a recombinase target DNA sequence for recombination with an artificial chromosome.
  • the lox sequence may be optimally modified to further contain a selectable marker which is inactive but can be activated by insertion of the lox recombinase recognition sequence into the artificial chromosome.
  • a promoterless marker gene or selectable marker gene linked to the recombinase recognition sequence which is first inserted into the chromosomes of a plant cell can be used to engineer a platform chromosome.
  • a promoter is linked to a recombinase recognition site, in an orientation that allows the promoter to control the expression of the marker or selectable marker gene upon recombination within the artificial chromosome.
  • a cell Upon a site-specific recombination event between a recombinase recognition site in a plant chromosome and the recombinase recognition site within the introduced artificial chromosome, a cell is derived with a recombined artificial chromosome, the artificial chromosome containing an active marker or selectable marker activity that permits the identification and or selection of the cell.
  • the artificial chromosomes can be transferred to other plant or animal species and the functionality of the new combinations tested.
  • the ability to conduct such an inter-chromosomal transfer of sequences has been demonstrated in the art.
  • the use of the Cre-lox recombinase system to cause a chromosome recombination event between two chromatids of different chromosomes has been shown.
  • Any number of recombination systems may be employed as described herein, such as, but not limited to, bacterially derived systems such as the att/int system of phage lambda, and the Gin/gix system.
  • More than one recombination system may be employed, including, for example, one recombinase system for the introduction of DNA into an artificial chromosome, and a second recombinase system for the subsequent transfer of the newly introduced DNA contained within an artificial chromosome into the naturally occurring chromosome of a second plant species.
  • the choice of the specific recombination system used will be dependent on the nature of the modification contemplated.
  • ACes, cell lines and methods for use in screening a new chromosomal combinations, deletions, truncations with eucaryotic genome that take advantage of the site-specific recombination systems incorporated onto platform ACes provided herein.
  • a cell line useful for making a library of ACes comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes.
  • Also provided herein is a method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into a cell line comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell's chromosomal DNA; and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within.
  • a library of cells useful for genomic screening comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein.
  • the library of cells can be from a different species and/or cell type than the chromosomal nucleic acid within the ACes.
  • a method of making one or more cell lines comprising
  • ACes, cell lines and methods utilize the site-specific recombination sites on platform ACes analogous YAC manipulation related to: the methods of generating terminal deletions in normal and artificial chromosomes (e.g., ACes; as described in Vollrath et al., 1988 , PNAS, USA, 85:6027-66031; and Pavan et al., PNAS, USA, 87:1300-1304); the methods of generating interstitial deletions in normal and artificial chromosomes (as described in Campbell et al., 1991 , PNAS, USA, 888:5744-5748); and the methods of detecting homologous recombination between two ACes (as described in Cellini et al., 1991 , Nuc. Acid Res., 19(5):997-1000).
  • ACes as described in Vollrath et al., 1988 , PNAS, USA, 85:6027-66031; and Pavan et al., PNAS, USA
  • the platform can be engineered via the IntR lambda integrase to carry reporter-linked constructs (reporter genes) that monitor changes in cellular physiology as measured by the particular reporter gene (or a series of different reporter genes) readout.
  • reporter linked constructs are designed to include a gene that can be detected (by for example fluorescence, drug resistance, immunohistochemistry, or transcript production, and the like) with well-known regulatory sequences that would control the expression of the detectable gene.
  • Exemplary regulatory promoter sequences are well-known in the art:
  • the ACes can be engineered to carry reporter-linked constructs that indicate a signal is being transduced through one or a number of pathways.
  • transcriptionally regulated promoters from genes at the end (or any other chosen point) of particular signal transduction pathways could be engineered on the ACes to express the appropriate readout (either by fluorescent protein production or drug resistance) when the pathway is activated (or down-regulated as well).
  • a number of reporters from different can be placed on a ACes chromosome. Cells (and/or whole animals) containing such a Reporter ACes could be exposed to a variety of drugs or compounds and monitored for the effects of the drugs or compounds upon the selected pathway(s) by the reporter gene(s).
  • drugs or compounds can be classified or identified by particular pathways they excite or down-regulate.
  • transcriptional profiles obtained from genomic array experiments can be biologically validated using the reporter ACes provided herein.
  • Environmental or man-made genotoxicants can be tested in cell lines carrying a number of reporter-genes platform ACes linked to promoters that are transcriptionally regulated in response to DNA damage, induced apoptosis or necrosis, and cell-cycle perturbations.
  • new drugs and/or compounds could be tested in a similar manner with the genotoxicant ACes reporter for their cellular/genetic toxicity by such a screen.
  • toxic compound testing could be carried out in whole transgenic animals carrying the ACes chromosome that measures genotoxicant exposure (“canary in a coal mine”).
  • the same or similar type ACes could be used for toxicity testing in either a cell-based or whole animal setting.
  • An example would include ACes that carry reporter-linked genes controlled by various cytochrome P450 profiled promoters and the like.
  • a common disease may arise via various mechanisms. In many instances there are multiple treatments available for a given disease. However, the success of a given treatment may depend upon the mechanism by which the disease originated and/or by the genetic background of the patient. In order to establish the most effective treatment for a given patient one could utilize the ACes reporters provided herein. ACes reporters can be used in patient cell samples to determine an individualized drug regimen for the patient. In addition, potential polymorphisms affecting the transcriptional regulation of an individual's particular gene can be assessed by this approach.
  • cancer cells arise via different mechanisms. Furthermore, as a cancerous cell propagates it may undergo genomic alterations. An ACes reporter transferred to cells of different patients having the same disease, i.e. similar cancers, could be used to categorize the particular cancer of each patient, thereby facilitating the identification of the most effective therapeutic regimen. Examples would include the validation of array profiling of certain classes of breast cancers. Subsequently, appropriate drug profiling could be carried out as described above.
  • ACes reporter as a “differentiation” sensor in stem cells or other progenitor cells in order to enrich by selection (either FACS based screening, drug selection and/or use of suicide gene) for a particular class of differentiated or undifferentiated cells.
  • this assay could also be used for compound screening for small molecule modifiers of cell differentiation.
  • any of the above Reporter ACes methods could be used in conjunction with whole-body imaging to monitor reporter genes within whole animals without sacrificing the animal. This would allow temporal and spatial analysis of expression patterns under a given set of conditions.
  • the conditions tested may include for example, normal differentiation of a stem cell, response to drug or compound treatment whether targeted to the diseased tissue or presented systemically, response to genotoxicants, and the like.
  • Cosmid pFK161 (SEQ ID NO: 118) was obtained from Dr. Gyula Hadlaczky and contains a 9 kb NotI insert derived from a murine rDNA repeat (see clone 161 described in PCT Application Publication No. WO97/40183 by Hadlaczky et al. for a description of this cosmid).
  • This cosmid referred to as clone 161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQ ID NO. 26. It was produced by inserting fragments of the megachromosome (see, U.S. Pat. No. 6,077,697 and International PCT application No. WO 97/40183).
  • H1D3 which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome into plasmid pWE15 (Stratagene, La Jolla, Calif.; SEQ ID No. 31) as follows.
  • Half of a 100 ⁇ l low melting point agarose block (mega-plug) containing isolated SATACs was digested with NotI overnight at 37° C. Plasmid pWE15 was similarly digested with NotI overnight.
  • the mega-plug was then melted and mixed with the digested plasmid, ligation buffer and T4 DNA ligase. Ligation was conducted at 16° C. overnight.
  • Bacterial DH5 ⁇ cells were transformed with the ligation product and transformed cells were plated onto LB/Amp plates. Fifteen to twenty colonies were grown on each plate for a total of 189 colonies. Plasmid DNA was isolated from colonies that survived growth on LB/Amp medium and analyzed by Southern blot hybridization for the presence of DNA that hybridized to a pUC19 probe. This screening methodology assured that all clones, even clones lacking an insert but yet containing the pWE15 plasmid, would be detected.
  • the clone was digested with NotI and BamHI and ligated with NotI/BamHI-digested pBluescript KS (Stratagene, La Jolla, Calif.). Two fragments of the insert of clone no. 161 were obtained: a 0.2-kb and a 0.7-kb insert fragment. To subclone the internal fragment of the insert of clone no. 161, the same digest was ligated with BamHI-digested pUC19. Three fragments of the insert of clone no. 161 were obtained: a 0.6-kb, a 1.8-kb and a 4.8-kb insert fragment.
  • the insert corresponds to an internal section of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 as set forth in GENBANK accession no. X82564, which is provided as SEQ ID NO. 18.
  • the sequence data obtained for the insert of clone no. 161 is set forth in SEQ ID NOS. 19-25.
  • the individual subclones corresponded to the following positions in GENBANK accession no. X82564 (SEQ ID NO:18) and in SEQ ID NOs. 19-25: Start End Subclone in X82564 Site SEQ ID No.
  • BamHI 19 161m5 7756 8494 BamHI 20 161m7 8495 10231
  • BamHI 21 shows only sequence corresponding to nt. 8495-8950
  • 22 shows only sequence corresponding to nt. 9851-10231
  • BamHI 23 shows only sequence corresponding to nt. 10232-10600
  • 24 shows only sequence corresponding to nt. 14267-15000
  • the rDNA insert from the clone was prepared by digesting the cosmid with NotI and BglII and was purified as described above. Growth and maintenance of bacterial stocks and purification of plasmids were performed using standard well known methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press), and plasmids were purified from bacterial cultures using Midi- and Maxi-preps Kits (Qiagen, Mississauga, Ontario).
  • This vector is available from Clontech (see SEQ ID No. 29) and encodes the red fluorescent protein (DsRed; Genbank accession no. AF272711; SEQ ID Nos. 39 and 40). DsRed, which has a vivid red fluorescence, was isolated from the Indo Pacific sea anemone relative Discosoma species.
  • the plasmid pDsRed1N1 constitutively expresses a human codon-optimized variant of the fluorescent protein under control of the CMV promoter.
  • this vector expresses high levels of DsRed1 and includes sites for creating N-terminal fusions by cloning proteins of interest into the multiple cloning site (MCS). It is Kan and Neo resistant for selection in bacterial or eukaryotic cells.
  • Plasmid pMG (InvivoGen, San Diego, Calif.; see SEQ. ID. NO. 27 for the nucleotide sequence of pMG) contains the hygromycin phosphotransferase gene under the control of the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A.
  • Vector pMG also contains two transcriptional units allowing for the coexpression of two heterologous genes from a single vector sequence.
  • the first transcriptional unit of pMG contains a multiple cloning site for insertion of a gene of interest, the hygromycin phosphotransferase gene (hph) and the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A (see, e.g., Chapman et al. (1991) Nuc. Acids Res. 19:3979-3986) located upstream of hph and the multiple cloning site, which drives the expression of hph and any gene of interest inserted into the multiple cloning site as a polycistronic mRNA.
  • hph hygromycin phosphotransferase gene
  • hCMV immediate-early human cytomegalovirus
  • the first transcriptional unit also contains a modified EMCV internal ribosomal entry site (IRES) upstream of the hph gene but downstream of the hCMV promoter and MCS for ribosomal entry in translation of the hph gene (see SEQ ID NO. 27, nucleotides 2736-3308).
  • the IRES is modified by insertion of the constitutive E. coli promoter (EM7) within an intron (IM7) into the end of the IRES.
  • EM7 constitutive E. coli promoter
  • IM7 intron
  • the E. coli promoter is treated as an intron and is spliced out of the transcript.
  • a polyadenylation signal from the bovine growth hormone (bGh) gene see, e.g., Goodwin and Rottman (1992) J. Biol.
  • the second transcriptional unit of pMG contains another multiple cloning site for insertion of a gene of interest and an EF-1 ⁇ /HTLV hybrid promoter located upstream of this multiple cloning site, which drives the expression of any gene of interest inserted into the multiple cloning site.
  • the hybrid promoter is a modified human elongation factor-1 alpha (EF-1 alpha) gene promoter (see, e.g., Kim et al. (1990) Gene 91:217-223) that includes the R segment and part of the U5 sequence (R-U5′) of the human T-cell leukemia virus (HTLV) type I long terminal repeat (see, e.g., Takebe et al. (1988) Mol. Cell.
  • Vector pMG contains a synthetic polyadenylation site for the first and second transcriptional units at the end of the transcriptional unit based on the rabbit, ⁇ -globin gene and containing the AATAAA hexanucleotide sequence and a GT/T-rich sequence with 22-23 nucleotides between them (see, e.g., Levitt et al. (1989) Genes Dev. 3:1019-1025).
  • a pause site derived from the C2 complement gene is also located at the 3′ end of the second transcriptional unit.
  • Vector pMG also contains an ori sequence (ori pMB1) located between the SV40 polyadenylation signal and the synthetic polyadenylation site.
  • ori sequence ori pMB1
  • a targeting vector derived from the vector pWE15 (GeneBank Accession #X65279) was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance containing fragment with the PvuII/BamHI (Klenow filled) puromycin resistance containing fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc. Palo Alto, Calif.; SEQ ID No. 30) resulting in plasmid pWEPuro.
  • a subclone was isolated containing an artificial chromosome, designated 5B11.12, which carries 4-8 copies of the puromycin resistance gene contained on the pWEPuro9K vector.
  • FISH analysis of the 5B11.12 subclone demonstrated the presence of telomeres and mouse minor on the ACes.
  • DOT PCR has been done on the 5B11.12 ACes revealing the absence of uncharacterized euchromatic regions on the ACes.
  • a recombination site such as an att or loxP engineering site or a plurality thereof, was introduced onto this ACes thereby providing a platform for site-specific introduction of heterologous nucleic acid.
  • a single sequence-specific recombination site is placed onto the platform chromosome via homologous recombination.
  • DNA sequences containing the site-specific recombination sequence can be flanked with DNA sequences of homology to the platform chromosome.
  • mouse rDNA sequences or mouse major satellite DNA can be used as homologous sequences to target onto the platform chromosome.
  • a vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the platform chromosome, the DNA is transfected into cells harboring the platform chromosome (FIG. 3).
  • site-specific cassettes that are targeted to the platform chromosome using either mouse rDNA or mouse major repeat DNA include the SV40-attP-hygro cassette and a red fluorescent protein (RFP) gene flanked by loxP sites (Cre/lox, see, e.g., U.S. Pat. No. 4,959,317 and description herein).
  • homologous recombination events onto the platform chromosome are subcloned and identified by FACS (e.g. screen and single cell subclone via expression of resistance or fluorescent marker) and PCR analysis.
  • a vector can be constructed containing regions of the mouse rDNA locus flanking a gene cassette containing the SV40 early reporter-bacteriophage lambda attP site-hygromycin selectable marker (see FIG. 4 and described below).
  • the use of the bacteriophage lambda attP site for lambda integrase-mediated site-specific recombination is described below.
  • Homologous recombination event of the SV40-attP-hygro cassette onto the platform chromosome was identified using PCR primers that detect the homologous recombination and further confirmed by FISH analysis.
  • cells carrying the platform chromosome with a single site-specific recombination site can now be engineered with site-specific recombinases (e.g. lambda INT, Cre) for integrating a target gene expression vector.
  • site-specific recombinases e.g. lambda INT, Cre
  • rDNA sequences were chosen as the target on the ACes since they represent a less frequent target than that of the satellite repeat sequences.
  • a targeting vector based on the rDNA gene segment in pWEPuro9K would have a higher probability of targeting to the ACes rather than to other LMTK ⁇ chromosomes. Accordingly, a targeting vector, pBSFKLoxDsRedLox, was designed and constructed based on the rDNA sequences contained in pWEPuro9K.
  • the plasmid pBSFKLoxDsRedLox was generated in 4 steps.
  • the DsRed gene from pDsRed1-N1 was then cloned into the polylinker between the loxP sites generating p193LoxDsRedLox.
  • a fragment consisting of the DsRed gene flanked by loxP sites was cloned into a unique NdeI within the rDNA insert of pBSFK generating pBSFKLoxDsRedLox.
  • a gel purified 11 Kb Pml/EcoRV fragment of pBSFKLoxDsRedLox was used for transfection.
  • PCR primers were designed from rDNA sequences within the 5′ NotI-PmlI fragment of pWEPuro9K that is not present on the targeting fragment (5′primer) and sequence within the LoxDsRedLox cassette (3′ primer). If the targeting DNA integrated correctly within the rDNA sequences, PCR amplification using these primers would give rise to a 2.3 Kb band.
  • PCR reactions containing 1-4 ⁇ l of genomic DNA were carried out according to the MasterTaq protocol (Eppendorf), using murine rDNA 5′ primer (5′-CGGACAATGCGGTTGTGCGT-3′; SEQ ID NO:72) and DsRed 3′primer (5′GGCCCCGTAATGCAGAAGAA-3′; SEQ ID NO:73) and PCR products were analyzed by agarose gel electrophoresis.
  • the RFP positive gated populations were recovered, diluted in medium supplemented with 1 ⁇ penicillin-streptomycin (Invitrogen), then plated and cultured as previously described. After 4 rounds of enrichment, the percentage of RFP positive cells reached levels of 50% or higher. DNA from populations was analyzed by PCR for evidence of targeted integration. Ultimately, single cell subclones were established from positive pools and were analyzed by PCR and PCR-positive clones confirmed by FISH as described below.
  • DNA was purified from pools or single cell clones using previously described methods set forth in Lahm et al., Transgenic Res., 1998; 7:131-134, or in some cases using a Wizard Genomic DNA purification kit (Promega).
  • a biotinylated DsRed gene probe was generated by PCR using DsRed specific primers and biotin-labeled dUTP (5′ RFP primer: 5′-GGTTTAAAGTGCGCTCCTCCAAGAACGTCATC-3′, SEQ ID NO:74; and 3′ RFP primer: 5′AGATCTAGAGCCGCCGCTACAGGAACAGGTGGTGGCGGCC-3′; SEQ ID NO:75).
  • Tyramide amplification was carried out according to the manufacturers protocols (NEN).
  • PCR screening with primers that amplify from a spacer region within the segment of the 45s pre-rRNA gene in pWEPuro9K to a specific anchor sequence within the DsRed gene in the targeting cassette would give rise to a diagnostic 2.3 Kb band.
  • confirmation of targeting to an ACes would require fluorescence in situ hybridization (FISH) analysis.
  • FISH fluorescence in situ hybridization
  • PCR analysis of genomic DNA isolated from the D11-C4 subclone gave rise to a 2.3 Kb band, indicative of a targeted integration into an rDNA locus.
  • Further analysis of the subclone by FISH analysis with a DsRed gene probe demonstrated integration of the LoxDsRedLox targeting cassette on the ACes co-localizing with one of the regions of rDNA staining seen on the 5B11-12 ACes, consistent with a targeted integration into an rDNA locus of the ACes, while integrations on other chromosomes were not observed. Since transfected cells were maintained as heterogeneous populations through several cycles of sorting and replating it was not possible to estimate the frequency of targeted events.
  • the number of loxP sites on the ACes can be reduced to a single site by in situ treatment with Cre recombinase, provided that the sites are co-linear.
  • Cre recombinase Such a process is described for multiple loxP-flanked integrations on a native mouse chromosome (Garrick et al., Nature Genet., 1998, Jan;18(1):56-59). Reduction to a single loxP site on the D11-C4 ACes would result in the loss of the DsRed gene, forming the basis of a useful screen for this event.
  • a Cre expression plasmid pCX-Cre/GFP III has been generated by first deleting the EcoRi fragment of pCX-eGFP (SEQ ID NO:71) containing the eGFP coding sequence and replacing it with that of a PCR amplified Cre recombinase coding sequence (SEQ ID NO:58), generating pCX-Cre.
  • LMTK ⁇ cell line D11-C4 (containing first generation platform ACes with multiple loxP-DsRED sites) and 5B11-12 cell line (containing ACes with no loxP-DsRED sites) are maintained in culture as described above.
  • D11C4 cells are transfected with 2 ⁇ g of plasmid pCX-Cre ⁇ GFP III or 2 ⁇ g pCX-CreRev ⁇ GFP III using Lipofectamine (Invitrogen) as previously described.
  • transfected D11-C4 cells are harvested and GFP positive cells are sorted by cell cytometry using a FACSta Vantage cell sorter (Beckton-Dickinson) as follows: All D11-C4 cells transfected with pCX-Cre ⁇ GFP III or control plasmid pCX-CreRev ⁇ GFP III that exhibit GFP fluorescent higher than the gate level established by untransfected cells are collected and placed in culture a further 7-14 days.
  • D11-C4 cells are harvested and analyzed by cell cytometry as follows: Untransfected D11-C4 cells are used to establish the gate that defines the RFP positive population, while 5B11-12 cells are used to set the RFP negative gate.
  • Untransfected D11-C4 cells are used to establish the gate that defines the RFP positive population, while 5B11-12 cells are used to set the RFP negative gate.
  • the GFP positive population of D11-C4 transfected with pCX-Cre ⁇ GFP III should show decreased red fluorescence compared to pCX-CreRev ⁇ GFP III transfected or untransfected control D11-C4 cells.
  • the cells exhibiting greatly decreased or no RFP expression are collected and single cell clones subsequently established. These clones will be expanded and analyzed by fluorescence in-situ hybridization and Southern blotting to confirm the removal of loxP-DsRed gene copies.
  • FIG. 4 An example of a selectable marker system for the creation of a chromosome-based platform is shown in FIG. 4.
  • This system includes a vector containing the SV40 early promoter immediately followed by (1) a 282 base pair (bp) sequence containing the bacteriophage lambda attP site and (2) the puromycin resistance marker.
  • bp 282 base pair
  • bp 282 base pair
  • a PvuII/StuI fragment containing the SV40 early promoter from plasmid pPUR (Clontech Laboratories, Inc., Palo Alto, Calif.; Seq ID No. 30) was subcloned into the EcoRI/CRI site of pNEB193 (a PUC19 derivative obtained from New England Biolabs, Beverly, Mass.; SEQ ID No.
  • GGCGCGCC A unique Ascl site is located between the BamHI site and the SmaI site, a unique PacI site (TTAATTAA) is located between the BamHI site and the XbaI site and a unique PmeI site (GTTTAAAC) is located between the PstI site and the SalI site.
  • AttP site was PCR amplified from lambda genome (GenBank Accession # NC 001416) using the following primers: attPUP: CCTTGCGCTAATGCTCTGTTACAGG SEQ ID No.1 attPDWN: CAGAGGCAGGGAGTGGGACAAAATTG SEQ ID No.2
  • the attP site was cloned into the SmaI site of pSV40193 and the orientation of the attP site was determined by DNA sequence analysis (plasmid pSV40193attP).
  • the gene encoding puromycin resistance (Puro) was isolated by digesting the plasmid pPUR (Clontech Laboratories, Inc. Palo Alto, Calif.) with AgeI/BamHI followed by filling in the overhangs with Klenow and subsequently cloned into the AscI site downstream of the attP site of pSV40193attP generating the plasmid pSV40193attPsensePUR (FIG. 4; SEQ ID NO:113)).
  • the plasmid pSV40193attPsensePUR was digested with ScaI and co-transfected with the plasmid pFK161 (SEQ ID NO: 118) into mouse LMtk ⁇ cells and platform artificial chromosomes were identified and isolated as described above.
  • the process for generating this exemplary platform ACes containing multiple site-specific recombination sites is summarized in FIG. 5.
  • One platform ACes resulting from this experiment is designated B19-18.
  • This platform ACes chromosome may subsequently be engineered to contain target gene expression nucleic acids using the lambda integrase mediated site-specific recombination system as described herein in Example 7 and 8.
  • a vector expressing the red fluorescent protein (RFP) was produced and recombined into the attP site residing on an artificial chromosome within LMTK ⁇ cells. This recombination is depicted in FIG. 7.
  • lambda integrase was PCR amplified from bacteriophage lambda DNA (c1857 ind Sam 7; New England Biolabs) using the following primers: Lamint1 TTCGAATTCATGGGAAGAAGGCGAAGTCATGAGCG) (SEQ ID No.3) Lamint2 (TTCGAATTGTTATTTGATTTCAATTTTGTCCCAC). (SEQ ID No.4)
  • Lambda integrase was mutated at amino acid position 174 using QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligos (generating a glutamic acid to arginine change at position 174): LambdaINTE174R (SEQ ID No.6) (CGCGCAGCAAAATCTAGAGTAAGGAGATCAAGACTTACGGCTGACG), LamintR174rev (SEQ ID No.7) (CGTCAGCCGTAAGTCTTGATCTCCTTACTCTAGATTTTGCTGCGCG).
  • the resulting site directed mutant was confirmed by sequence analysis.
  • the wildtype and mutant lambda genes were cloned into the EcoR I site of pCX creating pCX-Lamint (SEQ ID NO: 127) and pCXLamIntR (FIG. 8; SEQ ID NO: 112).
  • the plasmid pCX (SEQ ID No. 70) was derived from plasmid pCXeGFP (SEQ ID No. 71). Excision of the EcoRI fragment containing the eGFP marker generated pCX.
  • plasmid pCXLamINTR (SEQ ID NO: 112) an EcoRI fragment containing the lambda integrase El 74R (SEQ ID No. 37) mutation was cloned into the EcoRI site of pCX, and to generate plasmid pCX-LamINT, an EcoRI fragment containing the wildtype lambda integrase was cloned into the EcoRI site of pCX.
  • the plasmid pDsRedN1 (Clontech Laboratories, Palo Alto, Calif.; SEQ ID No. 29) was digested with Hpa I and ligated to the following annealed oligos: attB1 (TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA) (SEQ ID No.8) attB2 (TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA) (SEQ ID No.9)
  • LM(tk ⁇ ) cells containing the Prototype A ACes were co-transfected with pDsRedN1 or pDsRedN1-attB and either pCXLamInt (SEQ ID NO: 127) or pCXLamIntR (SEQ ID NO: 112) using Lipofectamine Plus Reagent (LifeTechnologies, Gaithersburg, Md.).
  • the transfected cells were grown in DMEM (LifeTechnologies, Gaithersburg, Md.) with 10% FBS (CanSera) and G418 (CalBiochem) at a concentration of 1 mg/ml.
  • the transfected cells were sorted using a FACs Vantage SE cell sorter (Becton Dickenson) to enrich for cells expressing DsRed.
  • the cells were excited with a 488 nm Argon laser at 200 watts and cells fluorescing in the 585/42 detection channel were collected.
  • the sorted cells were returned to growth medium for recovery and expansion.
  • single cell sorting into 96 well plates was performed using the same parameters. Duplicate plates of the single cell clones were made for PCR analysis.
  • the resulting PCR reactions were analyzed by gel electrophoresis and the potential individual clones containing the site-specific recombination event were identified by combining the PCR results of all of the pooled rows and columns for each 96 well plate.
  • the individual clones were then further analyzed by PCR using the following primers that flank the recombination junction. L1for and F1rev flank the attR junction whereas REDfor and L2rev flank the attL junction (see FIG.
  • PCR products spanning the recombination junction were Topo-cloned into pcDNA3.1D/V5His (Invitrogen Inc., San Diego, Calif.) and then sequenced by cycle-sequencing. The clones were confirmed to have the correct attR and attL junctions by cycle sequencing.
  • FISH Fluorescent In situ Hybridization
  • the cell lines containing the correct recombination junction sequence were further analyzed by fluorescent in situ hybridization (FISH) by probing with the DsRed coding region labeled with biotin and visualizing with the Tyramide Signal Amplification system (TSA; NEN Life Science Products). The results indicate that the RFP sequence is present on the ACes.
  • FISH fluorescent in situ hybridization
  • Genomic DNA was harvested from the cell lines containing an ACes with the correct recombinant event and digested with EcoR I. The digested DNAs were separated on a 0.7% agarose gel, transferred and fixed to a nylon membrane and probed with RFP coding sequences. The result showed that there is an integrated copy of RFP coding sequence in each clone.
  • the plasmid pIRESpuro2 (Clontech, Palo Alto, Calif.; SEQ ID NO: 88) was digested with EcoRI and NotI then ligated to the D2eGFP EcoRI-NotI fragment from pD2eGFP-N1 (Clontech, Palo Alto, Calif.) to create pD2eGFPIresPuro2. Subsequently, oligos encoding the attB site were annealed and ligated into the NruI site of pD2eGFPIresPuro2 to create pD2eGFPIresPuroattB. The orientation of attB in the NruI site was determined by PCR.
  • Twenty ⁇ l of the Plus reagent was added to the DNA and incubated at room temperature for 15 minutes.
  • a mixture of 30 ⁇ l of lipofectamine and 750 ⁇ l DMEM was added to the DNA mixture and incubated an additional 15 minutes at room temperature.
  • the DNA mixture was then added dropwise to approximately 3 million cells attached to a 10 cm dish in 5 mls of DMEM.
  • the cells were incubated 4 hours (37° C., 5% CO 2 ) with the DNA-lipid mixture, after which DMEM with 20% fetal bovine serum was added to the dishes to bring the culture medium to 10% fetal bovine serum.
  • the dishes were incubated at 37° C. with 5% CO 2 .
  • Plasmid pD2eGFPIresPuroattB has a puromycin gene transcriptionally linked to the GFP gene via an IRES element. Two days after the transfection the cells were placed in medium containing puromycin at 4 ⁇ g/ml to select for cells containing the pD2eGFPIresPuroattB plasmid integrated into the genome. Twenty-three clones were isolated after 17 days of selection with puromycin. These clones were expanded and then analyzed for the presence of the GFP gene on the ACes by 2-color (RFP/biotin & GFP/digoxigenin) TSA-FISH (NEN) according to the manufacturers protocol. Sixteen of the 23 clones produced a positive FISH signal on the ACes with a GFP probe.
  • Percent transfected cells was determined on a FACS Vantage flow cytometer with argon laser tuned to 488 nm at 200 mW and FITC fluorescence collected through a standard FITC 530/30 nm band pass filter. After 24 hours, IdUrd labeled ACes were delivered to human MSCs in the range of 30-50%, varying with transfection agent and dose. ACes delivery curves were generated from data collected in experiments that varyied the dose of the transfection reagents. Dose response curves of Superfect and LipofectAMINE PLUS, showing delivery of ACes into recipient hMSCs cells, were prepared, measured by transfer of IdUrd labeled ACes and detected by flow cytometry.
  • the hygromycin resistant population was then blocked in mitosis with colchicine and analyzed for presence of intact ACes by FISH.
  • Preliminary FISH results show approximately 2-8% of the hMSC-transfected population had an intact ACes. This compared to rat skeletal muscle myoblast clones, which were in the range of 60-95%.
  • an enrichment step can be utilized as described in Example 2C.
  • ACes artificial chromosomes
  • Targeting MSCs permits gene transfer into cells in an undifferentiated state where the cells are easier to expand and purify.
  • the genetically modified cells can then be differentiated in vitro or injected into a site in vivo where the microenvironment will induce transformation into specific cell lineages.
  • Platform ACes containing pSV40attPsensePURO were constructed as set forth in Examples 3 and 4.
  • the base vector p18attBZeo (3166 bp; SEQ ID NO: 114) was constructed by ligating the 1067 bp HindIII-SspI fragment containing attBZeo, obtained from pLITattBZeo (SEQ ID NO:91), into pUC18 (SEQ ID NO: 122) digested with HindIII and SspI.
  • p18attBZEO-eGFP (6119 bp; SEQ ID NO: 126) was constructed by inserting the 2977 bp. SpeI-HindIII fragment from pCXeGFP (SEQ ID NO:71; Okabe, et al. (1997) FEBS Lett 407:313-319) containing the eGFP gene into p18attBZeo (SEQ ID NO: 114) digested with HindIII and XbaI.
  • p18attBZEO-5′6XHS4eGFP (FIG. 10; 7631 bp; SEQ ID NO: 116) was constructed by ligating the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5′ of the chicken beta actin promoter and the polyadenylation signal into the HindIII site of p18attBZeo (SEQ ID NO: 114).
  • p18attBZEO-3′6XHS4eGFP (FIG. 11; 7600 bp; SEQ ID NO: 115) was created by removing the 5′6XHS4 element from p18attBZeo-(6XHS4)2eGFP (SEQ ID NO: 110).
  • p18attBZeo-(6XHS4)2eGFP was digested with EcoRV and SpeI, treated with Klenow and religated to form p18attBZeo3′6XHS4eGFP (SEQ ID NO: 115).
  • p18attBZEO-(6XHS4)2eGFP (FIG. 12; 9080 bp; SEQ ID NO: 110) was created in two steps. First, the EcoRI-SpeI fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains 6 copies of the HS4 core element was ligated into p18attBZeo (SEQ ID NO: 114) digested with EcoRI and XbaI to create p18attBZeo6XHS4 (4615 bp; SEQ ID NO: 117).
  • p18attBZeo6XHS4 was digested with HindIII and ligated to the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5′ of the chicken beta actin promoter and the polyadenylation signal.
  • the mouse cell line containing the 2 nd generation platform ACE, B19-38 (constructed as set forth in Example 3), was plated onto four 10 cm dishes at approximately 5 million cells per dish. The cells were incubated overnight in DMEM with 10% fetal calf serum at 37° C. and 5% CO 2 . The following day the cells were transfected with 5 ⁇ g of each of the 4 vectors listed in Example 7.A. above and 5 ⁇ g of pCXLamintR (SEQ ID NO: 112), for a total of 10 ⁇ g per 10 cm dish. Lipofectamine Plus reagent was used to transfect the cells according to the manufacturers protocol. Two days post-transfection zeocin was added to the medium at 500 ug/ml. The cells were maintained in selective medium until colonies formed. The colonies were then ring-cloned (see, e.g., McFarland, 2000 , Methods Cell Sci, Mar; 22(1):63-66).
  • Genomic DNA was isolated from each of the candidate clones with the Wizard kit (Promega) and following the manufacturers protocol. The following primer set was used to analyze the genomic DNA isolated from the zeocin resistant clones: 5PacSV40 5PacSV40- CTGTTAATTAACTGTGGAATGTGTGTCAGTTAGGGTG; (SEQ ID NO:76) Antisense Zeo- TGAACAGGGTCACGTCGTCC. (SEQ ID NO:77)
  • PCR primers are designed to contain an attB site at the 5′ end of one of the primers in the primer set.
  • the remaining primers which could be one or more than one primer, do not contain an attB site, but are complementary to sequences flanking the gene or genes of interest and any associated regulatory sequences.
  • 2 primers one containing an attB site are used to amplify a selective gene such as puromycin.
  • the primer set includes primers 1 & 2 that amplify the GFP gene without amplification of an upstream promoter.
  • Primer 1 contains the attB site at the 5′ end of the oligo.
  • Primers 3 & 4 are designed to amplify the IRES-blasticidin DNA sequences from the vector pIRESblasticidin.
  • the 5′end of primer 3 contains sequences complementary to the 5′ end of primer 2 such that annealing can occur between 5′ ends of the two primers.
  • the two PCR primers are combined with a puromycin DNA template such as pPUR (Clontech), a heat stable DNA polymerase and appropriate conditions for DNA amplification.
  • a puromycin DNA template such as pPUR (Clontech)
  • pPUR a heat stable DNA polymerase
  • the resulting PCR product is then then purified and self-ligated to form a circular molecule.
  • amplification of the GFP gene and IRES-blasticidin sequences is accomplished by combining primers 1 & 2 with DNA template pD2eGFP and primers 3 & 4 with template pIRESblasticidin under appropriate conditions to amplify the desired template.
  • primers 1 & 2 primers 1 & 2 with DNA template pD2eGFP and primers 3 & 4 with template pIRESblasticidin under appropriate conditions to amplify the desired template.
  • a second round of amplification using both of the PCR products from the first round of amplification together with primers 1 and 4 amplifies the fusion product attB-GFP-IRES-blasticidin (FIG. 13).
  • the circular PCR product is then be introduced to the platform ACes using the bacteriphage lambda integrase E174R.
  • the introduction can be performed in vivo by transfecting the pCXLamIntR (SEQ ID NO: 112) vector encoding the lambda integrase mutant E174R together with the circularized PCR product into a cell line containing the platform ACE.
  • the marker gene (in this case either puromycin, blasticidin or GFP) is used to enrich the population for cells containing the proper integration event.
  • a proper integration event in the second example juxtaposes a promoter residing on the platform ACes 5′ to the attB-GFP-IRES-Blasticidin PCR product, allowing for transcription of both GFP and blasticidin. If enrichment is done by drug selection, blasticidin is added to the medium on the transfected cells 24-48 hours post-transfection. Selection is maintained until colonies are formed on the plates. If enrichment is done by cell sorting, cells are sorted 2-4 days post-transfection to enrich for cells expressing the fluorescent marker (GFP in this case).
  • Genome Systems (IncyteGenomics) was supplied with the primers 5′HETS (GGGCCGAAACGATCTCAACCTATT; SEQ ID NO:78), and 3′HETS (CGCAGCGGCCCTCCTACTC; SEQ ID NO:79), which were used to amplify a 538 bp PCR product homologous to nt 9680-10218 of the human rDNA sequences (GenBank Accession No. U13369) and used as a probe to screen a human genomic PlAC (P1 Artificial Chromosome) library constructed in the vector pCYPAC2 (loannou et al. (1994) Nat. Genet. 6(1): 84-89).
  • 5′HETS GGGCCGAAACGATCTCAACCTATT; SEQ ID NO:78
  • CGCAGCGGCCCTCCTACTC SEQ ID NO:79
  • Genome Systems clone #18720 was isolated in this screen and contains three repeats of human rDNA as assessed by restriction analysis.
  • GS clone #18720 was digested with PmeI, a restriction enzyme unique to a single repeat of the human rDNA (45 Kbp), and then religated to form pPACrDNA (FIG. 15).
  • PmeI a restriction enzyme unique to a single repeat of the human rDNA (45 Kbp)
  • the pPACrDNA, rDNA sequences are homologous to Genbank Accession #U13369, containing an insert of about 45 kB comprising a single repeat beginning from the end of one repeat at ⁇ 33980 (relative to the Genbank sequence) through the beginning of the next repeat up to approximately 35120 (the repeat offset from that listed in the GenBank file).
  • the rDNA sequence is just over 1 copy of the repeat extending from 33980 (+/ ⁇ 10 bp) to the end of the first repeat (43 Kbp) and continuing into the second repeat to bp 35120 (+/ ⁇ 10 bp).
  • MSU1.1 cells Five hundred thousand MSU1.1 cells (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136; provided by Dr. Justin McCormick at Michigan State University) were plated per 6 cm plate (3 plates total) and allowed to grow overnight. The cells were 70-80% confluent the following day.
  • One plate was transfected with 15 ⁇ g pPACrDNA (linearized with Pme I) and 2 ⁇ g pSV40attPsensePuro (linearized with Sca I; see Example 3). The remaining plates were controls and were transfected with either 20 ⁇ g pBS (Stratagene) or 20 ⁇ g pSV40attBsensePuro (linearized with Sca I). All three plates were transfected using a CaPO 4 protocol.
  • FISH analysis was performed on the candidate clones to detect ACes formation. Metaphase spreads from the candidate clones were probed in multiple probe combinations. In one experiment, the probes used were biotin-labeled human alphoid DNA (pPACrDNA) and digoxigenin-labeled mouse major DNA (pFK161) as a negative control.
  • pPACrDNA biotin-labeled human alphoid DNA
  • pFK161 digoxigenin-labeled mouse major DNA
  • Candidate M2-2d was single cell subcloned by flow sorting and the candidate subclones were reanalyzed by FISH. Subclone 1B1 of M2-2d was determined to be a platform ACes and is also designated human Platform ACE 0.1.
  • the promoterless delivery method was used to deliver a promoterless blasticidin marker gene onto the human platform ACes with excellent results.
  • the human ACes platform with a promoterless blasticidin marker gene resulted in 21 of 38 blasticidin resistant clones displaying a PCR product of the expected size from the population co-transfected with pLIT38attBBSRpolyA10 and pCXLamintR (FIG. 8; SEQ ID NOs. 111 and 112).
  • the population transfected with pBlueScript resulted in 0 blasticidin resistant colonies.
  • the vector pLITMUS 38 (New England Biolabs; U.S. Pat. No. 5,691,140; SEQ ID NO: 119) was digested with EcoRV and ligated to two annealed oligomers, which form an attB site (attB15′-TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA-3′ (SEQ ID NO:8); attB2 5′-TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA-3′; SEQ ID NO:9). This ligation reaction resulted in the vector pLIT38attB (SEQ ID NO: 120).
  • the blasticidin resistance gene and SV40 polyA site was PCR amplified with primers: 5BSD (ACCATGAAAACATTTAACATTTCTCAACA; SEQ ID NO:80) and SV40polyA (TTTATTTGTGAAATTTGTGATGCTATTGC; SEQ ID NO:81) using pPAC4 (Frengen, E., et al. (2000) Genomics 68 (2), 118-126; GenBank Accession No. U75992) as template.
  • the blasticidin-SV 40polyA PCR product was then ligated into pLIT38attB at the BamHI site, which was Klenow treated following digestion with BamHI.
  • MSU1.1 cells containing human platform ACE 0.1 was expanded and plated to five 10 cm dishes with 1.3 ⁇ 10 6 cells per dish. The cells were incubated overnight in DMEM with 10% fetal bovine serum, at 37° C. and 5% CO 2 . The following day the cells were transfected with 5 ⁇ g of each plasmid as set forth in Table 3, for a total of 10 ⁇ g of DNA per plate of cells transfected (see Table 3) using ExGen 500 in vitro transfection reagent (MBI fermentas, cat. no. R0511). The transfection was performed according to the manufacturers protocol.
  • Genomic DNA was isolated from these clones with the Promega Wizard Genomic cDNA purification kit, digested with EcoRI and used as template in a PCR reaction with the following primers: 3BSP-TTAATTTCGGG TATATTTGAGTGGA (SEQ ID NO:82); 5PacSV40-CTGTTAATTAACTGTGGAA TGTGTGTCAGTTAGGGTG (SEQ ID NO:76).
  • the PCR conditions were as follows.
  • 10 ng of genomic DNA was amplified with 0.5 ul Herculase polymerase (Stratagene) in a 50 ul reaction that contained 12.5 pmole of each primer, 2.5 mM of each dNTP, and 1 ⁇ Herculase buffer (Stratagene).
  • the reactions were placed in a PerkinElmer thermocycler programmed as follows: Initial denaturation at 95° C. for 10 minutes; 35 cycles of 94° C. for 1 minute, 53° C. for 1 minute, 72° C. for 1 minute, and 72° C. for 1 minute; Final extension for 10 minutes at 72° C.; and 4° C. hold.
  • PCR amplification with the above primers should yield an 804 bp product. Twenty-one of the 38 clones from plate 3 produced a PCR product of the expected 804 bp size.
  • the erythropoietin cDNA was PCR amplified from a human cDNA library (E. Perkins et al., 1999 , Proc. Natl. Acad. Sci. USA 96(5): 2204-2209) using the following primers: EPO5XBA-TATCTAGAATGGGGGTGC ACGAATGTCCTGCC (SEQ ID NO: 83); EPO3BSI-TACGTACGTCATC TGTCCCCTGTCCTGCAGGC (SEQ ID NO: 84).
  • the cDNA was amplified through two successive rounds of PCR using the following conditions: heat denaturation at 95° C.
  • BIO-X-ACT (BIOLINE) was used to amplify the erythropoietin cDNA from 2.5 ng of the human cDNA library in the first round of amplification. Five ⁇ l of the first amplification product was used as template for the second round of amplification. Two PCR products were produced from the second amplification with Taq polymerase (Eppendorf), each product was cloned into pCR2.1-Topo (Invitrogen) and sequenced.
  • the larger PCR product contained the expected cDNA sequence for erythropoietin.
  • the erythropoietin cDNA was moved from pTopoEPO into p18attBZeo(6XHS4)2eGFP (SEQ ID NO: 110).
  • pTopoEPO was digested with BsiWI and XbaI to release a 588 bp EPO cDNA. BsrGI and BsiWI create compatable ends.
  • the eGFP gene was removed from p18attBZeo(6XHS4)2eGFP by digestion with BsiWI and XbaI, the 8.3 Kbp vector backbone was gel purified and ligated to the 588 bp EPO cDNA to create p18EPOcDNA (SEQ ID NO: 124).
  • the erythropoietin genomic clone was PCR amplified from a human genomic library (Clontech) using the following primers: GENEPO3BSI-CGTACGTCATCTGTCCCCT GTCCTGCA (SEQ ID NO: 85); GENEPO 5XBA-TCTAGAATGGGGGT GCACGGTGAGTACT (SEQ ID NO: 86).
  • the reaction conditions for the amplification were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C.
  • the erythropoietin genomic PCR product (2147 bp) was gel purified and cloned into pCR2.1Topo to create pTopogenEPO. Sequence analysis revealed 2 bp substitutions and insertions in the intronic sequences of the genomic clone of erythropoietin.
  • the erythropoietin genomic and cDNA genes were each moved onto the platform ACes B19-38 (constructed as set forth in Example 3) by co-transfecting with pCXLamIntR. Control transfections were also performed using pCXLamInt (SEQ ID NO: 127) together with either p18EPOcDNA (SEQ ID NO: 124) or p18genEPO (SEQ ID NO: 125).
  • Lipofectamine Plus was used to transfect the DNA's into B19-38 cells according to the manufacturer's protocol. The cells were placed in selective medium (DMEM with 10% FBS and Zeocin @500 ug/ml) 48 hours post-transfection and maintained in selective medium for 13 days. Clones were isolated 15 days post-transfection.
  • the negative control was a Zeocin resistant clone isolated from B19-38 cells transfected with p18attBZeo(6XHS4) (SEQ ID NO: 117; no insert control vector) and pCXLamIntR (SEQ ID NO: 112).
  • the preliminary ELISA assay was executed as follows: 1) Nunc-Immuno Plates (MaxiSorb 96-well, Catalogue # 439454) were coated with 75 ul of a 1/200 dilution (in Phosphate buffered Saline, pH 7.4 (PBS), Sigma Catalogue # P-3813) of monoclonal anti-human erythropoietin antibody overnight at 4° C.
  • step 7 Add 75 ul of polyclonal anti-human erythropoietin antibody (1/250 dilution in dilution buffer (0.5% BSA, 0.01% Tween 20, 1 ⁇ PBS, pH 7.4) and incubate 1 hour at 37° C. 8) Repeat washes of step 2. 9) Add 75 ul of goat anti-rabbit conjugated alkaline phosphatase diluted 1/4000 in dilution buffer and incubate 1 hour at 37° C. 10) Repeat washes of step 2.
  • the erythropoietin standard curve was derived from readings of diluted human recombinant Erythropoietin (Roche, catalogue # 1-120-166; dilution range 125-7.8 mUnits/ml). From this preliminary assay the 21 clones displaying the highest expression of erythropoietin were analyzed a second time in the same manner using medium supernatants that had been on the clones for 24 hours and a 1:3 dilution therof.
  • Genomic DNA was isolated from the 21 clones with the best expression (as assessed by the initial ELISA assay above) as well as the B19-38 cell line and used for PCR analysis. Genomic DNA was isolated using the Wizard genomic DNA purification kit (Promega) according to the manufacturers protocol. Amplification was performed on 10 ng of genomic DNA as template with MasterTaq DNA Polymerase (Eppendorf) and the primer set 5PacSV40-CTGTTAATTAACTGTGGAATGTGTG TCAGTTAGGGTG (SEQ ID NO: 76) and Antisense Zeo-TGAACAGGGTCACGTCGTCC (SEQ ID NO:77).
  • the amplification conditions were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C. per cycle to 50° C.), and 1 minutes extension (72° C.); 15 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (50° C.), and 1 minute extension (72° C.); the last cycle is followed by a 10 minute extension at 72° C.
  • PCR products were size separated by gel electrophoresis. Of the 21 clones analyzed 19 produced a PCR product of 650 bp as expected for a site-specific integration event.
  • Plant artificial chromosomes can be generated by introducing nucleic acid, such as DNA, which can include a targeting DNA, for example rDNA or lambda DNA, into a plant cell, allowing the cell to grow, and then identifying from among the resulting cells those that include a chromosome with a structure that is distinct from that of any chromosome that existed in the cell prior to introduction of the nucleic acid.
  • the structure of a PAC reflects amplification of chromosomal DNA, for example, segmented, repeat region-containing and heterochromatic structures. It is also possible to select cells that contain structures that are precursors to PACs, for example, chromosomes containing more than one centromere and/or fragments thereof, and culture and/or manipulate them to ultimately generate a PAC within the cell.
  • the nucleic acid can be introduced into a variety of plant cells.
  • the nucleic acid can include targeting DNA and/or a plant expressable DNA encoding one or multiple selectable markers (e.g., DNA encoding bialophos (bar) resistance) or scorable markers (e.g., DNA encoding GFP).
  • selectable markers e.g., DNA encoding bialophos (bar) resistance
  • scorable markers e.g., DNA encoding GFP.
  • targeting DNA include, but are not limited to, N. tabacum rDNA intergenic spacer sequence (IGS) and Arabidopsis rDNA such as the 18S, 5.8S, 26S rDNA and/or the intergenic spacer sequence.
  • the DNA can be introduced using a variety of methods, including, but not limited to Agrobacterium-mediated methods, PEG-mediated DNA uptake and electroporation using, for example, standard procedures according to Hartmann et al [(1998) Plant Molecular Biology 36:741].
  • the cell into which such DNA is introduced can be grown under selective conditions and can initially be grown under non-selective conditions and then transferred to selective media.
  • the cells or protoplasts can be placed on plates containing a selection agent to grow, for example, individual calli. Resistant calli can be scored for scorable marker expression.
  • Metaphase spreads of resistance cultures can be prepared, and the metaphase chromosomes examined by FISH analysis using specific probes in order to detect amplification of regions of the chromosomes.
  • Cells that have artificial chromosomes with functioning centromeres or artificial chromosomal intermediate structures including, but not limited to, dicentric chromosomes, formerly dicentric chromosomes, minichromosomes, heterochromatin structures (e.g. sausage chromosomes), and stable self-replicating artificial chromosomal intermediates as described herein, are identified and cultured. In particular, the cells containing self-replicating artificial chromosomes are identified.
  • the DNA introduced into a plant cell for the generation of PACs can be in any form, including in the form of a vector.
  • An exemplary vector for use in methods of generating PACs can be prepared as follows.
  • plant transformation vectors as exemplified by pAgIIa and pAgIIb, containing a selectable marker, a targeting sequence, and a scorable marker were constructed using procedures well known in the art to combine the various fragments.
  • the vectors can be prepared using vector pAg1 as a base vector and inserting the following DNA fragments into pAg1: DNA encoding ⁇ -glucoronidase under the control of the nopaline synthase (NOS) promoter fragment and flanked at the 3′ end by the NOS terminator fragment, a fragment of mouse satellite DNA and an N. tabacum rDNA intergenic spacer sequence (IGS).
  • NOS nopaline synthase
  • IGS N. tabacum rDNA intergenic spacer sequence
  • vector pAg2 can also be used as the base vector.
  • Vector pAg1 (SEQ. ID. NO: 89) is a derivative of the CAMBIA vector named pCambia 3300 (Center for the Application of Molecular Biology to International Agriculture, i.e., CAMBIA, Canberra, Australia; www.cambia.org), which is a modified version of vector pCambia 1300 to which has been added DNA from the bar gene confering resistance to phosphinothricin.
  • the nucleotide sequence of pCambia 3300 is provided in SEQ. ID. NO: 90.
  • pCambia 3300 also contains a lacZ alpha sequence containing a polylinker region.
  • pAg1 was constructed by inserting two new functional DNA fragments into the polylinker of pCambia 3300: one sequence containing an attB site and a promoterless zeomycin resistance-encoding DNA flanked at the 3′ end by a SV40 polyA signal sequence, and a second sequence containing DNA from the hygromycin resistance gene (hygromycin phosphotransferase) confering resistance to hygromycin for selection in plants.
  • hygromycin resistance gene hygromycin phosphotransferase
  • the zeomycin-SV40 polyA signal fusion is not expected to function in plant cells, it can be activated in mammalian cells by insertion of a functional promoter element into the attB site by site-specific recombination catalyzed by the Lambda att integrase.
  • the inclusion of the attB-zeomycin sequences allows for evaluation of functionality of plant artificial chromosomes in mammalian cells by activation of the zeomycin resistance-encoding DNA, and provides an att site for further insertion of new DNA sequences into plant artificial chromosomes formed as a result of using pAg1 for plant transformation.
  • the second functional DNA fragment allows for selection of plant cells with hygromycin.
  • pAg1 contains DNA from the bar gene confering resisance to phosphinothricin, DNA from the hygromycin resistance gene, both resistance-encoding DNAs under the control of a separate cauliflower mosaic virus (CaMV) 35S promoter, and the attB-promoterless zeomycin resistance-encoding DNA.
  • CaMV cauliflower mosaic virus
  • pAg1 is a binary vector containing Agrobacterium right and left T-DNA border sequences for use in Agrobacterium-mediated transformation of plant cells or protoplasts with the DNA located between the border sequences.
  • pAg1 also contains the pBR322 Ori for replication in E. coli.
  • pAg1 was constructed by ligating HindIII/PstI-digested p3300attBZeo with HindIII/PstI-digested pBSCaMV35SHyg as follows.
  • Plasmid pCambia 3300 was digested with PstI/Ecl136 II and ligated with PstI/StuI-digested pLITattBZeo (the nucleotide sequence of pLITattBZeo is provided in SEQ. ID. NO: 91. (containing DNA encoding the zeocin resistance gene and an attB Integrase recognition sequence) to generate p3300attBZeo which contains an attB site, a promoterless zeomycin resistance-encoding DNA flanked at the 3′ end by a SV40 polyA signal, and a reconstructed PstI site.
  • a DNA fragment containing DNA encoding hygromycin phosphotransferase flanked by the CaMV 35S promoter and the CaMV 35S polyA signal sequence was obtained by PCR amplification of plasmid pCambia 1302 (GenBank Accession No. AF234298 and SEQ. ID. NO: 92).
  • the primers used in the amplification reaction were as follows: CaMV35SpolyA: SEQ. ID. NO:93 5′-CTGAATTAACGCCGAATTAATTCGGGGGATCTG-3′
  • CaMV35Spr SEQ. ID. NO:94 5′-CTAGAGCAGCTTGCCAACATGGTGGAGCA-3′
  • pBSCaMV35SHyg was digested with HindIII/PstI and ligated with HindIII/PstI-digested p3300attBZeo.
  • pAg1 contains the pCambia 3300 backbone with DNA conferring resistance to phophinothricin and hygromycin under the control of separate CaMV 35S promoters, an attB-promoterless zeomycin resistance-encoding DNA recombination cassette and unique sites for adding additional markers, e.g., DNA encoding GFP.
  • the attB site can be used as decribed hereing for the addition of new DNA sequences to plant artificial chromosomes, including PACs formed as a result of using the pAg1 vector, or derivatives thereof, in the production of PACs.
  • the attB site provides a convenient site for recombinase-mediated insertion of DNAs containing a homologous att site.
  • the vector pAg2 (SEQ. ID. NO: 95) is a derivative of vector pAg1 formed by adding DNA encoding a green fluorescent protein (GFP), under the control of a NOS promoter and flanked at the 3′ end by a NOS polyA signal, to pAg1.
  • GFP green fluorescent protein
  • pAg2 was constructed as follows. A DNA fragment containing the NOS promoter was obtained by digestion of pGEM-T-NOS, or pGEMEasyNOS (SEQ. ID.
  • telomere sequence containing the NOS promoter in the cloning vector pGEM-T-Easy (Promega Biotech, Madison, Wis., U.S.A.), with XbaI/NcoI and was ligated to an XbaI/NcoI fragment of pCambia 1302 containing DNA encoding GFP (without the CaMV 35S promoter) to generate p1302NOS (SEQ. ID. NO: 97) containing GFP-encoding DNA in operable association with the NOS promoter.
  • Plasmid p1302NOS was digested with SmaI/BsiWI to yield a fragment containing the NOS promoter and GFP-encoding DNA.
  • pAg2 contains DNA from the bar gene confering resistance to phosphinothricin, DNA conferring resistance to hygromycin, both resistance-encoding DNAs under the control of a cauliflower mosaic virus 35S promoter, DNA encoding kanamycin resistance, a GFP gene under the control of a NOS promoter and the attB-zeomycin resistance-encoding DNA.
  • fragments can be used to generate the pAg1 and pAg2 derivatives and that other heterlogous DNA can be incorporated into pAg1 and pAg2 derivatives using methods well known in the art.
  • pAgIIa and pAgIIb transformation vectors Vectors pAgIIa and pAgIIb were constructed by inserting the following DNA fragments into pAgI: DNA encoding 8-glucoronidase, the nopaline synthase terminator fragment, the nopaline synthase (NOS) promoter fragment, a fragment of mouse satellite DNA and an N. tabacum rDNA intergenic spacer sequence (IGS). The construction of pAgIIa and pAgIIb was as follows.
  • An N. tabacum rDNA intergenic spacer (IGS) sequence (SEQ. ID. NO: 98; see also GenBank Accession No. Y08422; see also Borysyuk et al. (2000) Nature Biotechnology 18:1303-1306; Borysyuk et al. (1997) Plant Mol. Biol. 35:655-660; U.S. Pat. Nos. 6,100,092 and 6,355,860) was obtained by PCR amplification of tobacco genomic DNA.
  • the IGS can be used as a targeting sequence by virtue of its homology to tobacco rDNA genes; the sequence is also an amplification promoter sequence in plants.
  • NTIGS-FI SEQ ID No.99
  • NTIGS-RI SEQ ID No.100
  • pIGS-I A fragment of mouse satellite DNA (Msatl fragment; GenBank Accession No. V00846; and SEQ ID No. 101) was amplified via PCR from pSAT-1 using the following primers: MSAT-F1 (SEQ ID No.102) 5′-AAT ACC GCG GAA GCT TGA CCT GGA ATA TCG C-3′ and MSAT-Ri (SEQ ID No.103) 5′-ATA ACC GCG GAG TCC TTC AGT GTG CA T-3′
  • This amplification added a SacII and a HindIII site at the 5′end and a SacII site at the 3′ end of the PCR fragment. This fragment was then cloned into the SacII site in pIGS-1 to give pMIGS-1, providing a eukaryotic centromere-specific DNA and a convenient DNA sequence for detection via FISH.
  • a functional marker gene containing a NOS-promoter:GUS:NOS terminator fusion was then constructed containing the NOS promoter (GenBank Accession No. U09365; SEQ ID No. 104), E. coli ⁇ -glucuronidase coding sequence (from the GUS gene; GenBank Accession No. S69414; and SEQ ID No. 105), and the nopaline synthase terminator sequence (GenBank Accession No. U09365; SEQ ID No. 107).
  • the NOS promoter in pGEM-T-NOS was added to a promoterless GUS gene in pBlueScript (Stratagene, La Jolla, Calif., U.S.A.) using NotI/SpeI to form pNGN-1, which has the NOS promoter in the opposite orientation relative to the GUS gene.
  • pMIGS-1 was digested with NotI/SpeI to yield a fragment containing the mouse major satellite DNA and the tobacco IGS which was then added to NotI-digested pNGN-1 to yield pNGN-2.
  • the NOS promoter was then re-oriented to provide a functional GUS gene, yielding pNGN-3, by digestion and religation with SpeI.
  • Plasmid pNGN-3 was then digested with HindIII, and the HindIII fragment containing the ⁇ -glucuronidase coding sequence and the rDNA intergenic spacer, along with the Msat sequence, was added to pAG-1 to form pAgIIa (SEQ ID NO: 108), using the unique HindIII site in pAgI located near the right T-DNA border of pAgI, within the T-DNA region.
  • pAgIIb Another plasmid vector, referred to as pAgIIb, was also recovered, which contained the inserted HindIII fragment (SEQ ID NO: 108) in the opposite orientation relative to that observed in pAgIIa.
  • pAgIIa and pAgIIb differ only in the orientation of the HindIII fragment containing the mouse major satellite sequence, the GUS DNA sequence and the IGS sequence.
  • the nucleotide sequences of pAgIIa is provided in SEQ. ID. NOS: 109.

Abstract

Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome for a variety of applications.

Description

    RELATED APPLICATIONS
  • Benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application Serial No. 60/294,758, filed May 30, 2001, to Perkins, et al., entitled “CHROMOSOME-BASED PLATFORMS” and to U.S. provisional application Serial No. 60/366,891, filed Mar. 21, 2002, to Perkins, et al., entitled “CHROMOSOME-BASED PLATFORMS” is claimed. Where permitted, the subject matter of which are herein incorporated by reference in their entirety. [0001]
  • This application is related to Provisional Application No. 60/294,687, filed May 30, 2001, by CARL PEREZ AND STEVEN FABIJANSKI entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING PLANT ARTIFICIAL CHROMOSOMES and to U.S. Provisional Application No. 60/296,329, filed Jun. 4, 2001, by CARL PEREZ AND STEVEN FABIJANSKI entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING PLANT ARTIFICIAL CHROMOSOMES. This application also is related to U.S. Provisional Application No. 60/294,758, filed May 30, 2001, by EDWARD PERKINS et al. entitled CHROMOSOME-BASED PLATFORMS and to U.S. Provisional Application No. 60/366,891, filed Mar. 21, 2002, by by EDWARD PERKINS et al. entitled CHROMOSOME-BASED PLATFORMS. This application is also related to U.S. application Serial Nos. (attorney dkt nos. 24601-419 and 419PC), filed on the same day herewith, entitled PLANT ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS OF PREPARING PLANT ARTIFICIAL CHROMOSOMES to Perez et al. [0002]
  • This application is related to U.S. application Ser. No. 08/695,191, filed Aug. 7, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, now U.S. Pat. No. 6,025,155. This application is also related to U.S. application Ser. No. 08/682,080, filed Jul. 15, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES, now U.S. Pat. No. 6,077,697. This application is also related U.S. application Ser. No. 08/629,822, filed Apr. 10, 1996 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES (now abandoned), and is also related to copending U.S. application Ser. No. 09/096,648, filed Jun. 12, 1998, by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES and to U.S. application Ser. No. 09/835,682, Apr. 10, 1997 by GYULA HADLACZKY and ALADAR SZALAY, entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES (now abandoned). This application is also related to copending U.S. application Ser. No. 09/724,726, filed Nov. 28, 2000, U.S. application Ser. No. 09/724,872, filed Nov. 28, 2000, U.S. application Ser. No. 09/724,693, filed Nov. 28, 2000, U.S. application Ser. No. 09/799,462, filed Mar. 5, 2001, U.S. application Ser. No. 09/836,911, filed Apr. 17, 2001, and U.S. application Serial No. 10/125,767, filed Apr. 17, 2002, each of which is by GYULA HADLACZKY and ALADAR SZALAY, and is entitled ARTIFICIAL CHROMOSOMES, USES THEREOF AND METHODS FOR PREPARING ARTIFICIAL CHROMOSOMES. This application is also related to International PCT application No. WO 97/40183. The subject matter of each of these provisional applications, international applications, and applications is incorporated by reference in its entirety. [0003]
    • FIELD OF INVENTION
  • Artificial chromosomes, including ACes, that have been engineered to contain available sites for site-specific, recombination-directed integration of DNA of interest are provided. These artificial chromosomes permit tractable, efficient, rational engineering of the chromosome. [0004]
    • BACKGROUND
  • Artificial Chromosomes [0005]
  • A variety of artificial chromosomes for use in plants and animals, particularly higher plants and animals are available. In particular, U.S. Pat. Nos. 6,025,155 and 6,077,697 provide heterochromatic artificial chromosomes designated therein as satellite artificial chromosomes (SATACs) and now designated artificial chromosome expression systems (ACes). These chromosomes are prepared by introducing heterologous DNA into a selected plant or animal cell under conditions that result in integration into a region of the chromosome that leads to an amplification event resulting in production of a dicentric chromosome. Subsequent treatment and growth of cells with dicentric chromosomes, including further amplifications, ultimately results in the artificial chromosomes provided therein. In order to introduce a desired heterologous gene (or a plurality of heterologous genes) into the artificial chromosome, the process is repeated introducing the desired heterologous genes and nucleic acids in the initial targeting step. This process is time consuming and tedious. Hence, more tractable and efficient methods for introducing heterologous nucleic acid molecules into artificial chromosomes, particularly ACes, are needed. [0006]
  • Therefore, it is an object herein to provide engineered artificial chromosomes that permit tractable, efficient and rational engineering of artificial chromosomes. [0007]
    • SUMMARY OF THE INVENTION
  • Provided herein are artificial chromosomes that permit tractable, efficient and rational engineering thereof. In particular, the artificial chromosomes provided herein contain one or a plurality of loci (sites) for site-specific, recombination-directed integration of DNA. Thus, provided herein are platform artificial chromosome expression systems (“platform ACes”) containing single or multiple site-specific, recombination sites. [0008]
  • The artificial chromosomes and ACes artificial chromosomes include plant and animal chromosomes. Any recombinase system that effects site-specific recombination is contemplated for use herein. [0009]
  • In one embodiment, chromosomes, including platform ACes, are provided that contain one or more lambda att sites designed for recombination-directed integration in the presence of lambda integrase, and that are mutated so that they do not require additional factors. Methods for preparing such chromosomes, vectors for use in the methods, and uses of the resulting chromosomes are also provided. [0010]
  • Platform ACes containing the recombination site(s) and methods for introducing heterologous nucleic acid into such sites and vectors therefor, are provided. [0011]
  • Also provided herein is a bacteriophage lambda (A) integrase site-specific recombination system. [0012]
  • Methods using recombinase mediated recombination target gene expression vectors and/or genes for insertion thereof into platform chromosomes and the resulting chromosomes are provided. Combinations and kits containing the combinations of vectors encoding a recombinase and integrase and primers for introduction of the site recognized thereby are also provided. The kits optionally include instructions for performing site-directed integration or preparation of ACes containing such sites. [0013]
  • Also provided herein are mammalian and plant cells comprising the artificial chromosomes and ACes described herein. The cells can be nuclear donor cells, stem cells, such as a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell or an embryonic stem cell. [0014]
  • Also provide is a lamba-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-integrase3. Also provided are transgenic animals and methods for producing a transgenic animal, comprising introducing a ACes into an embryonic cell, such as a stem cell or embryo. The ACes can comprise heterologous nucleic acid that encodes a therapeutic product. The transgenic animal can be a fish, insect, reptile, amphibians, arachnid or mammal. In certain embodiments, the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer. [0015]
  • The platform ACes, including plant and animal ACes, such as MACs, provided herein can be introduced into cells, such as, but not limited to, animal cells, including mammalian cells, and into plant cells. Hence plant cells that contain platform MACs, animal cells that contain platform PACs and other combinations of cells and platform ACes are provided.[0016]
    • DESCRIPTION OF FIGURES
  • FIG. 1 provides a diagram depicting creation of an exemplary ACes artificial chromosome prepared using methods detailed in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183. In this exemplified embodiment, the nucleic acid is targeted to an acrocentric chromosome in an animal or plant, and the heterologous nucleic acid includes a sequence-specific recombination site and marker genes. [0017]
  • FIG. 2 provides a map of pWEPuro9K, which is a targeting vector derived from the vector pWE15 (GenBank Accession #X65279; SEQ ID No. 31). Plasmid pWE15 was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance encoding fragment with the PvuII/BamHI (Klenow filled) puromycin resistance-encoding fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc., Palo Alto, Calif.; GenBank Accession no. U07648; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb NotI fragment from the plasmid pFK161 (see Example 1, see, also Csonka et al. (2000) [0018] Journal of Cell Science 113:3207-32161; and SEQ ID NO: 118), containing a portion of the mouse rDNA region, was cloned into the NotI site of pWEPuro resulting in plasmid pWEPuro9K.
  • FIG. 3 depicts construction of an ACes platform chromosome with a single recombination site, such as loxP sites or an attP or attB site. This platform ACes chromosome is an exemplary artificial chromosome with a single recombination site. [0019]
  • FIG. 4 provides a map of plasmid pSV40-193attPsensePur. [0020]
  • FIG. 5 depicts a method for formation of a chromosome platform with multiple recombination integration sites, such as attP sites. [0021]
  • FIG. 6 sets forth the sequences of the core region of attP, attB, attL and attR (SEQ ID Nos. 33-36). [0022]
  • FIG. 7 depicts insertional recombination of a vector encoding a marker gene, DsRed and an attB site with an artificial chromosome containing an attP site. [0023]
  • FIG. 8 provides a map of plasmid pCXLamIntR (SEQ ID NO: 112), which includes the Lambda integrase (E174R)-encoding nucleic acid. [0024]
  • FIG. 9 diagrammatically summarizes the platform technology; [0025] marker 1 permits selection of the artificial chromosomes containing the integration site; marker 2, which is promoterless in the target gene expression vector, permits selection of recombinants. Upon recombination with the platform marker 2 is expressed under the control of a promoter resident on the platform.
  • FIG. 10 provides the vector map for the plasmid p18attBZEO-5′6XHS4eGFP (SEQ ID NO: 116). [0026]
  • FIG. 11 provides the vector map for the plasmid p18attBZEO-3′6XHS4eGFP (SEQ ID NO: 115). [0027]
  • FIG. 12 provides the vector map for the plasmid p18attBZEO-(6XHS4)2eGFP (SEQ ID NO: 110). [0028]
  • FIGS. [0029] 13 AND 14 depict the integration of a PCR product by site-specific recombination as set forth in Example 8.
  • FIG. 15 provides the vector map for the plasmid pPACrDNA as set forth in Example 9.A.[0030]
    • DETAILED DESCRIPTION OF THE INVENTION
  • A. Definitions [0031]
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. Where reference is made to a URL or other such indentifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information. [0032]
  • As used herein, nucleic acid refers to single-stranded and/or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. When referring to probes or primers, optionally labeled, with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that they are statistically unique and of low copy number (typically less than 5, preferably less than 3) for probing or priming a library. Generally a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides long. [0033]
  • As used herein, DNA is meant to include all types and sizes of DNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs. [0034]
  • As used herein, nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified-nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs. [0035]
  • As used herein, heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations and/or in amounts in a genome or cell that differ from that in which it occurs in nature. Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or prepared synthetically. Generally, although not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed. Any DNA or RNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA. Heterologous DNA and RNA may also encode RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes. [0036]
  • Examples of heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced for purposes of modification of the endogenous genes or for production of an encoded protein. For example, a heterologous or foreign gene may be isolated from a different species than that of the host genome, or alternatively, may be isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene. Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers traits including, but not limited to, herbicide, insect, or disease resistance; traits, including, but not limited to, oil quality or carbohydrate composition. Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced. [0037]
  • As used herein, operative linkage or operative association, or grammatical variations thereof, of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. [0038]
  • In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5′ untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation (i.e., start) codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites (see, e.g., Kozak (1991) [0039] J. Biol. Chem. 266:19867-19870) can be inserted immediately 5′ of the start codon and may enhance expression.
  • As used herein, a sequence complementary to at least a portion of an RNA, with reference to antisense oligonucleotides, means a sequence having sufficient complementarity to be able to hybridize with the RNA, preferably under moderate or high stringency conditions, forming a stable duplex. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. The longer the hybridizing nucleic acid, the more base mismatches it can contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. [0040]
  • As used herein, regulatory molecule refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a polypeptide that is capable of enhancing or inhibiting expression of a gene. [0041]
  • As used herein, recognition sequences are particular sequences of nucleotides that a protein, DNA, or RNA molecule, or combinations thereof, (such as, but not limited to, a restriction endonuclease, a modification methylase and a recombinase) recognizes and binds. For example, a recognition sequence for Cre recombinase (see, e.g., SEQ ID NO:58) is a 34 base pair sequence containing two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core and designated loxP (see, e.g., Sauer (1994) [0042] Current Opinion in Biotechnology 5:521-527). Other examples of recognition sequences, include, but are not limited to, attB and attP, attR and attL and others (see, e.g., SEQ ID Nos. 8, 41-56 and 72), that are recognized by the recombinase enzyme Integrase (see, SEQ ID Nos. 37 and 38 for the nucleotide and encoded amino acid sequences of an exemplary lambda phage integrase).
  • The recombination site designated attB is an approximately 33 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region; attP (SEQ ID No. 72) is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins IHF, FIS, and Xis (see, e.g., Landy (1993) [0043] Current Opinion in Biotechnology 3:699-7071 see, e.g., SEQ ID Nos. 8 and 72).
  • As used herein, a recombinase is an enzyme that catalyzes the exchange of DNA segments at specific recombination sites. An integrase herein refers to a recombinase that is a member of the lambda (λ) integrase family. [0044]
  • As used herein, recombination proteins include excisive proteins, integrative proteins, enzymes, co-factors and associated proteins that are involved in recombination reactions using one or more recombination sites (see, Landy (1993) [0045] Current Opinion in Biotechnology 3:699-707). The recombination proteins used herein can be delivered to a cell via an expression cassette on an appropriate vector, such as a plasmid, and the like. In other embodiments, the recombination proteins can be delivered to a cell in protein form in the same reaction mixture used to deliver the desired nucleic acid, such as a platform ACes, donor target vectors, and the like.
  • As used herein the expression “lox site” means a sequence of nucleotides at which the gene product of the cre gene, referred to herein as Cre, can catalyze a site-specific recombination event. A LoxP site is a 34 base pair nucleotide sequence from bacteriophage P1 (see, e.g., Hoess et al. (1982) [0046] Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402). The LoxP site contains two 13 base pair inverted repeats separated by an 8 base pair spacer region as follows: (SEQ ID NO. 57):
  • ATAACTTCGTATA ATGTATGC TATACGAAGTTAT [0047]
  • [0048] E. coliDH5Δlac and yeast strain BSY23 transformed with plasmid pBS44 carrying two loxP sites connected with a LEU2 gene are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53254 and ATCC 20773, respectively. The lox sites can be isolated from plasmid pBS44 with restriction enzymes EcoRI and SalI, or XhoI and BamHI. In addition, a preselected DNA segment can be inserted into pBS44 at either the SalI or BamHI restriction enzyme sites. Other lox sites include, but are not limited to, LoxB, LoxL, LoxC2 and LoxR sites, which are nucleotide sequences isolated from E. coli (see, e.g., Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398). Lox sites can also be produced by a variety of synthetic techniques (see, e.g., Ito et al. (1982) Nuc. Acid Res. 10:1755 and Ogilvie et al. (1981) Science 270:270).
  • As used herein, the expression “cre gene” means a sequence of nucleotides that encodes a gene product that effects site-specific recombination of DNA in eukaryotic cells at lox sites. One cre gene can be isolated from bacteriophage P1 (see, e.g., Abremski et al. (1983) [0049] Cell 32:1301-1311). E. coli DH1 and yeast strain BSY90 transformed with plasmid pBS39 carrying a cre gene isolated from bacteriophage P1 and a GALL regulatory nucleotide sequence are available from the American Type Culture Collection (ATCC) under accession numbers ATCC 53255 and ATCC 20772, respectively. The cre gene can be isolated from plasmid pBS39 with restriction enzymes XhoI and SalI.
  • As used herein, site-specific recombination refers to site-specific recombination that is effected between two specific sites on a single nucleic acid molecule or between two different molecules that requires the presence of an exogenous protein, such as an integrase or recombinase. [0050]
  • For example, Cre-lox site-specific recombination can include the following three events: [0051]
  • a. deletion of a pre-selected DNA segment flanked by lox sites; [0052]
  • b. inversion of the nucleotide sequence of a pre-selected DNA segment flanked by lox sites; and [0053]
  • c. reciprocal exchange of DNA segments proximate to lox sites located on different DNA molecules. [0054]
  • This reciprocal exchange of DNA segments can result in an integration event if one or both of the DNA molecules are circular. DNA segment refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source. [0055]
  • Since the lox site is an asymmetrical nucleotide sequence, two lox sites on the same DNA molecule can have the same or opposite orientations with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the gene product of the cre gene. Thus, the Cre-lox system can be used to specifically delete, invert, or insert DNA. The precise event is controlled by the orientation of lox DNA sequences, in cis the lox sequences direct the Cre recombinase to either delete (lox sequences in direct orientation) or invert (lox sequences in inverted orientation) DNA flanked by the sequences, while in trans the lox sequences can direct a homologous recombination event resulting in the insertion of a recombinant DNA. [0056]
  • As used herein, a chromosome is a nucleic acid molecule, and associated proteins, that is capable of replication and segregation within a cell upon cell division. Typically, a chromosome contains a centromeric region, replication origins, telomeric regions and a region of nucleic acid between the centromeric and telomeric regions. [0057]
  • As used herein, a centromere is any nucleic acid sequence that confers an ability to segregate to daughter cells through cell division. A centromere may confer stable segregation of a nucleic acid sequence, including an artificial chromosome containing the centromere, through mitotic or meiotic divisions, including through both mitotic and meiotic divisions. A particular centromere is not necessarily derived from the same species in which it is introduced, but has the ability to promote DNA segregation in cells of that species. [0058]
  • As used herein, euchromatin and heterochromatin have their recognized meanings. Euchromatin refers to chromatin that stains diffusely and that typically contains genes, and heterochromatin refers to chromatin that remains unusually condensed and that has been thought to be transcriptionally inactive. Highly repetitive DNA sequences (satellite DNA) are usually located in regions of the heterochromatin surrounding the centromere (pericentric or pericentromeric heterochromatin). Constitutive heterochromatin refers to heterochromatin that contains the highly repetitive DNA which is constitutively condensed and genetically inactive. [0059]
  • As used herein, an acrocentric chromosome refers to a chromosome with arms of unequal length. [0060]
  • As used herein, endogenous chromosomes refer to genomic chromosomes as found in a cell prior to generation or introduction of an artificial chromosome. [0061]
  • As used herein, artificial chromosomes are nucleic acid molecules, typically DNA, that stably replicate and segregate alongside endogenous chromosomes in cells and have the capacity to accommodate and express heterologous genes contained therein. It has the capacity to act as a gene delivery vehicle by accommodating and expressing foreign genes contained therein. A mammalian artificial chromosome (MAC) refers to chromosomes that have an active mammalian centromere(s). Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include centromeres that function in plant, insect and avian cells, respectively. A human artificial chromosome (HAC) refers to chromosomes that include centromeres that function in human cells. For exemplary artificial chromosomes, see, e.g., U.S. Pat. Nos. 6,025,155; 6,077,697; 5,288,625; 5,712,134; 5,695,967; 5,869,294; 5,891,691 and 5,721,118 and published International PCT application Nos, WO 97/40183 and WO 98/08964. Artificial chromosomes include those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183), minichromosomes that contain a de novo centromere (see, U.S. Pat. Nos. 5,712,134, 5,891,691 and 5,288,625), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA and in vitro assembled artificial chromosomes (see, copending U.S. provisional application Serial No. 60/294,687, filed on May 30, 2001). [0062]
  • As used herein, the term “satellite DNA-based artificial chromosome (SATAC)” is interchangable with the term “artificial chromosome expression system (ACes)”. These artificial chromosomes (ACes) include those that are substantially all neutral non-coding sequences (heterochromatin) except for foreign heterologous, typically gene-encoding nucleic acid, that is interspersed within the heterochromatin for the expression therein (see U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183), or that is in a single locus as provided herein. Also included are ACes that may include euchromatin and that result from the process described in U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183 and outlined herein. The delineating structural feature is the presence of repeating units, that are generally predominantly heterochromatin. The precise structure of the ACes will depend upon the structure of the chromosome in which the initial amplification event occurs; all share the common feature of including a defined pattern of repeating units. Generally ACes have more heterochromatin than euchromatin. Foreign nucleic acid molecules (heterologous genes) contained in these artificial chromosome expression systems can include any nucleic acid whose expression is of interest in a particular host cell. Such foreign nucleic acid molecules, include, but are not limited to, nucleic acid that encodes traceable marker proteins (reporter genes), such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively), other reporter genes, such as β-galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance. Other examples of heterologous nucleic acid molecules include, but are not limited to, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, DNA that encodes other types of proteins, such as antibodies, and DNA that encodes RNA molecules (such as antisense or siRNA molecules) that are not translated into proteins. [0063]
  • As used herein, an artificial chromosome platform, also referred to herein as a “platform ACes” or “ACes platform”, refers to an artificial chromosome that has been engineered to include one or more sites for site-specific, recombination-directed integration. In particular, ACes that are so-engineered are provided. Any sites, including but not limited to any described herein, that are suitable for such integration are contemplated. Plant and animal platform ACes are provided. Among the ACes contemplated herein are those that are predominantly heterochromatic (formerly referred to as satellite artificial chromosomes (SATACs); see, e.g., U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183), artificial chromosomes predominantly made up of repeating nucleic acid units and that contain substantially equivalent amounts of euchromatic and heterochromatic DNA resulting from an amplification event depicted in the referenced patent and herein. Included among the ACes for use in generating platforms, are artificial chromosomes that introduce and express heterologous nucleic acids in plants (see, copending U.S. provisional application Serial No. 60/294,687, filed on May 30, 2001). These include artificial chromosomes that have a centromere derived from a plant, and, also, artificial chromosomes that have centromeres that may be derived from other organisms but that function in plants. [0064]
  • As used herein a “reporter ACes” refers to a an ACes that comprises one or a plurality of reporter constructs, where the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds. [0065]
  • As used herein, amplification, with reference to DNA, is a process in which segments of DNA are duplicated to yield two or multiple copies of substantially similar or identical or nearly identical DNA segments that are typically joined as substantially tandem or successive repeats or inverted repeats. [0066]
  • As used herein, amplification-based artificial chromosomes are artificial chromosomes derived from natural or endogenous chromosomes by virtue of an amplification event, such as one initiated by introduction of heterologous nucleic acid into rDNA in a chromosome. As a result of such an event, chromosomes and fragments thereof exhibiting segmented or repeating patterns arise. Artificial chromosomes can be formed from these chromosomes and fragments. Hence, amplification-based artificial chromosomes refer to engineered chromosomes that exhibit an ordered segmentation that is not observed in naturally occurring chromosomes and that distinguishes them from naturally occurring chromosomes. The segmentation, which can be visualized using a variety of chromosome analysis techniques known to those of skill in the art, correlates with the structure of these artificial chromosomes. In addition to containing one or more centromeres, the amplification-based artificial chromosomes, throughout the region or regions of segmentation are predominantly made up of nucleic acid units also referred to as “amplicons”, that is (are) repeated in the region and that have a similar gross structure. Repeats of an amplicon tend to be of similar size and share some common nucleic acid sequences. For example, each repeat of an amplicon may contain a replication site involved in amplification of chromosome segments and/or some heterologous nucleic acid that was utilized in the initial production of the artificial chromosome. Typically, the repeating units are substantially similar in nucleic acid composition and may be nearly identical. [0067]
  • The amplification-based artificial chromosomes differ depending on the chromosomal region that has undergone amplification in the process of artificial chromosome formation. The structures of the resulting chromosomes can vary depending upon the initiating event and/or the conditions under which the heterologous nucleic acid is introduced, including modification to the endogenous chromosomes. For example, in some of the artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of heterochromatic DNA. In other artificial chromosomes provided herein, the region or regions of segmentation may be made up predominantly of euchromatic DNA or may be made up of similar amounts of heterochromatic and euchromatic DNA. [0068]
  • As used herein an amplicon is a repeated nucleic acid unit. In some of the artificial chromosomes described herein, an amplicon may contain a set of inverted repeats of a megareplicon. A megareplicon represents a higher order replication unit. For example, with reference to some of the predominantly heterochromatic artificial chromosomes, the megareplicon can contain a set of tandem DNA blocks (e.g., ˜7.5 Mb DNA blocks) each containing satellite DNA flanked by non-satellite DNA or may be made up of substantially rDNA. Contained within the megareplicon is a primary replication site, referred to as the megareplicator, which may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres. Within the megareplicon there may be smaller (e.g., 50-300 kb) secondary replicons. [0069]
  • In artificial chromosomes, such as those provided U.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT application No. WO 97/40183, the megareplicon is defined by two tandem blocks (˜7.5 Mb DNA blocks in the chromosomes provided therein). Within each artificial chromosome or among a population thereof, each amplicon has the same gross structure but may contain sequence variations. Such variations will arise as a result of movement of mobile genetic elements, deletions or insertions or mutations that arise, particularly in culture. Such variation does not affect the use of the artificial chromosomes or their overall structure as described herein. [0070]
  • As used herein, amplifiable, when used in reference to a chromosome, particularly the method of generating artificial chromosomes provided herein, refers to a region of a chromosome that is prone to amplification. Amplification typically occurs during replication and other cellular events involving recombination (e.g., DNA repair). Such regions include regions of the chromosome that contain tandem repeats, such as satellite DNA, rDNA, and other such sequences. [0071]
  • As used herein, a dicentric chromosome is a chromosome that contains two centromeres. A multicentric chromosome contains more than two centromeres. [0072]
  • As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments is a replicable chromosome. If one of the chromosomes undergoes amplification of primarily euchromatic DNA to produce a fully functional chromosome that is predominantly (at least more than 50%) euchromatin, it is a minichromosome. The remaining chromosome is a formerly dicentric chromosome. If one of the chromosomes undergoes amplification, whereby heterochromatin (such as, for example, satellite DNA) is amplified and a euchromatic portion (such as, for example, an arm) remains, it is referred to as a sausage chromosome. A chromosome that is substantially all heterochromatin, except for portions of heterologous DNA, is called a predominantly heterochromatic artificial chromosome. Predominantly heterochromatic artificial chromosomes can be produced from other partially heterochromatic artificial chromosomes by culturing the cell containing such chromosomes under conditions such as BrdU treatment that destabilize the chromosome and/or growth under selective conditions so that a predominantly heterochromatic artificial chromosome is produced. For purposes herein, it is understood that the artificial chromosomes may not necessarily be produced in multiple steps, but may appear after the initial introduction of the heterologous DNA. Typically, artificial chromosomes appear after about 5 to about 60, or about 5 to about 55, or about 10 to about 55 or about 25 to about 55 or about 35 to about 55 cell doublings after initiation of artificial chromosome generation, or they may appear after several cycles of growth under selective conditions and BrdU treatment. [0073]
  • As used herein, an artificial chromosome that is predominantly heterochromatic (i.e., containing more heterochromatin than euchromatin, typically more than about 50%, more than about 70%, or more than about 90% heterochromatin) may be produced by introducing nucleic acid molecules into cells, such as, for example, animal or plant cells, and selecting cells that contain a predominantly heterochromatic artificial chromosome. Any nucleic acid may be introduced into cells in such methods of producing the artificial chromosomes. For example, the nucleic acid may contain a selectable marker and/or optionally a sequence that targets nucleic acid to the pericentric, heterochromatic region of a chromosome, such as in the short arm of acrocentric chromosomes and nucleolar organizing regions. Targeting sequences include, but are not limited to, lambda phage DNA and rDNA for production of predominantly heterochromatic artificial chromosomes in eukaryotic cells. [0074]
  • After introducing the nucleic acid into cells, a cell containing a predominantly heterochromatic artificial chromosome is selected. Such cells may be identified using a variety of procedures. For example, repeating units of heterochromatic DNA of these chromosomes may be discerned by G-banding and/or fluorescence in situ hybridization (FISH) techniques. Prior to such analyses, the cells to be analyzed may be enriched with artificial chromosome-containing cells by sorting the cells on the basis of the presence of a selectable marker, such as a reporter protein, or by growing (culturing) the cells under selective conditions. It is also possible, after introduction of nucleic acids into cells, to select cells that have a multicentric, typically dicentric, chromosome, a formerly multicentric (typically dicentric) chromosome and/or various heterochromatic structures, such as a megachromosome and a sausage chromosome, that contain a centromere and are predominantly heterochromatic and to treat them such that desired artificial chromosomes are produced. Cells containing a new chromosome are selected. Conditions for generation of a desired structure include, but are not limited to, further growth under selective conditions, introduction of additional nucleic acid molecules and/or growth under selective conditions and treatment with destabilizing agents, and other such methods (see International PCT application No. WO 97/40183 and U.S. Pat. Nos. 6,025,155 and 6,077,697). [0075]
  • As used herein, a “selectable marker” is a nucleic acid segment, generally DNA, that allows one to select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds and compositions. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be identified, such as phenotypic markers, including β-galactosidase, red, blue and/or green fluorescent proteins (FPs), and cell surface proteins; (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos. 1-5 above (e.g., antisense oligonucleotides or siRNA molecules for use in RNA interference); (7) nucleic acid segments that bind products that modify a substrate (e.g. restriction endonucleases); (8) nucleic acid segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional, such as for PCR amplification of subpopulations of molecules; and/or (10) nucleic acid segments, which when absent, directly or indirectly confer sensitivity to particular compounds. Thus, for example, selectable markers include nucleic acids encoding fluorescent proteins, such as green fluorescent proteins, β-galactosidase and other readily detectable proteins, such as chromogenic proteins or proteins capable of being bound by an antibody and FACs sorted. Selectable markers such as these, which are not required for cell survival and/or proliferation in the presence of a selection agent, are also referred to herein as reporter molecules. Other selectable markers, e.g., the neomycin phosphotransferase gene, provide for isolation and identification of cells containing them by conferring properties on the cells that make them resistant to an agent, e.g., a drug such as an antibiotic, that inhibits proliferation of cells that do not contain the marker. [0076]
  • As another example, interference of gene expression by double stranded RNA has been shown in [0077] Caenorhabditis elegans, plants, Drosophila, protozoans and mammals. This method is known as RNA interference (RNAi) and utilizes short, double-stranded RNA molecules (siRNAs). The siRNAs are generally composed of a 19-22 bp double-stranded RNA stem, a loop region and a 1-4 bp overhang on the 3′ end. The reduction of gene expression has been accomplished by direct introduction of the siRNAs into the cell (Harborth J et al., 2001, J Cell Sci 114(pt 24):4557-65) as well as the introduction of DNA encoding and expressing the siRNA molecule. The encoded siRNA molecules are under the regulation of an RNA polymerase III promoter (see, e.g., Yu et al., 2002, Proc Natl Acad Sci USA 99(9);6047-52; Brummelkamp et al., 2002, Science 296(5567):550-3; Miyagishi et al., 2002, Nat Biotechnol 20(5):497-500; and the like). In certain embodiments, RNAi in mammalian cells may have advantages over other therapeutic methods. For example, producing siRNA molecules that block viral genetic activities in infected cells may reduce the effects of the virus. Platform ACes provided herein encoding siRNA molecule(s) are an additional utilization of the platform ACes technology. The platform ACes could be engineered to encode one or more siRNA molecules to create gene “knockdowns”. In one embodiment, a platform ACes can engineered to encode both the siRNA molecule and a replacement gene. For example, a mouse model or cell culture system could be generated using a platform ACes that has a knockdown of the endogenous mouse gene, by siRNA, and the human gene homolog expressing in place of the mouse gene. The placement of siRNA encoding sequences under the regulation of a regulatable or inducible promoter would allow one to temporally and/or spatially control the knockdown effect of the corresponding gene.
  • As used herein, a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Generally reporter genes are readily detectable. Examples of reporter genes include, but are not limited to nucleic acid encoding a fluorescent protein, CAT (chloramphenicol acetyl transferase) (Alton et al. (1979) [0078] Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984) Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158; Baldwin et al. (1984) Biochemistry 23:3663-3667); and alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
  • As used herein, growth under selective conditions means growth of a cell under conditions that require expression of a selectable marker for survival. [0079]
  • As used herein, an agent that destabilizes a chromosome is any agent known by those skilled in the art to enhance amplification events, and/or mutations. Such agents, which include BrdU, are well known to those skilled in the art. [0080]
  • In order to generate an artificial chromosome containing a particular heterologous nucleic acid of interest, it is possible to include the nucleic acid in the nucleic acid that is being introduced into cells to initiate production of the artificial chromosome. Thus, for example, a nucleic acid can be introduced into a cell along with nucleic acid encoding a selectable marker and/or a nucleic acid that targets to a heterochromatic region of a chromosome. For introducing a heterologous nucleic acid into the cell, it can be included in a fragment that includes a selectable marker or as part of a separate nucleic acid fragment and introduced into the cell with a selectable marker during the process of generating the artificial chromosomes. Alternatively, heterologous nucleic acid can be introduced into an artificial chromosome at a later time after the initial generation of the artificial chromosome. [0081]
  • As used herein, the minichromosome refers to a chromosome derived from a multicentric, typically dicentric, chromosome that contains more euchromatic than heterochromatic DNA. For purposes herein, the minichromosome contains a de novo centromere (e.g., a neocentromere). In some embodiments, for example, the minichromosome contains a centromere that replicates in animals, e.g., a mammalian centromere or in plants, e.g., a plant centromere. [0082]
  • As used herein, in vitro assembled artificial chromosomes or synthetic chromosomes can be either more euchromatic than heterochromatic or more heterochromatic than euchromatic and are produced by joining essential components of a chromosome in vitro. These components include at least a centromere, a megareplicator, a telomere and optionally secondary origins of replication. [0083]
  • As used herein, in vitro assembled plant or animal artificial chromosomes are produced by joining essential components (at least the centromere, telomere(s), megareplicator and optional secondary origins of replication) that function in plants or animals. In particular embodiments, the megareplicator contains sequences of rDNA, particularly plant or animal rDNA. [0084]
  • As used herein, a plant is a eukaryotic organism that contains, in addition to a nucleus and mitochondria, chloroplasts capable of carrying out photosynthesis. A plant can be unicellular or multicellular and can contain multiple tissues and/or organs. Plants can reproduce sexually or asexually and can be perennial or annual in growth. Plants can also be terrestrial or aquatic. The term “plant” includes a whole plant, plant cell, plant protoplast, plant calli, plant seed, plant organ, plant tissue, and other parts of a whole plant. [0085]
  • As used herein, stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95%, of the cells retain the chromosome. Stability is measured in the presence of a selective agent. Preferably these chromosomes are also maintained in the absence of a selective agent. Stable chromosomes also retain their structure during cell culturing, suffering no unintended intrachromosomal or interchromosomal rearrangements. [0086]
  • As used herein, de novo with reference to a centromere, refers to generation of an excess centromere in a chromosome as a result of incorporation of a heterologous nucleic acid fragment using the methods herein. [0087]
  • As used herein, BrdU refers to 5-bromodeoxyuridine, which during replication is inserted in place of thymidine. BrdU is used as a mutagen; it also inhibits condensation of metaphase chromosomes during cell division. [0088]
  • As used herein, ribosomal RNA (rRNA) is the specialized RNA that forms part of the structure of a ribosome and participates in the synthesis of proteins. Ribosomal RNA is produced by transcription of genes which, in eukaryotic cells, are present in multiple copies. In human cells, the approximately 250 copies of rRNA genes (i.e., genes which encode rRNA) per haploid genome are spread out in clusters on at least five different chromosomes (chromosomes 13, 14, 15, 21 and 22). In mouse cells, the presence of ribosomal DNA (rDNA, which is DNA containing sequences that encode rRNA) has been verified on at least 11 pairs out of 20 mouse chromosomes (chromosomes 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 19) (see e.g., Rowe et al. (1996) [0089] Mamm. Genome 7:886-889 and Johnson et al. (1993) Mamm. Genome 4:49-52). In Arabidopsis thaliana the presence of rDNA has been verified on chromosomes 2 and 4 (18S, 5.8S, and 25S rDNA) and on chromosomes 3,4, and 5 (5S rDNA)(see The Arabidopsis Genome Initiative (2000) Nature 408:796-815). In eukaryotic cells, the multiple copies of the highly conserved rRNA genes are located in a tandemly arranged series of rDNA units, which are generally about 40-45 kb in length and contain a transcribed region and a nontranscribed region known as spacer (i.e., intergenic spacer) DNA which can vary in length and sequence. In the human and mouse, these tandem arrays of rDNA units are located adjacent to the pericentric satellite DNA sequences (heterochromatin). The regions of these chromosomes in which the rDNA is located are referred to as nucleolar organizing regions (NOR) which loop into the nucleolus, the site of ribosome production within the cell nucleus.
  • As used herein, a megachromosome refers to a chromosome that, except for introduced heterologous DNA, is substantially composed of heterochromatin. Megachromosomes are made up of an array of repeated amplicons that contain two inverted megareplicons bordered by introduced heterologous DNA (see, e.g., FIG. 3 of U.S. Pat. No. 6,077,697 for a schematic drawing of a megachromosome). For purposes herein, a megachromosome is about 50 to 400 Mb, generally about 250-400 Mb. Shorter variants are also referred to as truncated megachromosomes (about 90 to 120 or 150 Mb), dwarf megachromosomes (˜150-200 Mb), and a micro-megachromosome (˜50-90 Mb, typically 50-60 Mb). For purposes herein, the term megachromosome refers to the overall repeated structure based on an array of repeated chromosomal segments (amplicons) that contain two inverted megareplicons bordered by any inserted heterologous DNA. The size will be specified. [0090]
  • As used herein, gene therapy involves the transfer or insertion of nucleic acid molecules into certain cells, which are also referred to as target cells, to produce specific products that are involved in preventing, curing, correcting, controlling or modulating diseases, disorders and deleterious conditions. The nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced. Alternatively, the nucleic acid may in some manner mediate expression of DNA that encodes a therapeutic product. This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. Expression of the nucleic acid by the target cells within an organism afflicted with a disease or disorder thereby provides for modulation of the disease or disorder. The nucleic acid encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. [0091]
  • For use in gene therapy, cells can be transfected in vitro, followed by introduction of the transfected cells into an organism. This is often referred to as ex vivo gene therapy. Alternatively, the cells can be transfected directly in vivo within an organism. [0092]
  • As used herein, therapeutic agents include, but are not limited to, growth factors, antibodies, cytokines, such as tumor necrosis factors and interleukins, and cytotoxic agents and other agents disclosed herein and known to those of skill in the art. Such agents include, but are not limited to, tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), pro-coagulants such as tissue factor and tissue factor variants, pro-apoptotic agents such FAS-ligand, fibroblast growth factors (FGF), nerve growth factor and other growth factors. [0093]
  • As used herein, a therapeutically effective product is a product that is encoded by heterologous DNA that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures the disease. [0094]
  • As used herein, transgenic plants and animals refer to plants and animals in which heterologous or foreign nucleic acid is expressed or in which the expression of a gene naturally present in the plant or animal has been altered by virtue of introduction of heterologous or foreign nucleic acid. [0095]
  • As used herein, IRES (internal ribosome entry site; see, e.g., SEQ ID No. 27 and nucleotides 2736-3308 SEQ ID No. 28) refers to a region of a nucleic acid molecule, such as an mRNA molecule, that allows internal ribosome entry sufficient to initiate translation, which initiation can be detected in an assay for cap-independent translation (see, e.g., U.S. Pat. No. 6,171,821). The presence of an IRES within an mRNA molecule allows cap-independent translation of a linked protein-encoding sequence that otherwise would not be translated. [0096]
  • Internal ribosome entry site (IRES) elements were first identified in picornaviruses, which elements are considered the paradigm for cap-independent translation. The 5′ UTRs of all picornaviruses are long and mediate translational initiation by directly recruiting and binding ribosomes, thereby circumventing the initial cap-binding step. IRES elements are frequently found in viral mRNA, they are rare in non-viral mRNA. Among non-viral mRNA molecules that contain functional IRES elements in their respective 5′ UTRs are those encoding immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) [0097] Nature 353:90-94); Drosophila Antennapedia (Oh et al. (1992) Genes Dev, 6:1643-1653); D. Ultrabithorax (Ye et al. (1997) Mol. Cell Biol. 17:1714-21); fibroblast growth factor 2 (Vagner et al. (1995) Mol. Cell Biol. 15:35-44); initiation factor eIF4G (Gan et al. (1998) J. Biol. Chem. 273:5006-5012); proto-oncogene c-myc (Nanbru et al. (1995) J. Biol. Chem. 272:32061-32066; Stoneley (1998) Oncogene 16:423-428); IRESH; from the 5′UTR of NRF1 gene (Oumard et al. (2000) Mol. and Cell Biol., 20(8):2755-2759); and vascular endothelial growth factor (VEGF) (Stein et al. (1998) Mol. Cell Biol. 18:3112-9).
  • As used herein, a promoter, with respect to a region of DNA, refers to a sequence of DNA that contains a sequence of bases that signals RNA polymerase to associate with the DNA and initiate transcription of RNA (such as pol 11 for mRNA) from a template strand of the DNA. A promoter thus generally regulates transcription of DNA into mRNA. A particular promoter provided herein is the Ferritin heavy chain promoter (excluding the Iron Response Element, located in the 5′UTR), which was joined to the 37 bp Fer-1 enhancer element. This promoter is set forth as SEQ ID NO:128. The endogenous Fer-1 enhancer element is located upstream of the Fer-1 promoter (e.g., a Fer-1 oligo was cloned proximal to the core promoter). [0098]
  • As used herein, isolated, substantially pure nucleic acid, such as, for example, DNA, refers to nucleic acid fragments purified according to standard techniques employed by those skilled in the art, such as that found in Sambrook et al. ((2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 3rd edition). [0099]
  • As used herein, expression refers to the transcription and/or translation of nucleic acid. For example, expression can be the transcription of a gene that may be transcribed into an RNA molecule, such as a messenger RNA (mRNA) molecule. Expression may further include translation of an RNA molecule and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA. With respect to an antisense construct, expression may refer to the transcription of the antisense DNA. [0100]
  • As used herein, vector or plasmid refers to discrete elements that are used to introduce heterologous nucleic acids into cells for either expression of the heterologous nucleic acid or for replication of the heterologous nucleic acid. Selection and use of such vectors and plasmids are well within the level of skill of the art. [0101]
  • As used herein, transformation/transfection refers to the process by which nucleic acid is introduced into cells. The terms transfection and transformation refer to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski et al. (1984) [0102] EMBO J. 3:2717-2722; Potrykus et al. (1985) Mol. Gen. Genet. 199:169-177; Reich et al. (1986) Biotechnology 4:1001-1004; Klein et al. (1987) Nature 327:70-73; U.S. Pat. No. 6,143,949; Paszkowski et al. (1989) in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J and Vasil, L. K. Academic Publishers, San Diego, Calif., p. 52-68; and Frame et al. (1994) Plant J. 6:941-948), direct uptake using calcium phosphate (CaPO4; see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376), polyethylene glycol (PEG)-mediated DNA uptake, lipofection (see, e.g., Strauss (1996) Meth. Mol. Biol. 54:307-327), microcell fusion (see, EXAMPLES, see, also Lambert (1991) Proc; Natl. Acad. Sci. U.S.A. 88:5907-5911; U.S. Pat. No. 5,396,767, Sawford et al. (1987) Somatic Cell Mol. Genet. 13:279-284; Dhar et al. (1984) Somatic Cell Mol. Genet. 10:547-559; and McNeill-Killary et al. (1995) Meth. Enzymol. 254:133-152), lipid-mediated carrier systems (see, e.g., Teifel et al. (1995) Biotechniques 19:79-80; Albrecht et al. (1996) Ann. Hematol. 72:73-79; Holmen et al. (1995) In Vitro Cell Dev. Biol. Anim. 31:347-351; Remy et al. (1994) Bioconjug. Chem. 5:647-654; Le Bolch et al. (1995) Tetrahedron Lett. 36:6681-6684; Loeffler et al. (1993) Meth. Enzymol. 217:599-618) or other suitable method. Methods for delivery of ACes are described in copending U.S. application Ser. No. 09/815,979. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a vector within the host cell.
  • As used herein, “delivery,” which is used interchangeably with “transfection,” refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids. [0103]
  • As used herein, injected refers to the microinjection, such as by use of a small syringe, needle, or pipette, for injection of nucleic acid into a cell. [0104]
  • As used herein, substantially homologous DNA refers to DNA that includes a sequence of nucleotides that is sufficiently similar to another such sequence to form stable hybrids, with each other or a reference sequence, under specified conditions. [0105]
  • It is well known to those of skill in this art that nucleic acid fragments with different sequences may, under the same conditions, hybridize detectably to the same “target” nucleic acid. Two nucleic acid fragments hybridize detectably, under stringent conditions over a sufficiently long hybridization period, because one fragment contains a segment of at least about 10, 14 or 16 or more nucleotides in a sequence that is complementary (or nearly complementary) to a substantially contiguous sequence of at least one segment in the other nucleic acid fragment. If the time during which hybridization is allowed to occur is held constant, at a value during which, under preselected stringency conditions, two nucleic acid fragments with complementary base-pairing segments hybridize detectably to each other, departures from exact complementarity can be introduced into the base-pairing segments, and base-pairing will nonetheless occur to an extent sufficient to make hybridization detectable. As the departure from complementarity between the base-pairing segments of two nucleic acids becomes larger, and as conditions of the hybridization become more stringent, the probability decreases that the two segments will hybridize detectably to each other. [0106]
  • Two single-stranded nucleic acid segments have “substantially the same sequence”, if (a) both form a base-paired duplex with the same segment, and (b) the melting temperatures of the two duplexes in a solution of 0.5× SSPE differ by less than 10° C. If the segments being compared have the same number of bases, then to have “substantially the same sequence”, they will typically differ in their sequences at fewer than 1 base in 10. Methods for determining melting temperatures of nucleic acid duplexes are well known (see, e.g., Meinkoth et al. (1984) [0107] Anal. Biochem. 138:267-284 and references cited therein).
  • As used herein, a nucleic acid probe is a DNA or RNA fragment that includes a sufficient number of nucleotides to specifically hybridize to DNA or RNA that includes complementary or substantially complementary sequences of nucleotides. A probe may contain any number of nucleotides, from as few as about 10 and as many as hundreds of thousands of nucleotides. The conditions and protocols for such hybridization reactions are well known to those of skill in the art as are the effects of probe size, temperature, degree of mismatch, salt concentration and other parameters on the hybridization reaction. For example, the lower the temperature and higher the salt concentration at which the hybridization reaction is carried out, the greater the degree of mismatch that may be present in the hybrid molecules. [0108]
  • To be used as a hybridization probe, the nucleic acid is generally rendered detectable by labeling it with a detectable moiety or label, such as [0109] 32P, 3H and 14C, or by other means, including chemical labeling, such as by nick-translation in the presence of deoxyuridylate biotinylated at the 5′-position of the uracil moiety. The resulting probe includes the biotinylated uridylate in place of thymidylate residues and can be detected (via the biotin moieties) by any of a number of commercially available detection systems based on binding of streptavidin to the biotin. Such commercially available detection systems can be obtained, for example, from Enzo Biochemicals, Inc. (New York, N.Y.). Any other label known to those of skill in the art, including non-radioactive labels, may be used as long as it renders the probes sufficiently detectable, which is a function of the sensitivity of the assay, the time available (for culturing cells, extracting DNA, and hybridization assays), the quantity of DNA or RNA available as a source of the probe, the particular label and the means used to detect the label.
  • Once sequences with a sufficiently high degree of homology to the probe are identified, they can readily be isolated by standard techniques (see, e.g., Sambrook et al. (2001) [0110] Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press).
  • As used herein, conditions under which DNA molecules form stable hybrids are considered substantially homologous, and a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a polypeptide. By the term “substantially homologous” is meant having at least 75%, preferably 80%, preferably at least 90%, most preferably at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function. [0111]
  • The terms “homology” and “identity” are often used interchangeably. In this regard, percent homology or identity may be determined, for example, by comparing sequence information using a GAP computer program. The GAP program utilizes the alignment method of Needleman and Wunsch ([0112] J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • By sequence identity, the number of conserved amino acids are determined by standard alignment algorithms programs, and are used with default gap penalties established by each supplier. Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Preferably the two molecules will hybridize under conditions of high stringency. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule. [0113]
  • Whether any two nucleic acid molecules have nucleotide sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determined using known computer algorithms such as the “FAST A” program, using for example, the default parameters as in Pearson and Lipman, [0114] Proc. Natl. Acad. Sci. USA 85:2444 (1988). Alternatively the BLAST function of the National Center for Biotechnology Information database may be used to determine relative sequence identity.
  • In general, sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: [0115] Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J Applied Math 48:1073 (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).
  • Therefore, as used herein, the term “identity” represents a comparison between a test and a reference polypeptide or polynucleotide. For example, a test polypeptide may be defined as any polypeptide that is 90% or more identical to a reference polypeptide. [0116]
  • As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides. [0117]
  • Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polynucleotide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons may be made between a test and reference polynucleotides. Such differences may be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they may be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10/100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, or deletions. [0118]
  • As used herein: stringency of hybridization in determining percentage mismatch encompass the following conditions or equivalent conditions thereto: [0119]
  • 1) high stringency: 0.1× SSPE or SSC, 0.1% SDS, 65° C. [0120]
  • 2) medium stringency: 0.2× SSPE or SSC, 0.1% SDS, 50° C. [0121]
  • 3) low stringency: 1.0× SSPE or SSC, 0.1% SDS, 50° C. [0122]
  • or any combination of salt and temperature and other reagents that result in selection of the same degree of mismatch or matching. Equivalent conditions refer to conditions that select for substantially the same percentage of mismatch in the resulting hybrids. Additions of ingredients, such as formamide, Ficoll, and Denhardt's solution affect parameters such as the temperature under which the hybridization should be conducted and the rate of the reaction. Thus, hybridization in 5× SSC, in 20% formamide at 42° C. is substantially the same as the conditions recited above hybridization under conditions of low stringency. The recipes for SSPE, SSC and Denhardt's and the preparation of deionized formamide are described, for example, in Sambrook et al. (1989) [0123] Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 8; see, Sambrook et al., vol. 3, p. B.13, see, also, numerous catalogs that describe commonly used laboratory solutions. It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. As used herein, all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.
  • As used herein, conservative amino acid substitutions, such as those set forth in Table 1, are those that do not eliminate biological activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. [0124] Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/Cummings Pub. co., p.224). Conservative amino acid substitutions are made, for example, in accordance with those set forth in TABLE 1 as follows:
    TABLE 1
    Original residue Conservative substitution
    Ala (A) Gly; Ser, Abu
    Arg (R) Lys, orn
    Asn (N) Gln; His
    Cys (C) Ser
    Gln (Q) Asn
    Glu (E) Asp
    Gly (G) Ala; Pro
    His (H) Asn; Gln
    Ile (I) Leu; Val; Met; Nle; Nva
    Leu (L) Ile; Val; Met; Nle; Nva
    Lys (K) Arg; Gln; Glu
    Met (M) Leu; Tyr; Ile; NLe Val
    Ornithine Lys; Arg
    Phe (F) Met; Leu; Tyr
    Ser (S) Thr
    Thr (T) Ser
    Trp (W) Tyr
    Tyr (Y) Trp; Phe
    Val (V) Ile; Leu; Met; Nle; Nva
  • Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions. [0125]
  • As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single-letter designations used routinely in the art. [0126]
  • As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA. [0127]
  • As used herein, a probe or primer based on a nucleotide sequence includes at least 10, 14, 16, 30 or 100 contiguous nucleotides from the reference nucleic acid molecule. [0128]
  • As used herein, recombinant production by using recombinant DNA methods refers to the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA. [0129]
  • As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in in vitro systems designed to test or use such activities. Thus, for purposes herein the biological activity of a luciferase is its oxygenase activity whereby, upon oxidation of a substrate, light is produced. [0130]
  • The terms substantially identical or similar varies with the context as understood by those skilled in the relevant art and generally means at least 40, 60, 80, 90, 95 or 98%. [0131]
  • As used herein, substantially identical to a product means sufficiently similar so that the property is sufficiently unchanged so that the substantially identical product can be used in place of the product. [0132]
  • As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers or isomers. In such instances, further purification might increase the specific activity of the compound. [0133]
  • As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. The vectors typically remain episomal, but may be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. An expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. [0134]
  • As used herein, protein-binding-sequence refers to a protein or peptide sequence that is capable of specific binding to other protein or peptide sequences generally, to a set of protein or peptide sequences or to a particular protein or peptide sequence. [0135]
  • As used herein, a composition refers to any mixture of two or more ingredients. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof. [0136]
  • As used herein, a combination refers to any association between two or more items. [0137]
  • As used herein, fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions. [0138]
  • As used herein, a cellular extract refers to a preparation or fraction that is made from a lysed or disrupted cell. [0139]
  • As used herein, the term “subject” refers to animals, plants, insects, and birds and other phyla, genera and species into which nucleic acid molecules may be introduced. Included are higher organisms, such as mammals, fish, insects and birds, including humans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, hamsters, cats, dogs, horses, chicken and others. [0140]
  • As used herein, flow cytometry refers to processes that use a laser based instrument capable of analyzing and sorting out cells and or chromosomes based on size and fluorescence. [0141]
  • As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) [0142] Biochem. 11:942-944).
  • B. Recombination Systems [0143]
  • Site-specific recombination systems typically contain three elements: a pair of DNA sequences (the site-specific recombination sequences) and a specific enzyme (the site-specific recombinase). The site-specific recombinase catalyzes a recombination reaction between two site-specific recombination sequences. [0144]
  • A number of different site-specific recombinase systems are available and/or known to those of skill in the art, including, but not limited to: the Cre/lox recombination system using CRE recombinase (see, e.g., SEQ ID Nos. 58 and 59) from the [0145] Escherichia coli phage P1 (see, e.g., Sauer (1993) Methods in Enzymology 225:890-900; Sauer et al. (1990) The New Biologist 2:441-449), Sauer (1994) Current Opinion in Biotechnology 5:521-527; Odell et al. (1990) Mol Gen Genet. 223:369-378; Lasko et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:6232-6236; U.S. Pat. No. 5,658,772), the FLP/FRT system of yeast using the FLP recombinase (see, SEQ ID Nos. 60 and 61) from the 2μ episome of Saccharomyces cerevisiae (Cox (1983) Proc. Natl. Acad. Sci. U.S.A. 80:4223; Falco et al. (1982) Cell 29:573-584; Golic et al. (1989) Cell 59:499-509; U.S. Pat. No. 5,744,336), the resolvases, including Gin recombinase of phage Mu (Maeser et al. (1991) Mol Gen Genet. 230:170-176; Klippel, A. et al (1993) EMBO J. 12:1047-1057; see, e.g., SEQ ID Nos. 64-67), Cin, Hin, αδ Tn3; the Pin recombinase of E. coli (see, e.g., SEQ ID Nos. 68 and 69; Enomoto et al. (1983) J Bacteriol. 6:663-668), the R/RS system of the pSR1 plasmid of Zygosaccharomyces rouxii (Araki et al. (1992) J. Mol. Biol. 225:25-37; Matsuzaki et al. (1990) J. Bacteriol. 172: 610-618) and site-specific recombinases from Kluyveromyces drosophilarium (Chen et al. (1986) Nucleic Acids Res. 314:4471-4481) and Kluyveromyces waltii (Chen et al. (1992) J. Gen. Microbiol. 138:337-345). Other systems are known to those of skill in the art (Stark et al. Trends Genet. 8:432-439; Utatsu et al. (1987) J. Bacteriol. 169:5537-5545; see, also, U.S. Pat. No. 6,171,861).
  • Members of the highly related family of site-specific recombinases, the resolvase family, such as γδ, Tn3 resolvase, Hin, Gin, and Cin are also available. Members of this family of recombinases are typically constrained to intramolecular reactions (e.g., inversions and excisions) and can require host-encoded factors. Mutants have been isolated that relieve some of the requirements for host factors (Maeser et al. (1991) [0146] Mol. Gen. Genet. 230:170-176), as well as some of the constraints of intramolecular recombination (see, U.S. Pat. No. 6,171,861).
  • The bacteriophage P1 Cre/lox and the yeast FLP/FRT systems are particularly useful systems for site-specific integration, inversion or excision of heterologous nucleic acid into, and out of, chromosomes, particularly ACes as provided herein. In these systems a recombinase (Cre or FLP) interacts specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences. The sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT). [0147]
  • The FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells (U.S. Pat. No. 5,744,386), and, thus, can be used for producing plant artificial chromosome platforms. In general, short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites. The system catalyzes intra- and intermolecular reactions, and, thus, can be used for DNA excision and integration reactions. The recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation. The site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event. [0148]
  • In the Cre-lox system, discovered in bacteriophage P1, recombination between loxP sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Pat. No. 5,658,772). This system can be used to insert, invert or excise nucleic acid located between two lox sites. Cre can be expressed from a vector. Since the lox site is an asymmetrical nucleotide sequence, lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites. In addition, reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region. [0149]
  • Any site-specific recombinase system known to those of skill in the art is contemplated for use herein. It is contemplated that one or a plurality of sites that direct the recombination by the recombinase are introduced into an artificial chromosome to produce platform ACes. The resulting platform ACes are introduced into cells with nucleic acid encoding the cognate recombinase, typically on a vector, and nucleic acid encoding heterologous nucleic acid of interest linked to the appropriate recombination site for insertion into the platform ACes. The recombinase-encoding-nucleic acid may be introduced into the cells on the same vector, or a different vector, encoding the heterologous nucleic acid. [0150]
  • An [0151] E. coli phage lambda integrase system for ACes platform engineering and for artificial chromosome engineering is provided (Lorbach et al. (2000) J. Mol. Biol 296:1175-1181). The phage lambda integrase (Landy, A. (1989) Annu. Rev. Biochem. 58:913-94) is adapted herein and the cognate att sites are provided. Chromosomes, including ACes, engineered to contain one or a plurality of att sites are provided, as are vectors encoding a mutant integrase that functions in the absence other factors. Methods using the modified chromosomes and vectors for introduction of heterologous nucleic acid are also provided.
  • For purposes herein, one or more of the sites (e.g., a single site or a pair of sites) required for recombination are introduced into an artificial chromosome, such as an ACes chromosome. The enzyme for catalyzing site-directed recombination is introduced with the DNA of interest, or separately, or is engineered onto the artificial chromosome under the control of a regulatable promoter. [0152]
  • As described herein, artificial chromosome platforms containing one or multiple recombination sites are provided. The methods and resulting products are exemplified with the lambda phage Att/Int system, but similar methods may be used for production of ACes platforms with other recombination systems. [0153]
  • The Att/Int system and vectors provided herein are not only intended for engineering ACes platforms, but may be used to engineer an Att/Int system into any chromosome. Introduction of att sites into a chromosome will permit engineering of natural chromosomes, such as by permitting targeted integration genes or regulatory regions, and by controlled excision of selected regions. For example, genes encoding a particular trait may be added to a chromosome, such as plant chromosome engineered to contain one or plurality of att sites. Such chromosomes may be used for screening DNA to identify genes. Large pieces of DNA can be introduced into cells and the cells screened phenotypically to select those having the desired trait. [0154]
  • C. Platforms [0155]
  • Provided herein are platform artificial chromosomes (platform ACes) containing single or multiple site-specific recombination sites. Chromosome-based platform technology permits efficient and tractable engineering and subsequent expression of multiple gene targets. Methods are provided that use DNA vectors and fragments to create platform artificial chromosomes, including animal, particularly mammalian, artificial chromosomes, and plant artificial chromosomes. The artificial chromosomes contain either single or multiple sequence-specific recombination sites suitable for the placement of target gene expression vectors onto the platform chromosome. The engineered chromosome-based platform ACes technology is applicable for methods, including cellular and transgenic protein production, transgenic plant and animal production and gene therapy. The platform ACes are also useful for producing a library of ACes comprising random portions of a given genome (e.g., a mammalian, plant or prokaryotic genome) for genomic screening; as well as a library of cells comprising different and/or mutually exclusive ACes therein. [0156]
  • Exemplary of artificial chromosome platforms are those based on ACes. ACes artificial chromosomes are non-viral, self-replicating nucleic acid molecules that function as a natural chromosome, having all the elements required for normal chromosomal replication and maintenance within the cell nucleus. ACes artificial chromosomes do not rely on integration into the genome of the cell to be effective, and they are not limited by DNA carrying capacity and as such the therapeutic gene(s) of interest, including regulatory sequences, can be engineered into the ACes. In addition, ACes are stable in vitro and in vivo and can provide predictable long-term gene expression. Once engineered and delivered to the appropriate cell or embryo, ACes work independently alongside host chromosomes, for ACes that are predominantly heterochromatin producing only the products (proteins) from the genes it carries. As provided herein ACes are modified by introduction of recombination site(s) to provide a platform for ready introduction of heterologous nucleic acid. The ACes platforms can be used for production of transgenic animals and plants; as vectors for genetic therapy; for use as protein production systems; for animal models to identify and target new therapeutics; in cell culture for the development and production of therapeutic proteins; and for a variety of other applications. [0157]
  • 1. Generation of Artificial Chromosomes [0158]
  • Artificial chromosomes may be generated by any method known to those of skill in the art. Of particular interest herein are the ACes artificial chromosomes, which contain a repeated unit. Methods for production of ACes are described in detail in U.S. Pat. Nos. 6,025,155 and 6,077,697, which, as with all patents, applications, publications and other disclosure, are incorporated herein in their entirety. [0159]
  • Generation of de novo ACes. [0160]
  • ACes can be generated by cotransfecting exogenous DNA—such as a mammary tissue specific DNA cassette including the gene sequences for a therapeutic protein, with a rDNA fragment and a drug resistance marker gene into the desired eukaryotic cell, such as plant or animal cells, such as murine cells in vitro. DNA with a selectable or detectable marker is introduced, and can be allowed to integrate randomly into pericentric heterochromatin or can be targeted to pericentric heterochromatin, such as that in rDNA gene arrays that reside on acrocentric chromosomes, such as the short arms of acrocentric chromosomes. This integration event activates the “megareplicator” sequence and amplifies the pericentric heterochromatin and the exogenous DNA, and duplicates a centromere. Ensuing breakage of this “dicentric” chromosome can result in the production of daughter cells that contain the substantially-original chromosome and the new artificial chromosome. The resulting ACes contain all the essential elements needed for stability and replication in dividing cells-centromere, origins of replications, and telomeres. ACes have been produced that express marker genes (lacZ, green fluorescent protein, neomycin-resistance, puromycin-resistance, hygromycin-resistance) and genes of interest. Isolated ACes, for example, have been successfully transferred intact to rodent, human, and bovine cells by electroporation, sonoporation, microinjection, and transfection with lipids and dendrimers. [0161]
  • To render the creation of ACes with desired genes more tractable and efficient, “platform” ACes (platform-ACes) can be produced that contain defined DNA sequences for enzyme-mediated homologous DNA recombination, such as by Cre or FLP recombinases (Bouhassira et al. (1996) [0162] Blood 88(supplement 1):190a; Bouhassira et al. (1997) Blood, 90:3332-3344; Siebler et al. (1997) Biochemistry: 36:1740-1747; Siebler et al. (1998) Biochemistry 37: 6229-6234; and Bethke et al. (1997) Nucl. Acids Res. 25:2828-2834), and as exemplified herein the lambda phage integrase. A lox site contains two 13 bp inverted repeats to which Cre-recombinase binds and an intervening 8 bp core region. Only pairs of sites having identity in the central 6 bp of the core region are proficient for recombination; sites having non-identical core sequences (heterospecific lox sites) do not efficiently recombine with each other (Hoess et al. (1986) Nucleic Acids Res. 14:2287-2300).
  • Generating Acrocentric Chromosomes for Plant Artificial Chromosome Formation. [0163]
  • In human and mouse cells de novo formation of a satellite DNA based artificial chromosome (SATAC, also referred to as ACes) can occur in an acrocentric chromosome where the short arm contains only pericentric heterochromatin, the rDNA array, and telomere sequences. Plant species may not have any acrocentric chromosomes with the same physical structure described, but “megareplicator” DNA sequences reside in the plant rDNA arrays, also known as the nucleolar organizing regions (NOR). A structure like those seen in acrocentric mammalian chromosomes can be generated using site-specific recombination between appropriate arms of plant chromosomes. [0164]
  • Approach [0165]
  • Qin et al. ((1994) [0166] Proc. Natl. Acad. Sci. U.S.A. 91:1706-1710, 1994) describes crossing two Nicotiana tabacum transgenic plants. One plant contains a construct encoding a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt), the other plant carries a construct containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre DNA recombinase coding region (35S-lox-cre). The constructs were introduced separately by infecting leaf explants with agrobacterium tumefaciens which carries the kanamycin-resistance gene (KanR). The resultant KanR transgenic plants were crossed. Plants that carried the appropriate DNA recombination event were identified by hygromycin-resistance.
  • Modification of the Above for Generation of ACes [0167]
  • The Kan[0168] R cultivars are initially screened, such as by FISH, to identify two sets of candidate transgenic plants. One set has one construct integrated in regions adjacent to the pericentric heterochromatin on the short arm of any chromosome. The second set of candidate plants has the other construct integrated in the NOR region of appropriate chromosomes. To obtain reciprocal translocation both sites must be in the same orientation. Therefore a series of crosses are required, KanR plants generated, and FISH analyses performed to identify the appropriate “acrocentric” plant chromosome for de novo plant ACes formation.
  • 2. Bacteriophage Lambda Integrase-Based Site-Specific Recombination System [0169]
  • An integral part of the platform technology includes a site-specific recombination system that allows the placement of selected gene targets or genomic fragments onto the platform chromosomes. Any such system may be used. In particular, a method is provided for insertion of additional DNA fragments into the platform chromosome residing in the cell via sequence-specific recombination using the recombinase activity of the bacteriophage lambda integrase. The lambda integrase system is exemplary of the recombination systems contemplated for ACes. Any known recombination system, including any described herein, particularly any that operates without the need for additional factors or that, by virtue of mutation, does not require additional factors, is contemplated. [0170]
  • As noted the lambda integrase system provided herein can be used with natural chromosomes and artificial chromosomes in addition to ACes. Single or a plurality of recombination sites, which may be the same or different, are introduced into artificial chromosomes to produce artificial chromosome platforms. [0171]
  • [0172] 3. Creation of Bacteriophage Lambda Integrase Site-Specific Recombination System
  • The lambda phage-encoded integrase (designated Int) is a prototypical member of the integrase family. Int effects integration and excision of the phage in and out of the [0173] E. coli genome via recombination between pairs of attachment sites designated attB/attP and attL/attR. Each att site contains two inverted 9 base pair core Int binding sites and a 7 base pair overlap region that is identical in wild-type att sites. Each site, except for attB contains additional Int binding sites. In flanking regions, there are recognition sequences for accessory DNA binding proteins, such as integration host factor (IHF), factor for inversion stimulation (FIS) and the phage encoded excision protein (XIS). Except for attB, Int is a heterobivalent DNA-binding protein and, with assistance from the accessory proteins and negative DNA supercoiling, binds simultaneously to core and arm sites within the same att site.
  • Int, like Cre and FLP, executes an ordered sequential pair of strand exchanges during integrative and excisive recombination. The natural pairs of target sequences for Int, attB and attP or attL and attR are located on the same or different DNA molecules resulting in intra or intermolecular recombination, respectively. For example, intramolecular recombination occurs between inversely oriented attB and attP, or between attL and attR sequences, respectively, leading to inversion of the intervening DNA segment. [0174]
  • Like the recombinase systems, such as Cre and FLP, Int directs site-specific recombination. Unlike the other systems, such Cre and FLP, Int generally requires additional protein factors for integrative and excisive recombination and negative supercoiling for integrative recombination. Hence, the Int system had not been used in eukaryotic targeting systems. [0175]
  • Mutant Int proteins, designated Int-h (El 74K) and a derivative thereof Int-h/218(E174K/E218K) do not require accessory proteins to perform intramolecular integrative and excisive recombination in co-transfection assays in human cells (Lorbach et al. (2000) [0176] J Mol. Biol. 296:1175-1181); wild-type Int does not catalyze intramolecular recombination in human cells harboring target sites attB and attP.
  • Hence it had been demonstrated that mutant Int can catalyze factor-independent recombination events in human cells. [0177]
  • There has been no demonstration by others that this system can be used for engineering of eukaryotic genomes or chromosomes. Provided herein are chromosomes, including artificial chromosomes, such as but not limited to ACes that contain att sites (e.g., platform ACes), and the use of such chromosomes for targeted integration of heterologous DNA into such chromosomes in eukaryotic cells, including animal, such as rodent and human, and plant cells. Mutant Int provided herein is shown to effect site-directed recombination between sites in artificial chromosomes and vectors containing cognate sites. [0178]
  • An additional component of the chromosome-based platform technology is the site-specific integration of target DNA sequences onto the platform. For this the native bacteriophage lambda integrase has been modified to carry out this sequence specific DNA recombination event in eukaryotic cells. The bacteriophage lambda integrase and its cognate DNA substrate att is a member of the site-specific recombinase family that also includes the bacteriophage P1 Cre/lox system as well as the [0179] Saccharomyces cerevisiae 2 micron based FLP/FRT system (see, e.g., Landy (1989) Ann. Rev. Biochem 58:913-949; Hoess et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:3398-3402; Broach et al. (1982) Cell 29:227-234).
  • By combining DNA endonuclease and DNA ligase activity these recombinases recognize and catalyze DNA exchanges between sequences flanking the recognition site. During the integration of lambda genome into the [0180] E. coli (lambda recombination) genome, the phage integrase (INT) in association with accessory proteins catalyzes the DNA exchange between the attP site of the phage genome and the attB site of the bacterial genome resulting in the formation of attL and attR sites (FIG. 6). The engineered bacteriophage lambda integrase has been produced herein to carry out an intermolecular DNA recombination event between an incoming DNA molecule (primarily on a vector containing the bacterial attB site) and the chromosome-based platform carrying the lambda attP sequence independent of lambda bacteriophage or bacterial accessory proteins.
  • In contrast to the bidirectional Cre/lox and FLP/FRT system, the engineered lambda recombination system derived for chromosome-based platform technology is advantageously unidirectional because accessory proteins, which are absent, are required for excision of integrated nucleic acid upon further exposure to the lambda Int recombinase. [0181]
  • 4. Creation of Platform Chromosome Containing Single or Multiple Sequence-Specific Recombination Sites [0182]
  • a. Multiple Sites [0183]
  • For the creation of a platform chromosome containing multiple, sequence-specific recombination sites, artificial chromosomes are produced as depicted in FIG. 5 and Example 3. As discussed above, artificial chromosomes can be produced using any suitable methodology, including those described in U.S. Pat. Nos. 5,288,625; 5,712,134; 5,891,691; 6,025,155. Briefly, to prepare artificial chromosomes containing multiple recombination (e.g., integration) sites, nucleic acid (either in the form a one or more plasmids, such as the plasmid pSV40193attPsensePUR set forth in Example 3) is targeted into an amplifiable region of a chromosome, such as the pericentric region of a chromosome. Among such regions are the rDNA gene loci in acrocentric mammalian chromosomes. Hence, targeting nucleic acid for integration into the rDNA region of mammalian acrocentric chromosomes can include the mouse rDNA fragments (for targeting into rodent cell lines) or large human rDNA regions on BAC/PAC vectors (or subclones thereof in standard vectors) for targeting into human acrocentric chromosomes, such as for human gene therapy applications. The targeting nucleic acid generally includes a detectable or selectable marker, such as antibiotic resistance, such as puromycin and hygromycin, a recombination site (such as attP, attB, attL, attR or the like), and/or human selectable markers as required for gene therapy applications. Cells are grown under conditions that result in amplification and ultimately production of ACes artificial chromosomes having multiple recombination (e.g., integration) sites therein. ACes having the desired size are selected for further engineering. [0184]
  • b. Creation of Platform Chromosome Containing a Single Sequence-Specific Recombination Site [0185]
  • In this method a mammalian platform artificial chromosome is generated containing a single sequence-specific recombination site. In the Example below, this approach is demonstrated using a puromycin resistance marker for selection and a mouse rDNA fragment for targeting into the rDNA locus on mouse acrocentric chromosomes. Other selection markers and targeting DNA sequences as desired and known to those of skill in the art can be used. Additional resistance markers include genes conferring resistance to the antibiotics neomycin, blasticidin, hygromycin and zeocin. For applications, such as gene therapy in which potentially immunogenic responses are to be avoided, host, such as human, derived selectable markers or markers detectable with monoclonal antibodies (MAb) followed by fluorescent activated cell sorting (FACS) can be used. Examples in this class include, but are not limited to: human nerve growth factor receptor (detection with MAb); truncated human growth factor receptor (detection with MAb); mutant human dihydrofolate reductase (DHFR; detectable using a fluorescent methotrexate substrate); secreted alkaline phosphatase (SEAP; detectable with fluorescent substrate); thymidylate synthase (TS; confers resistance to fluorodeoxyuridine); human CAD gene (confers resistance to N-phosphonacetyl-L-aspartate (PALA)). [0186]
  • To construct a platform artificial chromosome with a single site, an ACes artificial chromosome (or other artificial chromosome of interest) can be produced containing a selectable marker. A single sequence specific recombination site is targeted onto ACes via homologous recombination. For this, DNA sequences containing the site-specific recombination sequence are flanked with DNA sequences homologous to a selected sequence in the chromosome. For example, when using a chromosome containing rDNA or satellite DNA, such DNA can be used as homologous sequences to target the site-specific recombination sequence onto the chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the chromosome, the DNA is transfected into cells harboring the chromosome. After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified, for example by screening single cell subclones via expression of resistance or a fluorescent marker and PCR analysis. In one embodiment, a platform artificial chromosome, such as a platform ACes, that contains a single copy of the recombination site is selected. Examples 2B and 2D exemplify the process, and FIG. 3 provides a diagram depicting one method for the creation of a platform mammalian chromosome containing a single sequence-specific recombination site. [0187]
  • 5. Lambda Integrase Mediated Recombination of Target Gene Expression Vector onto Platform Chromosome [0188]
  • The third component of the chromosome-based platform technology involves the use of target gene expression vectors carrying, for example, genes for gene therapy, genes for transgenic animal or plant production, and those required for cellular protein production of interest. Using lambda integrase mediated site-specific recombination, or any other recombinase-mediated site-specific recombination, the target gene expression vectors are introduced onto the selected chromosome platform. The use of target gene expression vector permits use of the de novo generated chromosome-based platforms for a wide range of gene targets. Furthermore, chromosome platforms containing multiple attP sites provides the opportunity to incorporate multiple gene targets onto a single platform, thereby providing for expression of multiple gene targets, including the expression of cellular and genetic regulatory genes and the expression of all or parts of metabolic pathways. In addition to expressing small target genes, such as cDNA and hybrid cDNA/artificial intron constructs, the chromosome-based platform can be used for engineering and expressing large genomic fragments carrying target genes along with its endogenous genomic promoter sequences. This is of importance, for example, where the therapy requires precise cell specific expression and in instances where expression is best achieved from genomic clones rather than cDNA clones. FIG. 9 provides a diagram summarizing one embodiment of the chromosome-based technology. [0189]
  • A feature of the target gene expression vector that is of interest to include is a promoterless marker gene, which as exemplified (see, FIG. 9) contains an upstream attB site ([0190] marker 2 on FIG. 9). The nucleic acid encoding the marker is not expressed unless it is placed downstream from a promoter sequence. Using the recombinase technology provided herein, such as the lambda integrase technology (λINTE174R on FIG. 8) provided herein, site-specific recombination between the attB site on the vector and the promoter-attP site (in the “sense” orientation) on the chromosome-based platform results in the expression of marker 2 on the target gene expression vector, thereby providing a positive selection for the lambda INT mediated site-specific recombination event. Site-specific recombination events on the chromosome-based platform versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR. Examples of suitable marker 2 genes, include, but are not limited to, genes that confer resistance to toxic compounds or antibiotics, fluorescence activated cell sorting (FACS) sortable cell surface markers and various fluorescent markers. Examples of these genes include, but are not limited to, human L26aR (human homolog of Saccharomyces cerevisiae CYH8 gene), neomycin, puromycin, blasticidin, CD24 (see, e.g., U.S. Pat. Nos. 5,804,177 and 6,074,836), truncated CD4, truncated low affinity nerve growth factor receptor (LNGFR), truncated LDL receptor, truncated human growth hormone receptor, GFP, RFP, BFP.
  • The target gene expression vectors contain a gene (target gene) for expression from the chromosome platform. The target gene can be expressed using various constitutive or regulated promoter systems across various mammalian species. For the expression of multiple target genes within the same target gene expression vector, the expression of the multiple targets can be coordinately regulated via viral-based or human internal ribosome entry site (IRES) elements (see, e.g., Jackson et al. (1990) [0191] Trends Biochem Sci. 15: 477-83; Oumard et al. (2000) Mol. Cell. Biol. 20: 2755-2759). Furthermore, using IRES type elements linked to a downstream fluorescent marker, e.g., green, red or blue fluorescent proteins (GFP, RFP, BFP) allows for the identification of high expressing clones from the integrated target gene expression vector.
  • In certain embodiments described herein, the promoterless marker can be transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein. In addition, the selectable marker and the heterologous nucleic acid can be transcriptionally linked by the presence of a IRES between them. As set forth herein the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic or fluorescent. Expression from the target gene expression vector integrated onto the chromosome-based platform can be further enhanced using genomic insulator/boundary elements. The incorporation of insulator sequences into the target gene expression vector helps define boundaries in chromatin structure and thus minimizes influence of chromatin position effects/gene silencing on the expression of the target gene (Bell et al. (1999) [0192] Current Opinion in Genetics and Development 9:191-198; Emery et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:9150-9155). Examples of insulator elements that can be included onto target gene expression vector in order to optimize expression include, but are not limited to:
  • 1) chicken β-globin HS4 element (Prioleau et al. (1999) [0193] EMBO J 18: 4035-4048);
  • 2) matrix attachment regions (MAR; see, e.g., Ramakrishnan et al. (2000) [0194] Mol Cell. Biol. 20:868-877);
  • 3) scaffold attachment regions (SAR; see, e.g., Auten et al. (1999) [0195] Human Gene Therapy 10:1389-1399); and
  • 4) universal chromatin opening elements (UCOE; WO/0005393 and WO/0224930) [0196]
  • The copy number of the target gene can be controlled by sequentially adding multiple target gene expression vectors containing the target gene onto multiple integration sites on the chromosome platform. Likewise, the copy number of the target gene can be controlled within an individual target gene expression vector by the addition of DNA sequences that promote gene amplification. For example, gene amplification can be induced utilizing the dihydrofolate reductase (DHFR) minigene with subsequent selection with methotrexate (see, e.g., Schimke (1984) [0197] Cell 37:705-713) or amplification promoting sequences from the rDNA locus (see, e.g., Wegner et al. (1989) Nucl. Acids Res. 17: 9909-9932).
  • 6. Platforms with Other Recombinase System Sites [0198]
  • A “double lox” targeting strategy mediated by Cre-recombinase (Bethke et al. (1997) [0199] Nucl. Acids Res. 25:2828-2834) can be used. This strategy employs a pair of heterospecific lox sites-loxA and loxB, which differ by one nucleotide in the 8 bp spacer region. Both sites are engineered into the artificial chromosome and also onto the targeting DNA vector. This allows for a direct site-specific insertion of a commercially relevant gene or genes by a Cre-catalyzed double crossover event. In essence a platform ACes is engineered with a hygromycin-resistance gene flanked by the double lox sites generating lox-ACes, which is maintained in the thymidine kinase deficient cell, LMtk(−). The gene of interest, for example, for testing purposes, the green fluorescence protein gene, GFP and a HSV thymidine kinase gene (tk) marker, are engineered between the appropriate lox sites of the targeting vector. The vector DNA is cotransfected with plasmid pBS185 (Life Technologies) encoding the Cre recombinase gene into mammalian cells maintaining the dual-/ox artificial chromosome. Transient expression of the Cre recombinase catalyzes the site-specific insertion of the gene and the tk-gene onto the artificial chromosome. The transfected cells are grown in HAT medium that selects for only those cells that have integrated and expressed the thymidine kinase gene. The HATR colonies are screened by PCR analyses to identify artificial chromosomes with the desired insertion.
  • To generate the lox-ACes, Lambda-Hyg[0200] R-lox DNA is transfected into the LMtk(−) cell line harboring the precursor ACes. Hygromycin-resistant colonies are analyzed by FISH and Southern blotting for the presence of a single copy insert on the ACes.
  • To demonstrate the gene replacement technology, cell lines containing candidate lox-ACes are cotransfected with pTK-GFP-lox and pBS185 (encoding the Cre recombinase gene) DNA. After transfection, transient expression of plasmid pBS185 will provide sufficient burst of Cre recombinase activity to catalyze DNA recombination at the lox sites. Thus, a double crossover event between the ACes target and the exogenous targeting plasmid carrying the loxA and loxB permits the simple replacement of the hygromycin-resistance gene on the lox-ACes for the tk-GFP cassette from the targeting plasmid, with no integration of vector DNA. Transfected cells are grown in HAT-media to select for tk-expression. Correct targeting will result in the generation of HAT[0201] R, hygromycin sensitive, and green fluorescent cells. The desired integration event is verified by Southern and PCR analyses. Specific PCR primer sets are used to amplify DNA sequences flanking the individual loxA and loxB sites on the lox-ACes before and after homologous recombination.
  • D. Exemplary Applications of the Platform ACes [0202]
  • Platform ACes are applicable and tractable for different/optimized cell lines. Those that include a fluorescent marker, for example, can be purified and isolated using fluorescent activated cell sorting (FACS), and subsequently delivered to a target cell. Those with selectable markers provide for efficient selection and provide a growth advantage. Platform ACes allow multiple payload delivery of donor target vectors via a positive-selection site-specific, recombination system, and they allow for the inclusion of additional genetic factors that improve protein production and protein quality. [0203]
  • The construction and use of the platform ACes as provided for each application may be similarly applied to other applications. Particular descriptions are for exemplification. [0204]
  • 1. Cellular Protein Production Platform ACes (CPP ACes) [0205]
  • As described herein, ACes can be produced from acrocentric chromosomes in rodent (mouse, hamster) cell lines via megareplicator induced amplification of heterochromatin/rDNA sequences. Such ACes are ideal for cellular protein production as well as other applications described herein and known to those of skill in the art. ACes platforms that contain a plurality of recombination sites are particularly suitable for engineering as cellular protein production systems. [0206]
  • In one embodiment, CPP ACes involve a two-component system: the platform chromosome containing multiple engineering sites and the donor target vector containing a platform-specific recombination site with designed expression cassettes (see FIG. 9). [0207]
  • The platform ACes can be produced from any artificial chromosome, particularly the amplification-based artificial chromosomes. For exemplification, they are produced from rodent artificial chromosomes produced from acrocentric chromosomes using the technology of U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183, in which nucleic acid is targeted to the pericentric heterochromatic, and, particularly into rDNA to initiate the replication event(s). The ACes can be produced directly in the chosen cellular protein production cell lines, such as, but not limited to, CHO cells, hybridomas, plant cells, plant tissues, plant protoplasts, stem cells and plant calli. [0208]
  • a. Platform Construction [0209]
  • In the exemplary embodiment, the initial de novo platform construction requires co-transfecting with excess targeting DNA, such as, rDNA or lambda DNA without an attP region, and an engineered selectable marker. The engineered selectable marker should contain promoter, generally a constitutive promoter, such as human, viral, i.e., adenovirus or SV40 promoter, including the human ferritin heavy chain promoter (SEQ ID NO:128), SV40 and EF1α promoters, to control expression of a marker gene that provides a selective growth advantage to the cell. An example of such a marker gene is the [0210] E. coli hisD gene (encoding histidinol dehydrogenase) which is homologous and analogous to the S. typhimurium hisD a dominant marker selection system for mammalian cells previously described (see, Hartman et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8047-8051). Since histidine is an essential amino acid in mammals and a nutritional requirement in cell culture, the E. coli hisD gene can be used to select for histidine prototrophy in defined media. Furthermore more stringent selection can be placed on the cells by including histinol in the medium. Histidinol is itself permeable and toxic to cells. The hisD provides a means of detoxification.
  • Placed between the promoter and the marker gene is the bacteriophage lambda attP site to use the bacteriophage lambda integrase dependent site-specific recombination system (described herein). The insertion of an attP site downstream of a promoter element provide forward selection of site-specific recombination events onto the platform ACes. [0211]
  • b. Donor Target Vector Construction [0212]
  • A second component of the CPP platform ACes system involves the construction of donor target vectors containing a gene product(s) of interest for the CPP platform ACes. Individual donor target vectors can be designed for each gene product to be expressed thus enabling maximum usage of a de novo constructed platform ACes, so that one or a few CPP platform ACes will be required for many gene targets. [0213]
  • A key feature of the donor vector target is the promoterless marker gene containing an upstream attB site ([0214] marker 2 on FIG. 9). Normally the marker would not be expressed unless it is placed downstream of a promoter sequence. As discussed above, using the lambda integrase technology (AINTE174R on FIG. 8 and FIG. 9), site-specific recombination between the attB site on the vector and the promoter-attP site on the CPP platform ACes result in the expression of the donor target vector marker providing positive selection for the site-specific event. Site-specific recombination events on the CPP ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR. In addition, since the lambda integrase reaction is unidirectional, i.e. excision reaction is not possible, a number of unique targets can be loaded onto the CPP platform ACes limited only by the number of markers available.
  • Additional features of the donor target vector include gene target expression cassettes flanked by either chromatin insulator regions, matrix attachment regions (MAR) or scaffold attachment regions (SAR). The use of these regions will provide a more “open” chromatin environment for gene expression and help alleviate silencing. An example of such a cassette for expressing a monoclonal antibody is described. For this purpose, a strong constitutive promoter, e.g. chicken β-actin or RNA Poll, is used to drive the expression of the heavy and light chain open reading frames. The heavy and light chain sequences flank a nonattenuated human IRES (IRES[0215] H; from the 5′UTR of NRF1 gene; see Oumard et al., 2000, Mol. and Cell Biol., 20(8):2755-2759) element thereby coordinating transcription of both heavy and light chain sequence. Distal to the light chain open reading frame resides an additional viral encoded IRES (IRESV modified ECMV internal ribosomal entry site (IRES)) element attenuating the expression of the fluorescent marker gene hrGFP from Renilla (Stratagene). By linking the hrGFP with an attenuated IRES, the heavy and light chains along with the hrGFP are monocistronic. Thus, the identification of hrGFP fluorescing cells will provide a means to detect protein producing cells. In addition, high producing cell lines can be identified and isolated by FACS thereby decreasing the time frame in finding high expressers. Functional monoclonal antibody will be confirmed by ELISA.
  • C. Additional Components in Cellular Protein Production Platform ACes (CPP Aces) [0216]
  • In addition to the aforementioned CPP ACes system, other genetic factors can be included to enhance the yield and quality of the expressed protein. Again to provide maximum flexibility, these additional factors can be inserted onto the CPP platform ACes by λINTE174R dependent site-specific recombination. Other factors that could be used with a CPP Platform ACes include for example, adenovirus E1a transactivation system which upregulates both cellular and viral promoters (see, e.g., Svensson and Akusjarvi (1984) EMBO 3:789-794; and U.S. Pat. Nos. 5,866,359; 4,775,630 and 4,920,211). [0217]
  • d. Targets for CHO-ACes Engineering to Enhance Cell Growth, such as CHO Cell Growth and Protein Production/Quality [0218]
  • If adding these additional factors onto the CPP ACes is not prudent or desired, the host cell, CHO cells, can be engineered to express these factors (see, below, targets for CHO-ACes engineering to enhance CHO cell growth and protein production/quality). Additional factors to consider including are addition of insulin or IGF-1 to sustain viabililty; human sialyltransferases or related factors to produce more human-like glycoproteins; expression of factors to decrease ammonium accumulation during cell growth; expression of factors to inhibit apoptosis; expression of factors to improve protein secretion and protein folding; and expression of factors to permit serum-free transfection and selection. [0219]
  • 1) Addition of Insulin or IGF-1 to Sustain Viabililty [0220]
  • Stimulatory factors and/or their receptors are expressed to set up an autocrine loop, to improve cell growth, such as CHO cell growth. Two exemplary candidates are insulin and IGF-1 (see, Biotechnol Prog 2000 Sep;16(5):693-7). Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures. Insulin and IGF-1 analog (LongR(3) serve as growth and viability factors for CHO cells. [0221]
  • CHO cells were modified to produce higher levels of essential nutrients and factors. A serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44 cells) was prepared. Chinese hamster ovary cells (DG44 cells), which are normally maintained in 10% serum medium, were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium (see, Kim et al. (199) In [0222] Vitro Cell Dev Biol Anim 35(4):178-82). A SF medium (SF-DG44) was formulated by supplementing the basal medium with these components; basal medium was prepared by supplementing Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Development of a SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.
  • 2) Human Sialyltransferases or Related Factors to Produce More Human-Like Glycoproteins [0223]
  • CHO cells have been modified by increasing their ability to process protein via addition of complex carbohydrates. This has been achieved by overexpression of relevant processing enzymes, or in some cases, reducing expression of relevant enzymes (see, Bragonzi et al. (2000) [0224] Biochim Biophys Acta 1474(3):273-282; see, also Weikert et al. (1999) Nature biotech. 17:1116-11121; Ferrari J et al. (1998) Biotechnol Bioeng 60(5):589-95). A CHO cell line expressing alpha2,6-sialyltransferase was developed for the production of human-like sialylated recombinant glycoproteins. The sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA into the cells. Glycoproteins produced by such CHO cells display alpha2,6-and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins.
  • As another example for improving the production of human-like sialylated recombinant glycoproteins, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA (see, Ferrari J et al. (1998) [0225] Biotechnol Bioeng 60(5):589-95). Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5′ 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values.
  • Oligosaccharide biosynthesis pathways in mammalian cells have been engineered for generation of recombinant glycoproteins (see, e.g., Sburlati (1998) [0226] Biotechnol Prog 14(2):189-92), which describes a Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins. This cell line was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III) (see, also, Prati et al. (1998) Biotechnol Bioeng 59(4):445-50, which describes antisense strategies for glycosylation engineering of CHO cells).
  • 3) Expression of Factors to Decrease Ammonium Accumulation During Cell Growth [0227]
  • Excess ammonium, which is a by-product of CHO cell metabolism can have detrimental effects on cell growth and protein quality (see, Yang et al. (2000) [0228] Biotechnol Bioeng 68(4):370-80). To solve this problem ammonium levels were modified by overexpressing carbamoyl phosphate synthetase I and ornithine transcarbamoylase or glutamine synthetase in CHO cells. Such modification resulted in reduced ammonium levels observed and an increase in the growth rate (see Kim et al. (2000) J Biotechnol 81(2-3):129-40; and Enosawa et al. (1997) Cell Transplant 6(5):537-40).
  • 4) Expression of Factors to Improve Protein Secretion and Protein Folding [0229]
  • Overexpression of relevant enzymes can be engineered into the ACes to improve protein secretion and folding. [0230]
  • 5) Expression of Factors to Permit Serum-Free Transfection and Selection [0231]
  • It is advantageous to have the ability to convert CHO cells in suspension growing in serum free medium to adherence with out having to resort to serum addition. Laminin or fibronectin addition is sufficient to make cells adherent (see, e.g., Zaworski et al. (1993) [0232] Biotechniques 15(5):863-6) so that expressing either of these genes in CHO cells under an inducible promoter should allow for reversible shift to adherence without requiring serum addition.
  • 2. Platform ACes and Gene Therapy [0233]
  • The platform ACes provided herein are contemplated for use in mammalian gene therapy, particularly human gene therapy. Human ACes can be derived from human acrocentric chromosomes from human host cells, in which the amplified sequences are heterochromatic and/or human rDNA. Different platform ACes applicable for different tissue cell types are provided. The ACes for gene therapy can contain a single copy of a therapeutic gene inserted into a defined location on platform ACes. Therapeutic genes include genomic clones, cDNA, hybrid genes and other combinations of sequences. Preferred selectable markers are those from the mammalian host, such as human derived factors so that they are non-immunogenic, non-toxic and allow for efficient selection, such as by FACS and/or drug resistance. [0234]
  • Platform ACes, useful for gene therapy and other applications, as noted herein, can be generated by megareplicator dependent amplification, such as by the methods in U.S. Pat. Nos. 6,077,697 and 6,025,155 and published International PCT application No. WO 97/40183. In one embodiment, human ACes are produced using human rDNA constructs that target rDNA arrays on human acrocentric chromosomes and induce the megareplicator in human cells, particularly in primary cell lines (with sufficient number of doublings to form the ACes) or stem cells (such as hematopoietic stem cells, mesenchymal stem cells, adult stem cells or embryonic stem cells) to avoid the introduction of potentially harmful rearranged DNA sequences present in many transformed cell lines. Megareplicator induced ACes formation can result in multiple copies of targeting DNA/selectable markers in each amplification block on both chromosomal arms of the platform ACes. [0235]
  • In view of the considerations regarding immunogenicity and toxicity, the production of human platform ACes for gene therapy applications employs a two component system analogous to the platform ACes designed for cellular protein production (CPP platform ACes). The system includes a platform chromosome of entirely human DNA origin containing multiple engineering sites and a gene target vector carrying the therapeutic gene of interest. [0236]
  • a. Platform Construction [0237]
  • The initial de novo construction of the platform chromosome employs the co-transfection of excess targeting DNA and a selectable marker. In one embodiment, the DNA is targeted to the rDNA arrays on the human acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22). For example, two large human rDNA containing PAC clones 18714 and 18720 and the human PAC clone 558F8 are used for targeting (Genome Research (ML) now Incyte, BACPAC Resources, 747 52nd Street, Oakland Calif.). The mouse rDNA clone pFK161 (SEQ ID NO: 118), which was used to make the human SATAC from the 94-3 hamster/human hybrid cell line (see, e.g., published International PCT application No. WO 97/40183 and Csonka, et al, [0238] Journal of Cell Science 113:3207-32161 and Example 1 for a description of pFK161) can also be used.
  • For animal applications, selectable markers should be non-immunogenic in the animal, such as a human, and include, but are not limited to: human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAb), mutant human dihyrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+ cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2a; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectable by Ara-C). [0239]
  • Since megareplicator induced amplification generates multiple copies of the selectable marker, a second consideration for the selection of the human marker is the resulting dose of the expressed marker after ACes formation. High level of expression of certain markers may be detrimental to the cell and/or result in autoimmunity. One method to decrease the dose of the marker protein is by shortening its half-life, such as via the fusion of the well-conserved human ubiquitin tag (a 76 amino acid sequence) thus leading to increased turnover of the selectable marker. This has been used successfully for a number of reporter systems including DHFR (see, e.g., Stack et al. (2000) [0240] Nature Biotechnology 18:1298-1302 and references cited therein).
  • Using the ubiquitin tagged protein, a human selectable marker system analogous to the CPP ACes described herein is constructed. Briefly, a tagged selectable marker, such as for example one of those described herein, is cloned downstream of an attP site and expressed from a human promoter. Exemplary promoters contemplated for use herein include, but are not limited to, the human ferritin heavy chain promoter (SEQ ID NO:128); RNA Poll; EF1α; TR; glyceraldehyde-3-phosphate dehydrogenase core promoter (GAP); a GAP core promoter including a proximal insulin inducible element the intervening GAP sequence; phosphofructokinase promoter; and phosphoglycerate kinase promoter. Also contemplated herein is an aldolase A promoter H1 & H2 (representing closely spaced transcriptional start sites) along with the proximal H enhancer. There are 4 promoters (e.g., transcriptional start sites) for this gene, each having different regulatory and tissue activity. The H (most proximal 2) promoters are ubiquitously expressed off the H enhancer. This resulting marker can then be co-transfected along with excess human rDNA targeting sequence into the host cells. An important criteria for the selection of the recipient cells is sufficient number of cell doublings for the formation and detection of ACes. Accordingly, the co-transfections should be attempted in human primary cells that can be cultured for long periods of time, such as for example, stem cells (e.g., hematopoietic, mesenchymal, adult or embryonic stem cells), or the like. Additional cell types, include, but are not limited to: single gene transfected cells exhibiting increased life-span; over-expressing c-myc cells, e.g. MSU1.1 (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136); over-expressing telomerase lines, such as TERT cells; SV40 large T-antigen transfected lines; tumor cell lines, such as HT1080; and hybrid human cell lines, such as the 94-3 hamster/human hybrid cell line. [0241]
  • b. Gene Target Vector [0242]
  • The second component of the GT platform ACes (GT ACes) system involves the use of engineered target vectors carrying the therapeutic gene of interest. These are introduced onto the GT platform ACes via site-specific recombination. As with the CPP ACes, the use of engineered target vectors maximizes the use of the de novo generated GT platform ACes for most gene targets. Furthermore, using lambda integrase technology, GT platform ACes containing multiple attP sites permits the opportunity to incorporate multiple therapeutic targets onto a single platform. This could be of value in cases where a defined therapy requires multiple gene targets, a single therapeutic target requires an additional gene regulatory factor or a GT ACes requires a “kill” switch. [0243]
  • Similar to the CPP ACes, a feature of the gene target vector is the promoterless marker gene containing an upstream attB site ([0244] marker 2 on FIG. 9). Normally, the marker (in this case, a cell surface antigen that can be sorted by FACS would be ideal) would not be expressed unless it is placed downstream of a promoter sequence. Using the lambda integrase technology (λINTE174R on FIG. 9), site-specific recombination between the attB site on the vector and the promoter-attP site on the GT platform ACes results in the expression of marker#2 on the gene target vector, i.e. positive selection for the site-specific event. Site-specific recombination events on the GT ACes versus random integrations next to a promoter in the genome (false positive) can be quickly screened by designing primers to detect the correct event by PCR.
  • For expression of the therapeutic gene, human specific promoters, such as a ferritin heavy chain promoter (SEQ ID NO:128); EF1α or RNA Poll, are used. These promoters are for high level expression of a cDNA encoded therapeutic protein. In addition to expressing cDNA (or even hybrid cDNA/artificial intron constructs), the GT platform ACes are used for engineering and expressing large genomic fragments carrying therapeutic genes of interest expressed from native promoter sequences. This is of importance in situations where the therapy requires precise cell specific expression or in instances where expression is best achieved from genomic clones versus cDNA. [0245]
  • 3. Selectable Markers for Use, for Example, in Gene Therapy (GT) [0246]
  • The following are selectable markers that can be incorporated into human ACes and used for selection. [0247]
  • Dual Resistance to 4-Hydroperoxycyclophosphamide and Methotrexate by Retroviral Transfer of the Human [0248] Aldehyde Dehydrogenase Class 1 Gene and a Mutated Dihydrofolate Reductase Gene
  • The genetic transfer of drug resistance to hematopoietic cells is one approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors, a retroviral vector containing a human cytosolic ALDH-1-encoding DNA clone and a human doubly mutated DHFR-encoding clone (Phe22/Ser3l; termed F/S in the description of constructs) to generate increased resistance to CTX and MTX were constructed (Takebe et al. (2001) [0249] Mol Ther 3(1):88-96). This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression. ACes can be similarly constructed.
  • Multiple Mechanisms of N-phosphonacetyl-L-aspartate Resistance in Human Cell Lines: Carbamyl-P Synthetase/Aspartate Transcarbamylase/Dihydro-Orotase Gene Amplification is Frequent only when [0250] Chromosome 2 is Rearranged
  • Rodent cells resistant to N-phosphonacetyl-L-aspartate (PALA) invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells (Smith et al. (1997) [0251] Proc. Natl. Acad. Sci. U.S.A. 94:1816-21). In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD. These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts. Thus, ACes with multiple copies of the CAD gene would provide PALA resistance.
  • Retroviral Coexpression of Thymidylate Synthase and Dihydrofolate Reductase Confers Fluoropyrimidine and Antifolate Resistance [0252]
  • Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. Fantz et al. ((1998) [0253] Biochem Biophys Res Comm 243(1):6-12) have shown that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents. Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold). Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30to 140-fold). A L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA that encodes TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents. ACes can be similarly constructed.
  • Human CD34+Cells do not Express Glutathione S-transferases Alpha [0254]
  • The expression of glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+cells and bone marrow was studied using RT-PCR and immunoblotting (Czerwinski M, Kiem et al. (1997) [0255] Gene Ther 4(3):268-70). The GSTA1 protein conjugates glutathione to the stem cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body. Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow. Similarly, baboon CD34+cells and dog bone marrow do not express GSTA1. Thus, human GSTA1 is a chemoprotective selectable marker in human stem cell gene therapy and could be employed in ACes construction.
  • Selection of Retrovirally Transduced Hematopoietic Cells Using CD24 as a Marker of Gene Transfer [0256]
  • Pawliuk et al. ((1994) [0257] Blood 84(9):2868-2877) have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony-forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. These results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker permits rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and provides a tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells. ACes could be similarly constructed.
  • DeltahGHR, a Biosafe Cell Surface-Labeling Molecule for Analysis and Selection of Genetically Transduced Human Cells [0258]
  • A selectable marker for retroviral transduction and selection of human and murine cells is known (see, Garcia-Ortiz et al. (2000) [0259] Hum Gene Ther 11(2):333-46). The molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering. The engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding. This new marker, has a wide application spectrum, since hGHR in the human adult is highly expressed only in liver cells, and lower levels have been reported in certain lymphocyte cell populations. The deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans. The differential binding properties of several monoclonal antibodies (MAbs) are used in a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population is recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium. Such a system could be used to identify ACes containing cells.
  • 4. Transgenic Models for Evaluation of Genes and Discovery of New Traits in Plants [0260]
  • Of interest is the use of plants and plant cells containing artificial chromosomes for the evaluation of new genetic combinations and discovery of new traits. Artificial chromosomes, by virtue of the fact that they can contain significant amounts of DNA can also therefore encode numerous genes and accordingly a multiplicity of traits. It is contemplated here that artificial chromosomes, when formed from one plant species, can be evaluated in a second plant species. The resultant phenotypic changes observed, for example, can indicate the nature of the genes contained within the DNA contained within the artificial chromosome, and hence permit the identification of novel genetic activities. Artificial chromosomes containing euchromatic DNA or partially containing euchromatic DNA can serve as a valuable source of new traits when transferred to an alien plant cell environment. For example, it is contemplated that artificial chromosomes derived from dicot plant species can be introduced into monocot plant species by transferring a dicot artificial chromosome. The dicot artificial chromosome possessing a region of euchromatic DNA containing expressed genes. [0261]
  • The artificial chromosomes can be designed to allow the artificial chromosome to recombine with the naturally occurring plant DNA in such a fashion that a large region of naturally occurring plant DNA becomes incorporated into the artificial chromosome. This allows the artificial chromosome to contain new genetic activities and hence carry novel traits. For example, an artificial chromosome can be introduced into a wild relative of a crop plant under conditions whereby a portion of the DNA present in the chromosomes of the wild relative is transferred to the artificial chromosome. After isolation of the artificial chromosome, this naturally occurring region of DNA from the wild relative, now located on the artificial chromosome can be introduced into the domesticated crop species and the genes encoded within the transferred DNA expressed arid evaluated for utility. New traits and gene systems can be discovered in this fashion. The artificial chromosome can be modified to contain sequences that promote homologous recombination within plant cells, or be modified to contain a genetic system that functions as a site-specific recombination system. [0262]
  • Artificial chromosomes modified to recombine with plant DNA offer many advantages for the discovery and evaluation of traits in different plant species. When the artificial chromosome containing DNA from one plant species is introduced into a new plant species, new traits and genes can be introduced. This use of an artificial chromosome allows for the ability to overcome the sexual barrier that prevents transfer of genes from one plant species to another species. Using artificial chromosomes in this fashion allows for many potentially valuable traits to be identified including traits that are typically found in wild species. Other valuable applications for artificial chromosomes include the ability to transfer large regions of DNA from one plant species to another, such as DNA encoding potentially valuable traits such as altered oil, carbohydrate or protein composition, multiple genes encoding enzymes capable of producing valuable plant secondary metabolites, genetic systems encoding valuable agronomic traits such as disease and insect resistance, genes encoding functions that allow association with soil bacterium such as growth promoting bacteria or nitrogen fixing bacteria, or genes encoding traits that confer freezing, drought or other stress tolerances. In this fashion, artificial chromosomes can be used to discover regions of plant DNA that encode valuable traits. [0263]
  • The artificial chromosome can also be designed to allow the transfer and subsequent incorporation of these valuable traits now located on the artificial chromosome into the natural chromosomes of a plant species. In this fashion the artificial chromosomes can be used to transfer large regions of DNA encoding traits normally found in one plant species into another plant species. In this fashion, it is possible to derive a plant cell that no longer needs to carry an artificial chromosome to posses the novel trait. Thus, the artificial chromosome would serve as the transfer mechanism to permit the formation of plants with greater degree of genetic diversity. [0264]
  • The design of an artificial chromosome to accomplish the aforementioned purposes can include within the artificial chromosome the presence of specific DNA sequences capable of acting as sites for homologous recombination to take place. For example, the DNA sequence of Arabidopsis is now known. To construct an artificial chromosome capable of recombining with a specific region of Arabidopsis DNA, a sequence of Arabidopsis DNA, normally located near a chromosomal location encoding genes of potential interest can be introduced into an artificial chromosome by methods provided herein. It may be desirable to include a second region of DNA within the artificial chromosome that provides a second flanking sequence to the region encoding genes of potential interest, to promote a double recombination event which would ensure transfer of the entire chromosomal region, encoding genes of potential interest, to the artificial chromosome. The modified artificial chromosome, containing the DNA sequences capable of homologous recombination region, can then be introduced into Arabidopsis cells and the homologous recombination event selected. [0265]
  • It is convenient to include a marker gene to allow for the selection of a homologous recombination event. The marker gene is preferably inactive unless activated by an appropriate homologous recombination event. For example, U.S. Pat. No. 5,272,071, describes a method where an inactive plant gene is activated by a recombination event such that desired homologous recombination events can be easily scored. Similarly, U.S. Pat. No. 5,501,967 describes a method for the selection of homologous recombination events by activation of a silent selection gene first introduced into the plant DNA, the gene being activated by an appropriate homologous recombination event. Both of these methods can be applied to enable a selective process to be included to select for recombination between an artificial chromosome and a plant chromosome. Once the homologous recombination event is detected, the artificial chromosome, once selected, is isolated and introduced into a recipient cell, for example, tobacco, corn, wheat or rice, and the expression of the newly introduced DNA sequences evaluated. [0266]
  • Phenotypic changes in the recipient plant cells containing the artificial chromosome, or in regenerated plants containing the artificial chromosome, allows for the evaluation of the nature of the traits encoded by the Arabidopsis DNA, under conditions naturally found in plant cells, including the naturally occurring arrangement of DNA sequences responsible for the developmental control of the traits in the normal chromosomal environment. [0267]
  • Traits such as durable fungal or bacterial disease resistance, new oil and carbohydrate compositions, valuable secondary metabolites such as phytosterols, flavonoids, efficient nitrogen fixation or mineral utilization, resistance to extremes of drought, heat or cold are all found within different populations of plant species and are often governed by multiple genes. The use of single gene transformation technologies does not permit the evaluation of the multiplicity of genes controlling many valuable traits. Thus, incorporation of these genes into artificial chromosomes allows the rapid evaluation of the utility of these genetic combinations in heterologous plant species. [0268]
  • The large scale order and structure of the artificial chromosome provides a number of unique advantages in screening for new utilities or novel phenotypes within heterologous plant species. The size of new DNA that can be carried by an artificial chromosome can be millions of base pairs of DNA, representing potentially numerous genes that may have novel utility in a heterologous plant cell. The artificial chromosome is a “natural” environment for gene expression, the problems of variable gene expression and silencing seen for genes transferred by random insertion into a genome should not be observed. Similarly, there is no need to engineer the genes for expression, and the genes inserted would not need to be recombinant genes. Thus, one expects the expression from the transferred genes to be temporal and spatial, as observed in the species from where the genes were initially isolated. A valuable feature for these utilities is the ability to isolate the artificial chromosomes and to further isolate, manipulate and introduce into other cells artificial chromosomes carrying unique genetic compositions. [0269]
  • Thus, the use of artificial chromosomes and homologous recombination in plant cells can be used to isolate and identify many valuable crop traits. [0270]
  • In addition to the use of artificial chromosomes for the isolation and testing of large regions of naturally occurring DNA, methods for the use of artificial chromosomes and cloned DNA are also contemplated. Similar to that described above, artificial chromosomes can be used to carry large regions of cloned DNA, including that derived from other plant species. [0271]
  • The ability to incorporate novel DNA elements into an artificial chromosome as it is being formed allows for the development of artificial chromosomes specifically engineered as a platform for testing of new genetic combinations, or “genomic” discoveries for model species such as Arabidopsis. It is known that specific “recombinase” systems can be used in plant cells to excise or re-arrange genes. These-same systems can be used to derive new gene combinations contained on an artificial chromosome. [0272]
  • The artificial chromosomes can be engineered as platforms to accept large regions of cloned DNA, such as that contained in Bacterial Artificial Chromosomes (BACs) or Yeast Artificial Chromosomes (YACs). It is further contemplated, that as a result of the typical structure of artificial chromosomes containing tandemly repeated DNA blocks, that sequences other than cloned DNA sequence can be introduced by recombination processes. In particular recombination within a predefined region of the tandemly repeated DNA within the artificial chromosome provides a mechanism to “stack” numerous regions of cloned DNA, including large regions of DNA contained within BACs or YACs clones. Thus, multiple combinations of genes can be introduced onto artificial chromosomes and these combinations tested for functionality. In particular, it is contemplated that multiple YACs or BACs can be stacked onto an artificial chromosomes, the BACs or YACs containing multiple genes of complex pathways or multiple genetic pathways. The BACs or YACs are typically selected based on genetic information available within the public domain, for example from the Arabidopsis Information Management System (http://aims.cps.msu.edu/aims/index.html) or the information related to the plant DNA sequences available from the Institute for Genomic Research (http://www.tigr.org) and other sites known to those skilled in the art. Alternatively, clones can be chosen at random and evaluated for functionality. It is contemplated that combinations providing a desired phenotype can be identified by isolation of the artificial chromosome containing the combination and analyzing the nature of the inserted cloned DNA. [0273]
  • In this regard, it is contemplated that the use of site-specific recombination sequences can have considerable utility in developing artificial chromosomes containing DNA sequences recognized by recombinase enzymes and capable of accepting DNA sequences containing same. The use of site-specific recombination as a means to target an introduced DNA to a specific locus has been demonstrated in the art and such methods can be employed. The recombinase systems can also be used to transfer the cloned DNA regions contained within the artificial chromosome to the naturally occurring plant or mammalian chromosomes. [0274]
  • As noted herein, many site-specific recombinases are known and can be identified (Kilby et al. (1993) [0275] Trends in Genetics 9:413-418). The three recombinase systems that have been extensively employed include: an activity identified as R encoded by the pSR1 plasmid of Zygosaccharomyes rouxii, FLP encoded for the 2um circular plasmid from Saccharomyces cerevisiae and Cre-lox from the phage P1.
  • The integration function of site-specific recombinases is contemplated as a means to assist in the derivation of genetic combinations on artificial chromosomes. In order to accomplish this, it is contemplated that a first step of introducing site-specific recombinase sites into the genome of a plant cell in an essentially random manner is conducted, such that the plant cell has one or more site-specific recombinase recognition sequences on one or more of the plant chromosomes. An artificial chromosome is then introduced into the plant cell, the artificial chromosome engineered to contain a recombinase recognition site (e.g., integration site) capable of being recognized by a site-specific recombinase. Optionally, a gene encoding a recombinase enzyme is also included, preferably under the control of an inducible promoter. Expression of the site-specific recombinase enzyme in the plant cell, either by induction of a inducible recombinase gene, or transient expression of a recombinase sequence, causes a site-specific recombination event to take place, leading to the insertion of a region of the plant chromosomal DNA (containing the recombinase recognition site) into the recombinase recognition site of the artificial chromosome, and forming an artificial chromosome containing plant chromosomal DNA. The artificial chromosome can be isolated and introduced into a heterologous host, preferably a plant host, and expression of the newly introduced plant chromosomal DNA can be monitored and evaluated for desirable phenotypic changes. Accordingly, carrying out this recombination with a population of plant cells wherein the chromosomally located recombinase recognition site is randomly scattered throughout the chromosomes of the plant, can lead to the formation of a population of artificial chromosomes, each with a different region of plant chromosomal DNA, and each potentially representing a novel genetic combination. [0276]
  • This method requires the precise site-specific insertion of chromosomal DNA into the artificial chromosome. This precision has been demonstrated in the art. For example, Fukushige and Sauer ((1992) Proc. Natl. Acad. Sci. USA, 89:7905-7909) demonstrated that the Cre-lox homologous recombination system could be successfully employed to introduce DNA into a predefined locus in a chromosome of mammalian cells. In this demonstration a promoter-less antibiotic resistance gene modified to include a lox sequence at the 5′ end of the coding region was introduced into CHO cells. Cells were re-transformed by electroporation with a plasmid that contained a promoter with a lox sequence and a transiently expressed Cre recombinase gene. Under the conditions employed, the expression of the Cre enzyme catalyzed the homologous recombination between the lox site in the chromosomally located promoter-less antibiotic resistance gene, and the lox site in the introduced promoter sequence, leading to the formation of a functional antibiotic resistance gene. The authors demonstrated efficient and correct targeting of the introduced sequence, 54 of 56 lines analyzed corresponded to the predicted single copy insertion of the DNA due to Cre catalyzed site-specific homologous recombination between the lox sequences. [0277]
  • Accordingly a lox sequence may be first added to a genome of a plant species capable of being transformed and regenerated to a whole plant to serve as a recombinase target DNA sequence for recombination with an artificial chromosome. The lox sequence may be optimally modified to further contain a selectable marker which is inactive but can be activated by insertion of the lox recombinase recognition sequence into the artificial chromosome. [0278]
  • A promoterless marker gene or selectable marker gene linked to the recombinase recognition sequence, which is first inserted into the chromosomes of a plant cell can be used to engineer a platform chromosome. A promoter is linked to a recombinase recognition site, in an orientation that allows the promoter to control the expression of the marker or selectable marker gene upon recombination within the artificial chromosome. Upon a site-specific recombination event between a recombinase recognition site in a plant chromosome and the recombinase recognition site within the introduced artificial chromosome, a cell is derived with a recombined artificial chromosome, the artificial chromosome containing an active marker or selectable marker activity that permits the identification and or selection of the cell. [0279]
  • The artificial chromosomes can be transferred to other plant or animal species and the functionality of the new combinations tested. The ability to conduct such an inter-chromosomal transfer of sequences has been demonstrated in the art. For example, the use of the Cre-lox recombinase system to cause a chromosome recombination event between two chromatids of different chromosomes has been shown. [0280]
  • Any number of recombination systems may be employed as described herein, such as, but not limited to, bacterially derived systems such as the att/int system of phage lambda, and the Gin/gix system. [0281]
  • More than one recombination system may be employed, including, for example, one recombinase system for the introduction of DNA into an artificial chromosome, and a second recombinase system for the subsequent transfer of the newly introduced DNA contained within an artificial chromosome into the naturally occurring chromosome of a second plant species. The choice of the specific recombination system used will be dependent on the nature of the modification contemplated. [0282]
  • By having the ability to isolate an artificial chromosome, in particular, artificial chromosomes containing plant chromosomal DNA introduced via site-specific recombination, and re-introduce the chromosome into other mammalian or plant cells, particularly plant cells, these new combinations can be evaluated in different crop species without the need to first isolate and modify the genes, or carry out multiple transformations or gene transfers to achieve the same combination isolation and testing combinations of the genes in plants. The use of a site-specific recombinase also allows the convenient recovery of the plant chromosomal region into other recombinant DNA vectors and systems, such as mammalian or insect systems, for manipulation and study. [0283]
  • Also contemplated herein are ACes, cell lines and methods for use in screening a new chromosomal combinations, deletions, truncations with eucaryotic genome that take advantage of the site-specific recombination systems incorporated onto platform ACes provided herein. For example, provided herein is a cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes. Also provided herein is a method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into a cell line comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell's chromosomal DNA; and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within. Also provided herein is a library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein. The library of cells can be from a different species and/or cell type than the chromosomal nucleic acid within the ACes. Also provided is a method of making one or more cell lines, comprising [0284]
  • a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites, [0285]
  • b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell's endogenous chromosomal DNA; [0286]
  • c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein; [0287]
  • d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells; [0288]
  • e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines. [0289]
  • These ACes, cell lines and methods utilize the site-specific recombination sites on platform ACes analogous YAC manipulation related to: the methods of generating terminal deletions in normal and artificial chromosomes (e.g., ACes; as described in Vollrath et al., 1988[0290] , PNAS, USA, 85:6027-66031; and Pavan et al., PNAS, USA, 87:1300-1304); the methods of generating interstitial deletions in normal and artificial chromosomes (as described in Campbell et al., 1991, PNAS, USA, 888:5744-5748); and the methods of detecting homologous recombination between two ACes (as described in Cellini et al., 1991, Nuc. Acid Res., 19(5):997-1000).
  • 5. Use of Plateform ACes in Pharmacogenomic/Toxicology Applications (Development of “Reporter ACes”) [0291]
  • In addition to the placement of genes onto ACes chromosomes for therapeutic protein production or gene therapy, the platform can be engineered via the IntR lambda integrase to carry reporter-linked constructs (reporter genes) that monitor changes in cellular physiology as measured by the particular reporter gene (or a series of different reporter genes) readout. The reporter linked constructs are designed to include a gene that can be detected (by for example fluorescence, drug resistance, immunohistochemistry, or transcript production, and the like) with well-known regulatory sequences that would control the expression of the detectable gene. Exemplary regulatory promoter sequences are well-known in the art: [0292]
  • A) Reporter ACes for Drug Pathway Screening [0293]
  • The ACes can be engineered to carry reporter-linked constructs that indicate a signal is being transduced through one or a number of pathways. For example, transcriptionally regulated promoters from genes at the end (or any other chosen point) of particular signal transduction pathways could be engineered on the ACes to express the appropriate readout (either by fluorescent protein production or drug resistance) when the pathway is activated (or down-regulated as well). In one embodiment, a number of reporters from different can be placed on a ACes chromosome. Cells (and/or whole animals) containing such a Reporter ACes could be exposed to a variety of drugs or compounds and monitored for the effects of the drugs or compounds upon the selected pathway(s) by the reporter gene(s). Thus, drugs or compounds can be classified or identified by particular pathways they excite or down-regulate. Similarly, transcriptional profiles obtained from genomic array experiments can be biologically validated using the reporter ACes provided herein. [0294]
  • B) Reporter ACes for Toxic Compound Testing [0295]
  • Environmental or man-made genotoxicants can be tested in cell lines carrying a number of reporter-genes platform ACes linked to promoters that are transcriptionally regulated in response to DNA damage, induced apoptosis or necrosis, and cell-cycle perturbations. Furthermore, new drugs and/or compounds could be tested in a similar manner with the genotoxicant ACes reporter for their cellular/genetic toxicity by such a screen. Likewise, toxic compound testing could be carried out in whole transgenic animals carrying the ACes chromosome that measures genotoxicant exposure (“canary in a coal mine”). Thus, the same or similar type ACes could be used for toxicity testing in either a cell-based or whole animal setting. An example would include ACes that carry reporter-linked genes controlled by various cytochrome P450 profiled promoters and the like. [0296]
  • C) Reporter ACes for Individualized Pharmacogenomics/Drug Profiling [0297]
  • A common disease may arise via various mechanisms. In many instances there are multiple treatments available for a given disease. However, the success of a given treatment may depend upon the mechanism by which the disease originated and/or by the genetic background of the patient. In order to establish the most effective treatment for a given patient one could utilize the ACes reporters provided herein. ACes reporters can be used in patient cell samples to determine an individualized drug regimen for the patient. In addition, potential polymorphisms affecting the transcriptional regulation of an individual's particular gene can be assessed by this approach. [0298]
  • D) Reporter ACes for Classification of Similar Patient Tumors [0299]
  • As with other diseases as described in 5.C) above, cancer cells arise via different mechanisms. Furthermore, as a cancerous cell propagates it may undergo genomic alterations. An ACes reporter transferred to cells of different patients having the same disease, i.e. similar cancers, could be used to categorize the particular cancer of each patient, thereby facilitating the identification of the most effective therapeutic regimen. Examples would include the validation of array profiling of certain classes of breast cancers. Subsequently, appropriate drug profiling could be carried out as described above. [0300]
  • E) Reporter ACes as a “Differentiation” Sensor [0301]
  • Using the ACes reporter as a “differentiation” sensor in stem cells or other progenitor cells in order to enrich by selection (either FACS based screening, drug selection and/or use of suicide gene) for a particular class of differentiated or undifferentiated cells. For example, in one embodiment, this assay could also be used for compound screening for small molecule modifiers of cell differentiation. [0302]
  • F) Whole Animal Studies with Reporter ACes [0303]
  • Finally, with whole-body fluorescence imaging technology (Yang et al. (2000) PNAS 97:12278) any of the above Reporter ACes methods could be used in conjunction with whole-body imaging to monitor reporter genes within whole animals without sacrificing the animal. This would allow temporal and spatial analysis of expression patterns under a given set of conditions. The conditions tested may include for example, normal differentiation of a stem cell, response to drug or compound treatment whether targeted to the diseased tissue or presented systemically, response to genotoxicants, and the like. [0304]
  • The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [0305]
    • EXAMPLE 1
  • pFK161 [0306]
  • Cosmid pFK161 (SEQ ID NO: 118) was obtained from Dr. Gyula Hadlaczky and contains a 9 kb NotI insert derived from a murine rDNA repeat (see clone 161 described in PCT Application Publication No. WO97/40183 by Hadlaczky et al. for a description of this cosmid). This cosmid, referred to as clone 161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQ ID NO. 26. It was produced by inserting fragments of the megachromosome (see, U.S. Pat. No. 6,077,697 and International PCT application No. WO 97/40183). For example, H1D3, which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome into plasmid pWE15 (Stratagene, La Jolla, Calif.; SEQ ID No. 31) as follows. Half of a 100 μl low melting point agarose block (mega-plug) containing isolated SATACs was digested with NotI overnight at 37° C. Plasmid pWE15 was similarly digested with NotI overnight. The mega-plug was then melted and mixed with the digested plasmid, ligation buffer and T4 DNA ligase. Ligation was conducted at 16° C. overnight. Bacterial DH5α cells were transformed with the ligation product and transformed cells were plated onto LB/Amp plates. Fifteen to twenty colonies were grown on each plate for a total of 189 colonies. Plasmid DNA was isolated from colonies that survived growth on LB/Amp medium and analyzed by Southern blot hybridization for the presence of DNA that hybridized to a pUC19 probe. This screening methodology assured that all clones, even clones lacking an insert but yet containing the pWE15 plasmid, would be detected. [0307]
  • Liquid cultures of all 189 transformants were used to generate cosmid minipreps for analysis of restriction sites within the insert DNA. Six of the original 189 cosmid clones contained an insert. These clones were designated as follows: 28 (˜9-kb insert), 30 (˜9-kb insert), 60 (˜4-kb insert), 113 (˜9-kb insert), 157 (˜9-kb insert) and 161 (˜9-kb insert). Restriction enzyme analysis indicated that three of the clones (113, 157 and 161) contained the same insert. For sequence analysis the insert of cosmid clone no. 161 was subcloned as follows. To obtain the end fragments of the insert of clone no. 161, the clone was digested with NotI and BamHI and ligated with NotI/BamHI-digested pBluescript KS (Stratagene, La Jolla, Calif.). Two fragments of the insert of clone no. 161 were obtained: a 0.2-kb and a 0.7-kb insert fragment. To subclone the internal fragment of the insert of clone no. 161, the same digest was ligated with BamHI-digested pUC19. Three fragments of the insert of clone no. 161 were obtained: a 0.6-kb, a 1.8-kb and a 4.8-kb insert fragment. [0308]
  • The insert corresponds to an internal section of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 as set forth in GENBANK accession no. X82564, which is provided as SEQ ID NO. 18. The sequence data obtained for the insert of clone no. 161 is set forth in SEQ ID NOS. 19-25. Specifically, the individual subclones corresponded to the following positions in GENBANK accession no. X82564 (SEQ ID NO:18) and in SEQ ID NOs. 19-25: [0309]
    Start End
    Subclone in X82564 Site SEQ ID No.
    161k1 7579 7755 NotI, BamHI 19
    161m5 7756 8494 BamHI 20
    161m7 8495 10231 BamHI 21 (shows only sequence
    corresponding to nt.
    8495-8950),
    22 (shows only sequence
    corresponding to nt.
    9851-10231)
    161m12 10232 15000 BamHI 23 (shows only sequence
    corresponding to nt.
    10232-10600),
    24 (shows only sequence
    corresponding to nt.
    14267-15000)
    161k2 15001 15676 NotI, BamHI 25
  • The sequence set forth in SEQ ID NOs. 19-25 diverges in some positions from the sequence presented in positions 7551-15670 of GENBANK accession no. X82564. Such divergence may be attributable to random mutations between repeat units of rDNA. [0310]
  • For use herein, the rDNA insert from the clone was prepared by digesting the cosmid with NotI and BglII and was purified as described above. Growth and maintenance of bacterial stocks and purification of plasmids were performed using standard well known methods (see, e.g., Sambrook et al. (1989) [0311] Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press), and plasmids were purified from bacterial cultures using Midi- and Maxi-preps Kits (Qiagen, Mississauga, Ontario).
  • pDsRed1N1 [0312]
  • This vector is available from Clontech (see SEQ ID No. 29) and encodes the red fluorescent protein (DsRed; Genbank accession no. AF272711; SEQ ID Nos. 39 and 40). DsRed, which has a vivid red fluorescence, was isolated from the IndoPacific sea anemone relative Discosoma species. The plasmid pDsRed1N1 (Clontech; SEQ ID No. 29) constitutively expresses a human codon-optimized variant of the fluorescent protein under control of the CMV promoter. Unmodified, this vector expresses high levels of DsRed1 and includes sites for creating N-terminal fusions by cloning proteins of interest into the multiple cloning site (MCS). It is Kan and Neo resistant for selection in bacterial or eukaryotic cells. [0313]
  • Plasmid pMG [0314]
  • Plasmid pMG (InvivoGen, San Diego, Calif.; see SEQ. ID. NO. 27 for the nucleotide sequence of pMG) contains the hygromycin phosphotransferase gene under the control of the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A. Vector pMG also contains two transcriptional units allowing for the coexpression of two heterologous genes from a single vector sequence. [0315]
  • The first transcriptional unit of pMG contains a multiple cloning site for insertion of a gene of interest, the hygromycin phosphotransferase gene (hph) and the immediate-early human cytomegalovirus (hCMV) enhancer/promoter with intron A (see, e.g., Chapman et al. (1991) [0316] Nuc. Acids Res. 19:3979-3986) located upstream of hph and the multiple cloning site, which drives the expression of hph and any gene of interest inserted into the multiple cloning site as a polycistronic mRNA. The first transcriptional unit also contains a modified EMCV internal ribosomal entry site (IRES) upstream of the hph gene but downstream of the hCMV promoter and MCS for ribosomal entry in translation of the hph gene (see SEQ ID NO. 27, nucleotides 2736-3308). The IRES is modified by insertion of the constitutive E. coli promoter (EM7) within an intron (IM7) into the end of the IRES. In mammalian cells, the E. coli promoter is treated as an intron and is spliced out of the transcript. A polyadenylation signal from the bovine growth hormone (bGh) gene (see, e.g., Goodwin and Rottman (1992) J. Biol. Chem. 267:16330-16334) and a pause site derived from the 3′ flanking region of the human a2 globin gene (see, e.g., Enriquez-Harris et al. (1991) EMBO J. 10:1833-1842) are located at the end of the first transcription unit. Efficient polyadenylation is facilitated by inserting the flanking sequence of the bGh gene 3′ to the standard AAUAAA hexanucleotide sequence.
  • The second transcriptional unit of pMG contains another multiple cloning site for insertion of a gene of interest and an EF-1α/HTLV hybrid promoter located upstream of this multiple cloning site, which drives the expression of any gene of interest inserted into the multiple cloning site. The hybrid promoter is a modified human elongation factor-1 alpha (EF-1 alpha) gene promoter (see, e.g., Kim et al. (1990) [0317] Gene 91:217-223) that includes the R segment and part of the U5 sequence (R-U5′) of the human T-cell leukemia virus (HTLV) type I long terminal repeat (see, e.g., Takebe et al. (1988) Mol. Cell. Biol 8:466-472). The Simian Virus 40 (SV40) late polyadenylation signal (see Carswell and Alwine (1989) Mol. Cell. Biol. 9:4248-4258) is located downstream of the multiple cloning site. Vector pMG contains a synthetic polyadenylation site for the first and second transcriptional units at the end of the transcriptional unit based on the rabbit, β-globin gene and containing the AATAAA hexanucleotide sequence and a GT/T-rich sequence with 22-23 nucleotides between them (see, e.g., Levitt et al. (1989) Genes Dev. 3:1019-1025). A pause site derived from the C2 complement gene (see, Moreira et al. (1995) EMBO J. 14:3809-3819) is also located at the 3′ end of the second transcriptional unit.
  • Vector pMG also contains an ori sequence (ori pMB1) located between the SV40 polyadenylation signal and the synthetic polyadenylation site. [0318]
    • EXAMPLE 2
  • A. Construction of Targeting Vector and Transfection into LMtk− Cells for the Generation of Platform Chromosomes [0319]
  • A targeting vector derived from the vector pWE15 (GeneBank Accession #X65279) was modified by replacing the SalI (Klenow filled)/SmaI neomycin resistance containing fragment with the PvuII/BamHI (Klenow filled) puromycin resistance containing fragment (isolated from plasmid pPUR, Clontech Laboratories, Inc. Palo Alto, Calif.; SEQ ID No. 30) resulting in plasmid pWEPuro. Subsequently a 9 Kb NotI fragment from the plasmid pFK161 (SEQ ID NO: 118) containing a portion of the mouse rDNA region was cloned into the NotI site of pWEPuro resulting in plasmid pWEPuro9K (FIG. 2). The vector pWEPuro9K was digested with SpeI to linearize and transfected into LMtk− mouse cells. Puromycin resistant colonies were isolated and subsequently tested for artificial chromosome formation via fluorescent in situ hybridization (FISH) (using mouse major and minor DNA repeat sequences, the puromycin gene and telomeres sequences as probes), and fluorescent activated cell sorting (FACS). From this sort, a subclone was isolated containing an artificial chromosome, designated 5B11.12, which carries 4-8 copies of the puromycin resistance gene contained on the pWEPuro9K vector. FISH analysis of the 5B11.12 subclone demonstrated the presence of telomeres and mouse minor on the ACes. DOT PCR has been done on the 5B11.12 ACes revealing the absence of uncharacterized euchromatic regions on the ACes. A recombination site, such as an att or loxP engineering site or a plurality thereof, was introduced onto this ACes thereby providing a platform for site-specific introduction of heterologous nucleic acid. [0320]
  • B. Targeting a Single Sequence Specific Recombination Site onto Platform Chromosomes [0321]
  • After the generation of the 5B11.12 platform, a single sequence-specific recombination site is placed onto the platform chromosome via homologous recombination. For this, DNA sequences containing the site-specific recombination sequence can be flanked with DNA sequences of homology to the platform chromosome. For example, using the platform chromosome made from the pWEPuro9K vector, mouse rDNA sequences or mouse major satellite DNA can be used as homologous sequences to target onto the platform chromosome. A vector is designed to have these homologous sequences flanking the site-specific recombination site and, after the appropriate restriction enzyme digest to generate free ends of homology to the platform chromosome, the DNA is transfected into cells harboring the platform chromosome (FIG. 3). Examples of site-specific cassettes that are targeted to the platform chromosome using either mouse rDNA or mouse major repeat DNA include the SV40-attP-hygro cassette and a red fluorescent protein (RFP) gene flanked by loxP sites (Cre/lox, see, e.g., U.S. Pat. No. 4,959,317 and description herein). After transfection and integration of the site-specific cassette, homologous recombination events onto the platform chromosome are subcloned and identified by FACS (e.g. screen and single cell subclone via expression of resistance or fluorescent marker) and PCR analysis. [0322]
  • For example, a vector can be constructed containing regions of the mouse rDNA locus flanking a gene cassette containing the SV40 early reporter-bacteriophage lambda attP site-hygromycin selectable marker (see FIG. 4 and described below). The use of the bacteriophage lambda attP site for lambda integrase-mediated site-specific recombination is described below. Homologous recombination event of the SV40-attP-hygro cassette onto the platform chromosome was identified using PCR primers that detect the homologous recombination and further confirmed by FISH analysis. After identifying subcloned colonies containing the platform chromosome with a single site-specific recombination site, cells carrying the platform chromosome with a single site-specific recombination site can now be engineered with site-specific recombinases (e.g. lambda INT, Cre) for integrating a target gene expression vector. [0323]
  • C. Targeting a Red Fluorescent Protein (RFP) Gene Flanked by loxP Sites onto 5B11.12 Platform [0324]
  • As another example, while loxP recombination sites could have been introduced onto the ACes during de novo biosynthesis, it was thought that this might result in multiple segments of the ACes containing a high number of loxP sites, potentially leading to instability upon Cre-mediated recombination. A gene targeting approach was therefore devised to introduce a more limited number of loxP recombination sites into a locus of the 5B11-12 ACes containing introduced and possibly co-amplified endogenous rDNA sequences. Although there are more than 200 copies of rDNA genes in the haploid mouse genome distributed amongst 5-11 chromosomes (depending on strain), rDNA sequences were chosen as the target on the ACes since they represent a less frequent target than that of the satellite repeat sequences. Moreover, having observed much stronger pWEPuro9K hybridization to the 5B11-12 ACes than to other LMTK[0325] − chromosomes and in light of the observation that the transcribed spacer sequences within the rDNA may be less conserved than the rRNA coding regions, it was contemplated that a targeting vector based on the rDNA gene segment in pWEPuro9K would have a higher probability of targeting to the ACes rather than to other LMTK− chromosomes. Accordingly, a targeting vector, pBSFKLoxDsRedLox, was designed and constructed based on the rDNA sequences contained in pWEPuro9K.
  • The plasmid pBSFKLoxDsRedLox was generated in 4 steps. First, the NotI rDNA insert of pWEPuro9K (FIG. 2) was inserted into pBS SK-(Stratagene) giving rise to pBSFK. Second, a loxP polylinker cassette was generated by PCR amplification of pNEB193 (SEQ ID NO:32; New England Biolabs) using primers complementary to the M13 forward and reverse priming sites at their 3′end and a 34 bp 5′ extension comprising a LoxP site. This cassette was reinserted into pNEB193 generating p193LoxMCSLox. Third, the DsRed gene from pDsRed1-N1 (SEQ ID NO:29; Clontech) was then cloned into the polylinker between the loxP sites generating p193LoxDsRedLox. Fourth, a fragment consisting of the DsRed gene flanked by loxP sites was cloned into a unique NdeI within the rDNA insert of pBSFK generating pBSFKLoxDsRedLox. [0326]
  • A gel purified 11 Kb Pml/EcoRV fragment of pBSFKLoxDsRedLox was used for transfection. To detect targeted integration, PCR primers were designed from rDNA sequences within the 5′ NotI-PmlI fragment of pWEPuro9K that is not present on the targeting fragment (5′primer) and sequence within the LoxDsRedLox cassette (3′ primer). If the targeting DNA integrated correctly within the rDNA sequences, PCR amplification using these primers would give rise to a 2.3 Kb band. PCR reactions containing 1-4 μl of genomic DNA were carried out according to the MasterTaq protocol (Eppendorf), using murine rDNA 5′ primer (5′-CGGACAATGCGGTTGTGCGT-3′; SEQ ID NO:72) and [0327] DsRed 3′primer (5′GGCCCCGTAATGCAGAAGAA-3′; SEQ ID NO:73) and PCR products were analyzed by agarose gel electrophoresis.
  • 1.5×10[0328] 6 5B11-12 LMTK− cells were transfected with 2 μg of the pBSFKLoxDsRedLox targeting DNA described above using Lipofectamine Plus (Invitrogen). For flow sorting, harvested cells were suspended in medium and applied to the Becton Dickinson Vantage SE cell sorter, equipped with 488 nm lasers for excitation and 585/42 bandpass filter for optimum detection of RFP fluorescence. Cells were sorted using dPBS as sheath buffer. Negative control parental 5B11-12 cells and a positive control LMTK− cell line stably transfected with DsRed were used to establish the selection gates. The RFP positive gated populations were recovered, diluted in medium supplemented with 1× penicillin-streptomycin (Invitrogen), then plated and cultured as previously described. After 4 rounds of enrichment, the percentage of RFP positive cells reached levels of 50% or higher. DNA from populations was analyzed by PCR for evidence of targeted integration. Ultimately, single cell subclones were established from positive pools and were analyzed by PCR and PCR-positive clones confirmed by FISH as described below.
  • DNA was purified from pools or single cell clones using previously described methods set forth in Lahm et al., [0329] Transgenic Res., 1998; 7:131-134, or in some cases using a Wizard Genomic DNA purification kit (Promega). For FISH analysis, a biotinylated DsRed gene probe was generated by PCR using DsRed specific primers and biotin-labeled dUTP (5′ RFP primer: 5′-GGTTTAAAGTGCGCTCCTCCAAGAACGTCATC-3′, SEQ ID NO:74; and 3′ RFP primer: 5′AGATCTAGAGCCGCCGCTACAGGAACAGGTGGTGGCGGCC-3′; SEQ ID NO:75). To maximize the signal intensity of the DsRed probe, Tyramide amplification was carried out according to the manufacturers protocols (NEN).
  • The process of testing the feasibility of a more general targeting strategy that would not rely on enrichment via drug selection of stably transfected clones can be summarized as follows. A red fluorescent protein gene (RFP; encoded by the DsRed gene) was inserted between the loxP sites of the targeting vector to form pBSFKLoxDsRedLox. After transfection with PBSFKLoxDsRedLox, sequential rounds of high speed flow sorting and expansion of sorted cells in culture could then be used to enrich for stable transformants expressing RFP. In the event of targeted integration, PCR screening with primers that amplify from a spacer region within the segment of the 45s pre-rRNA gene in pWEPuro9K to a specific anchor sequence within the DsRed gene in the targeting cassette would give rise to a diagnostic 2.3 Kb band. However, as rDNA clusters are found on several chromosomes, confirmation of targeting to an ACes would require fluorescence in situ hybridization (FISH) analysis. Finally, the flanking of the DsRed gene by loxP sites would allow for its removal and subsequent replacement with other genes of interest. [0330]
  • After transfection of the targeting sequence into 5B11-12 cells, enrichment for targeted clones was carried out using a combination of flow cytometry to detect red-fluorescing cells and PCR screening. Ultimately 17 single cell subclones were identified as potential targeted clones by PCR and of these 16 were found by FISH to contain the DsRed integration event into the ACes. These subclones are referred to herein as D11-C4, D11-C12, D11-H3, C9-C9, C9-B9, C9-F4, C9-H8, C9-F2, C9-G8, C9-B6, C9-G3, C9-E 12, C9-A 11, C 11-E3, C 11-A9 and C 11-H4. PCR analysis of genomic DNA isolated from the D11-C4 subclone gave rise to a 2.3 Kb band, indicative of a targeted integration into an rDNA locus. Further analysis of the subclone by FISH analysis with a DsRed gene probe demonstrated integration of the LoxDsRedLox targeting cassette on the ACes co-localizing with one of the regions of rDNA staining seen on the 5B11-12 ACes, consistent with a targeted integration into an rDNA locus of the ACes, while integrations on other chromosomes were not observed. Since transfected cells were maintained as heterogeneous populations through several cycles of sorting and replating it was not possible to estimate the frequency of targeted events. In most mammalian cell lines the frequency of gene targeting via homologous recombination is roughly 10[0331] −5-10−7 treated cells. Despite the low frequency of these events in mammalian cells, it is clear that an RFP expression based screening paradigm, coupled with PCR analysis, can effectively detect and enrich for such infrequent events in a large population. In instances where drug selection is not possible or not desirable, such a system may provide a useful alternative. It was also verified that the modified ACes in subclone D11-C4 could be purified by flow cytometry. The results indicate that the flow karyogram of the D11-C4 subclone was unaltered from that of the 5B11-12 cell line. Thus, the D11-C4 ACes can be purified in high yield from native chromosomes of the host cell line.
  • D. Reduction of LoxP on ACes to a Single Site. [0332]
  • The strong hybridization signal detected by FISH on the ACes using the DsRed gene probe suggests that several copies of the targeting cassette may be present on the ACes in the D11-C4 line. This also suggests that multiple rDNA genes have been correctly targeted. [0333]
  • Accordingly, in certain embodiments where necessary, the number of loxP sites on the ACes can be reduced to a single site by in situ treatment with Cre recombinase, provided that the sites are co-linear. Such a process is described for multiple loxP-flanked integrations on a native mouse chromosome (Garrick et al., [0334] Nature Genet., 1998, Jan;18(1):56-59). Reduction to a single loxP site on the D11-C4 ACes would result in the loss of the DsRed gene, forming the basis of a useful screen for this event.
  • For this purpose, a Cre expression plasmid pCX-Cre/GFP III has been generated by first deleting the EcoRi fragment of pCX-eGFP (SEQ ID NO:71) containing the eGFP coding sequence and replacing it with that of a PCR amplified Cre recombinase coding sequence (SEQ ID NO:58), generating pCX-Cre. Next, the AseI/SspI fragment of pD2eGFP-N1 (containing the CMV promoter driving the D2EGFP gene with SV40 polyA signal; Clontech; SEQ ID NO:87) was inserted into the filled HindIII site of pCX-Cre, generating pCX-Cre\GFP III. Control plasmid pCX-CreRev\GFP III was generated in similar fashion except that the Cre recombinase coding sequence was inserted in the antisense orientation. LMTK[0335] − cell line D11-C4 (containing first generation platform ACes with multiple loxP-DsRED sites) and 5B11-12 cell line (containing ACes with no loxP-DsRED sites) are maintained in culture as described above. D11C4 cells are transfected with 2 μg of plasmid pCX-Cre\GFP III or 2 μg pCX-CreRev\GFP III using Lipofectamine (Invitrogen) as previously described.
  • Forty-eight to seventy-two hours after transfection, transfected D11-C4 cells are harvested and GFP positive cells are sorted by cell cytometry using a FACSta Vantage cell sorter (Beckton-Dickinson) as follows: All D11-C4 cells transfected with pCX-Cre\GFP III or control plasmid pCX-CreRev\GFP III that exhibit GFP fluorescent higher than the gate level established by untransfected cells are collected and placed in culture a further 7-14 days. After 7-14 days the initial D11-C4 cells are harvested and analyzed by cell cytometry as follows: Untransfected D11-C4 cells are used to establish the gate that defines the RFP positive population, while 5B11-12 cells are used to set the RFP negative gate. The GFP positive population of D11-C4 transfected with pCX-Cre\GFP III should show decreased red fluorescence compared to pCX-CreRev\GFP III transfected or untransfected control D11-C4 cells. The cells exhibiting greatly decreased or no RFP expression are collected and single cell clones subsequently established. These clones will be expanded and analyzed by fluorescence in-situ hybridization and Southern blotting to confirm the removal of loxP-DsRed gene copies. [0336]
    • EXAMPLE 3
  • Construction of Targeting Vector and Transfection into LMtk− Cells for the Generation of Platform Chromosomes Containing Multiple Site-Specific Recombination Sites [0337]
  • An example of a selectable marker system for the creation of a chromosome-based platform is shown in FIG. 4. This system includes a vector containing the SV40 early promoter immediately followed by (1) a 282 base pair (bp) sequence containing the bacteriophage lambda attP site and (2) the puromycin resistance marker. Initially a PvuII/StuI fragment containing the SV40 early promoter from plasmid pPUR (Clontech Laboratories, Inc., Palo Alto, Calif.; Seq ID No. 30) was subcloned into the EcoRI/CRI site of pNEB193 (a PUC19 derivative obtained from New England Biolabs, Beverly, Mass.; SEQ ID No. 32) generating the plasmid pSV40193. The only differences between pUC19 and pNEB193 are in the polylinker region. A unique Ascl site (GGCGCGCC) is located between the BamHI site and the SmaI site, a unique PacI site (TTAATTAA) is located between the BamHI site and the XbaI site and a unique PmeI site (GTTTAAAC) is located between the PstI site and the SalI site. [0338]
  • The attP site was PCR amplified from lambda genome (GenBank Accession # NC 001416) using the following primers: [0339]
    attPUP: CCTTGCGCTAATGCTCTGTTACAGG SEQ ID No.1
    attPDWN: CAGAGGCAGGGAGTGGGACAAAATTG SEQ ID No.2
  • After amplification and purification of the resulting fragment, the attP site was cloned into the SmaI site of pSV40193 and the orientation of the attP site was determined by DNA sequence analysis (plasmid pSV40193attP). The gene encoding puromycin resistance (Puro) was isolated by digesting the plasmid pPUR (Clontech Laboratories, Inc. Palo Alto, Calif.) with AgeI/BamHI followed by filling in the overhangs with Klenow and subsequently cloned into the AscI site downstream of the attP site of pSV40193attP generating the plasmid pSV40193attPsensePUR (FIG. 4; SEQ ID NO:113)). [0340]
  • The plasmid pSV40193attPsensePUR was digested with ScaI and co-transfected with the plasmid pFK161 (SEQ ID NO: 118) into mouse LMtk− cells and platform artificial chromosomes were identified and isolated as described above. The process for generating this exemplary platform ACes containing multiple site-specific recombination sites is summarized in FIG. 5. One platform ACes resulting from this experiment is designated B19-18. This platform ACes chromosome may subsequently be engineered to contain target gene expression nucleic acids using the lambda integrase mediated site-specific recombination system as described herein in Example 7 and 8. [0341]
    • EXAMPLE 4
  • Lambda Integrase Mediated Site-Specific Recombination of a RFP Expressing Vector onto Artificial Chromosomes [0342]
  • In this example, a vector expressing the red fluorescent protein (RFP) was produced and recombined into the attP site residing on an artificial chromosome within LMTK− cells. This recombination is depicted in FIG. 7. [0343]
  • A. Construction of Expression Vectors Containing Wildtype and Mutant Lambda Integrase [0344]
  • Mutations at the glutamic acid at position 174 in the lambda integrase protein relaxes the requirement for the accessory protein IHF during recombination and DNA supercoiling in vitro (see, Miller et al. (1980) [0345] Cell 20:721-729; Lange-Gustafson et al. (1984) J. Biol. Chem. 259:12724-12732). Mutations at this site promote attP, attB intramolecular recombination in mammalian cells (Lorbach et al. (2000) J. Mol. Biol 296:1175-1181).
  • To construct nucleic acid encoding the mutant, lambda integrase was PCR amplified from bacteriophage lambda DNA (c1857 ind Sam 7; New England Biolabs) using the following primers: [0346]
    Lamint1
    TTCGAATTCATGGGAAGAAGGCGAAGTCATGAGCG) (SEQ ID No.3)
    Lamint2
    (TTCGAATTGTTATTTGATTTCAATTTTGTCCCAC). (SEQ ID No.4)
  • The resulting PCR product was digested with EcoR I and cloned into the EcoR I site of pUC19. Lambda integrase was mutated at amino acid position 174 using QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligos (generating a glutamic acid to arginine change at position 174): [0347]
    LambdaINTE174R (SEQ ID No.6)
    (CGCGCAGCAAAATCTAGAGTAAGGAGATCAAGACTTACGGCTGACG),
    LamintR174rev (SEQ ID No.7)
    (CGTCAGCCGTAAGTCTTGATCTCCTTACTCTAGATTTTGCTGCGCG).
  • The resulting site directed mutant was confirmed by sequence analysis. The wildtype and mutant lambda genes were cloned into the EcoR I site of pCX creating pCX-Lamint (SEQ ID NO: 127) and pCXLamIntR (FIG. 8; SEQ ID NO: 112). [0348]
  • The plasmid pCX (SEQ ID No. 70) was derived from plasmid pCXeGFP (SEQ ID No. 71). Excision of the EcoRI fragment containing the eGFP marker generated pCX. To generate plasmid pCXLamINTR (SEQ ID NO: 112) an EcoRI fragment containing the lambda integrase El 74R (SEQ ID No. 37) mutation was cloned into the EcoRI site of pCX, and to generate plasmid pCX-LamINT, an EcoRI fragment containing the wildtype lambda integrase was cloned into the EcoRI site of pCX. [0349]
  • B. Construction of Integration Vector Containing attB and DsRed [0350]
  • The plasmid pDsRedN1 (Clontech Laboratories, Palo Alto, Calif.; SEQ ID No. 29) was digested with Hpa I and ligated to the following annealed oligos: [0351]
    attB1
    (TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA) (SEQ ID No.8)
    attB2
    (TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA) (SEQ ID No.9)
  • The resulting vector (pDsRedN1-attB) was confirmed by PCR and sequence analysis. [0352]
  • C. Transfection into LMtk− Cells [0353]
  • LM(tk−) cells containing the Prototype A ACes (L1-18; Chromos Molecular Systems Inc., Burnaby, BC Canada) were co-transfected with pDsRedN1 or pDsRedN1-attB and either pCXLamInt (SEQ ID NO: 127) or pCXLamIntR (SEQ ID NO: 112) using Lipofectamine Plus Reagent (LifeTechnologies, Gaithersburg, Md.). The transfected cells were grown in DMEM (LifeTechnologies, Gaithersburg, Md.) with 10% FBS (CanSera) and G418 (CalBiochem) at a concentration of 1 mg/ml. [0354]
  • D. Enrichment by Cell Sorting [0355]
  • The transfected cells were sorted using a FACs Vantage SE cell sorter (Becton Dickenson) to enrich for cells expressing DsRed. The cells were excited with a 488 nm Argon laser at 200 watts and cells fluorescing in the 585/42 detection channel were collected. The sorted cells were returned to growth medium for recovery and expansion. After three successive enrichments for cells expressing DsRed, single cell sorting into 96 well plates was performed using the same parameters. Duplicate plates of the single cell clones were made for PCR analysis. [0356]
  • E. PCR Analysis of Single Cell Clones [0357]
  • Pools of cells from each row and column of the 96 well plate were used for DNA isolation. DNA was prepared using a Wizard Genomic DNA purification kit (Promega Inc, Madison, Wis.). Nested PCR analysis on the DNA pools was performed to confirm the site-specific recombination event using the following primer sets: [0358]
    attPdwn2
    (TCTTCTCGGGCATAAGTCGGACACC) (SEQ ID No.10)
    CMVen
    (CTCACGGGGATTTCCAAGTCTCCAC) (SEQ ID No.11)
    followed by:
    attPdwn
    (CAGAGGCAGGGAGTGGGACAAAATTG) (SEQ ID No.12)
    CMVen2
    (CAACTCCGCCCCATTGACGCAAATG). (SEQ ID No.13)
  • The resulting PCR reactions were analyzed by gel electrophoresis and the potential individual clones containing the site-specific recombination event were identified by combining the PCR results of all of the pooled rows and columns for each 96 well plate. The individual clones were then further analyzed by PCR using the following primers that flank the recombination junction. L1for and F1rev flank the attR junction whereas REDfor and L2rev flank the attL junction (see FIG. 7): [0359]
    L1for
    AGTATCGCCGAACGATTAGCTCTTCA (SEQ ID No.14)
    F1rev
    GCCGATTTCGGCCTATTGGTTAAA (SEQ ID No.15)
    REDfor
    CCGCCGACATCCCCGACTACAAGAA (SEQ ID No.16)
    L2rev
    TTCCTTCGAAGGGGATCCGCCTACC. (SEQ ID No.17)
  • F. Sequence Analysis of Recombination Junctions [0360]
  • PCR products spanning the recombination junction were Topo-cloned into pcDNA3.1D/V5His (Invitrogen Inc., San Diego, Calif.) and then sequenced by cycle-sequencing. The clones were confirmed to have the correct attR and attL junctions by cycle sequencing. [0361]
  • G. Fluorescent In Situ Hybridization (FISH) [0362]
  • The cell lines containing the correct recombination junction sequence were further analyzed by fluorescent in situ hybridization (FISH) by probing with the DsRed coding region labeled with biotin and visualizing with the Tyramide Signal Amplification system (TSA; NEN Life Science Products). The results indicate that the RFP sequence is present on the ACes. [0363]
  • H. Southern Analysis [0364]
  • Genomic DNA was harvested from the cell lines containing an ACes with the correct recombinant event and digested with EcoR I. The digested DNAs were separated on a 0.7% agarose gel, transferred and fixed to a nylon membrane and probed with RFP coding sequences. The result showed that there is an integrated copy of RFP coding sequence in each clone. [0365]
    • EXAMPLE 5
  • Delivery of a Second Gene Encoding GFP onto the RFP Platform ACes [0366]
  • A. Construction of Integration Vector Containing attB and GFP (pD2eGFPIresPuroattB). [0367]
  • The plasmid pIRESpuro2 (Clontech, Palo Alto, Calif.; SEQ ID NO: 88) was digested with EcoRI and NotI then ligated to the D2eGFP EcoRI-NotI fragment from pD2eGFP-N1 (Clontech, Palo Alto, Calif.) to create pD2eGFPIresPuro2. Subsequently, oligos encoding the attB site were annealed and ligated into the NruI site of pD2eGFPIresPuro2 to create pD2eGFPIresPuroattB. The orientation of attB in the NruI site was determined by PCR. [0368]
  • B. Transfection of LMtk− Cells [0369]
  • The LMtk− cells containing the RFP platform ACes produced in Example 4, which has multiple attP sites, were co-transfected with pCXLamIntR and pD2eGFPIresPuroattB using LipofectAMINE PLUS reagent. Five μg of each vector was placed into a tube containing 750 μl of DMEM (Dulbecco's modified Eagles Medium). Twenty μl of the Plus reagent was added to the DNA and incubated at room temperature for 15 minutes. A mixture of 30 μl of lipofectamine and 750 μl DMEM was added to the DNA mixture and incubated an additional 15 minutes at room temperature. The DNA mixture was then added dropwise to approximately 3 million cells attached to a 10 cm dish in 5 mls of DMEM. The cells were incubated 4 hours (37° C., 5% CO[0370] 2) with the DNA-lipid mixture, after which DMEM with 20% fetal bovine serum was added to the dishes to bring the culture medium to 10% fetal bovine serum. The dishes were incubated at 37° C. with 5% CO2.
  • Plasmid pD2eGFPIresPuroattB has a puromycin gene transcriptionally linked to the GFP gene via an IRES element. Two days after the transfection the cells were placed in medium containing puromycin at 4 μg/ml to select for cells containing the pD2eGFPIresPuroattB plasmid integrated into the genome. Twenty-three clones were isolated after 17 days of selection with puromycin. These clones were expanded and then analyzed for the presence of the GFP gene on the ACes by 2-color (RFP/biotin & GFP/digoxigenin) TSA-FISH (NEN) according to the manufacturers protocol. Sixteen of the 23 clones produced a positive FISH signal on the ACes with a GFP probe. [0371]
    • EXAMPLE 6
  • Delivery Of ACes into human Mesenchymal Stem Cells (hMSC) [0372]
  • A. Transfection [0373]
  • Transfection conditions for the most efficient delivery of the ACes into hMSCs (Cambrex BioWhittaker Product Code PT-2501, lot#F0658, East Rutherford, N.J.) were assayed using LipofectAMINE PLUS and Superfect. One million prototype B ACes, which is a murine derived 60 Mb ACes having primarily murine pericentric heterochromatin, and carrying a “payload” containing a hygromycin B selectable marker gene and a lacZ reporter gene (see , Telenius et al., 1999[0374] , Chrom. Res., 7:3-7; and Kereso et al., 1996, Chrom. Res., 4:226-239; each of which is incorporated herein by reference in its entirety), were combined with 1-12 μl of the transfection agent. In the case of LipofectAMINE PLUS, the PLUS reagent was combined with the ACes for 15 minutes followed by LipofectAMINE for a further 15 minutes. Superfect was complexed for 10 minutes at a ratio of 2 μl Superfect per 1 million ACes. The ACes/transfection agent complex was then applied to 0.5 million recipient cells and the transfection was allowed to proceed according to the manufacturer's protocol. Percent transfected cells was determined on a FACS Vantage flow cytometer with argon laser tuned to 488 nm at 200 mW and FITC fluorescence collected through a standard FITC 530/30 nm band pass filter. After 24 hours, IdUrd labeled ACes were delivered to human MSCs in the range of 30-50%, varying with transfection agent and dose. ACes delivery curves were generated from data collected in experiments that varyied the dose of the transfection reagents. Dose response curves of Superfect and LipofectAMINE PLUS, showing delivery of ACes into recipient hMSCs cells, were prepared, measured by transfer of IdUrd labeled ACes and detected by flow cytometry. Superfect shows maximum delivery in the range of 30-50% at doses greater than 2 μl per million ACes. LipofectAMINE PLUS has a 42-48% delivery peak around 5-8 μl per million ACes. These dose curves were then correlated with toxicity data to determine the transfection conditions that will allow for highest potential transfection efficiency. Toxicity was determined by a modified plating efficiency assay (de Jong et al., 2001, Chrom. Research, 9:475-485). The population's normalized plating efficiency (at maximum % delivery doses) was in the range of 0.2-0.4 for Superfect and 0.5-0.6 with LipofectAMINE PLUS.
  • Due to the transfected population consisting of mixed cell types, flow cytometry allowed for the assessment of ACes delivery into each sub-population and the purification of the target population. Flow profiles showing forward scatter (cell size) and side scatter (internal cell granularity) revealed three distinct hMSC populations that were gated into three regions: R3 (small cell region), R4 (medium cell region), R5 (large cell region). Transfection conditions were further optimized by re-analyzing delivery curves and assessing the differences in delivery to each sub-population. Dose response curves of Superfect and LipofectAMINE were prepared showing % delivery to each sub-population represented by the gating on basis of cell size and granularity properties of the mixed population. Three distinct hMSC populations were gated and % delivery dose curves generated. Using Superfect and LipofectAMINE PLUS the overall % delivery increased with cell size (80-90% delivery in large cells). LipofectAMINE PLUS at high doses (8-12 μl per 1 million ACes) shows an increase in the overall proportion of chromosome transfer to the small population (10-20%). This suggests an advantage to using this transfection agent if the small-undifferentiated cell population is the desired target host cell. [0375]
  • B. Expression from Genes on ACes IN hMSCs [0376]
  • Following the delivery screening process conducted in section (A) above, the most promising results were subjected to further analyses to monitor expression and verify the presence of structurally intact ACes. The transfection conditions employed for these experiments were exactly the same as those that had been used during the screening process. Short-term expression was monitored by transfecting hMSCs with ACes containing a RFP gene (red fluorescent protein) set forth in Example 2C as “D11C4”. The unselected population was harvested at 72-96 hours post transfection and % positive fluorescent cells measured by flow cytometry. RFP expression was in the range of 1-20%. [0377]
  • Long term-gene expression was assayed by selecting for hygromycin B resistant cells over a period of 7-10 days. Cytogenetic analysis was done to detect presence of intact ACes by Fluorescent In Situ hybridization (FISH), where metaphase chromosomes were hybridized to a mouse major satellite-DNA probe (targeting murine pericentric heterochromatin) and a lambda probe (hybridizing to the lacZ gene). The human mesenchymal transfected culture could not undergo standard sub-cloning as diffuse colonies form with limited doublings available for expansion. Cytogenetic analysis was performed on the entire population, sampling over a period of 3-10 days post-transfection. The hygromycin resistant population was then blocked in mitosis with colchicine and analyzed for presence of intact ACes by FISH. Preliminary FISH results show approximately 2-8% of the hMSC-transfected population had an intact ACes. This compared to rat skeletal muscle myoblast clones, which were in the range of 60-95%. To increase the % of intact ACes in the hMSC-transfected population an enrichment step can be utilized as described in Example 2C. [0378]
  • C. Differentiation of The hMSCs [0379]
  • In initial experiments where transfected hMSCs cells have been induced to differentiate into adipose or osteocytes, the results indicate that the transfected cells appear to be differentiating at a rate comparable to the untransfected controls and the cultures are lineage specific as tested by microscopic examination, FISH, Oil Red O staining (adipocyte assay), and calcium secretion (osteocyte assay). [0380]
  • Accordingly, these results indicate that the artificial chromosomes (ACes) provided herein can be successfully transferred into hMSC target cells. Targeting MSCs (such as hMSCs) permits gene transfer into cells in an undifferentiated state where the cells are easier to expand and purify. The genetically modified cells can then be differentiated in vitro or injected into a site in vivo where the microenvironment will induce transformation into specific cell lineages. [0381]
    • EXAMPLE 7
  • Delivery of a Promoterless Marker Gene to a Platform ACes [0382]
  • Platform ACes containing pSV40attPsensePURO (FIG. 4) were constructed as set forth in Examples 3 and 4. [0383]
  • A. Construction of Targeting Vectors. [0384]
  • The base vector p18attBZeo (3166 bp; SEQ ID NO: 114) was constructed by ligating the 1067 bp HindIII-SspI fragment containing attBZeo, obtained from pLITattBZeo (SEQ ID NO:91), into pUC18 (SEQ ID NO: 122) digested with HindIII and SspI. [0385]
  • 1. p18attBZEO-eGFP (6119 bp; SEQ ID NO: 126) was constructed by inserting the 2977 bp. SpeI-HindIII fragment from pCXeGFP (SEQ ID NO:71; Okabe, et al. (1997) [0386] FEBS Lett 407:313-319) containing the eGFP gene into p18attBZeo (SEQ ID NO: 114) digested with HindIII and XbaI.
  • 2. p18attBZEO-5′6XHS4eGFP (FIG. 10; 7631 bp; SEQ ID NO: 116) was constructed by ligating the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5′ of the chicken beta actin promoter and the polyadenylation signal into the HindIII site of p18attBZeo (SEQ ID NO: 114). [0387]
  • 3. p18attBZEO-3′6XHS4eGFP (FIG. 11; 7600 bp; SEQ ID NO: 115) was created by removing the 5′6XHS4 element from p18attBZeo-(6XHS4)2eGFP (SEQ ID NO: 110). p18attBZeo-(6XHS4)2eGFP was digested with EcoRV and SpeI, treated with Klenow and religated to form p18attBZeo3′6XHS4eGFP (SEQ ID NO: 115). [0388]
  • 4. p18attBZEO-(6XHS4)2eGFP (FIG. 12; 9080 bp; SEQ ID NO: 110) was created in two steps. First, the EcoRI-SpeI fragment from pCXeGFPattB(6XHS4)2 (SEQ ID NO: 123) which contains 6 copies of the HS4 core element was ligated into p18attBZeo (SEQ ID NO: 114) digested with EcoRI and XbaI to create p18attBZeo6XHS4 (4615 bp; SEQ ID NO: 117). Next, p18attBZeo6XHS4 was digested with HindIII and ligated to the 4465 bp HindIII fragment from pCXeGFPattB(6XHS4)2 which contains the eGFP gene, under the regulation of the chicken beta actin promoter, 6 copies of the HS4 core element located 5′ of the chicken beta actin promoter and the polyadenylation signal. [0389]
    TABLE 2
    No. zeocin No. clones with No. clones with correct
    resistant expected PCR sequence at
    Targeting plasmid clones product size recombination junction
    p18attBZEO- 12 12 NT*
    eGFP
    p18attBZEO- 11 11 NT
    5′6XHS4eGFP
    p18attBZEO- 11 11 NT
    3′6XHS4eGFP
    p18attBZEO- 9 9 4/4
    (6XHS4)2eGFP
  • B. Transfection and Selection with Drug. [0390]
  • The mouse cell line containing the 2[0391] nd generation platform ACE, B19-38 (constructed as set forth in Example 3), was plated onto four 10 cm dishes at approximately 5 million cells per dish. The cells were incubated overnight in DMEM with 10% fetal calf serum at 37° C. and 5% CO2. The following day the cells were transfected with 5 μg of each of the 4 vectors listed in Example 7.A. above and 5 μg of pCXLamintR (SEQ ID NO: 112), for a total of 10 μg per 10 cm dish. Lipofectamine Plus reagent was used to transfect the cells according to the manufacturers protocol. Two days post-transfection zeocin was added to the medium at 500 ug/ml. The cells were maintained in selective medium until colonies formed. The colonies were then ring-cloned (see, e.g., McFarland, 2000, Methods Cell Sci, Mar;22(1):63-66).
  • C. Analysis of Clones (PCR, SEQUENCING). [0392]
  • Genomic DNA was isolated from each of the candidate clones with the Wizard kit (Promega) and following the manufacturers protocol. The following primer set was used to analyze the genomic DNA isolated from the zeocin resistant clones: 5PacSV40 [0393]
    5PacSV40- CTGTTAATTAACTGTGGAATGTGTGTCAGTTAGGGTG; (SEQ ID NO:76)
    Antisense Zeo- TGAACAGGGTCACGTCGTCC. (SEQ ID NO:77)
  • amplification with the above primers and genomic DNA from the site-specific integration of any of the 4 zeocin vectors would result in a 673 bp PCR product. [0394]
  • As set forth in Table 2, of the 4 zeocin resistant candidate clones thusfar analyzed by PCR, all 4 exhibit the correct sequence for a site-specific integration event. [0395]
    • EXAMPLE 8
  • Integration of a PCR product by site-specific recombination. In this example a gene is integrated onto the platform ACes by site-specific recombination without cloning said gene into a vector. [0396]
  • A. PCR Primer Design. [0397]
  • PCR primers are designed to contain an attB site at the 5′ end of one of the primers in the primer set. The remaining primers, which could be one or more than one primer, do not contain an attB site, but are complementary to sequences flanking the gene or genes of interest and any associated regulatory sequences. In first example, 2 primers (one containing an attB site) are used to amplify a selective gene such as puromycin. [0398]
  • In a second example as shown in FIG. 13, the primer set includes [0399] primers 1 & 2 that amplify the GFP gene without amplification of an upstream promoter. Primer 1 contains the attB site at the 5′ end of the oligo. Primers 3 & 4 are designed to amplify the IRES-blasticidin DNA sequences from the vector pIRESblasticidin. The 5′end of primer 3 contains sequences complementary to the 5′ end of primer 2 such that annealing can occur between 5′ ends of the two primers.
  • B. PCR Reaction and Subsequent Ligation to Create Circular Molecules from the PCR Product [0400]
  • In the first example set forth above in Section A, the two PCR primers are combined with a puromycin DNA template such as pPUR (Clontech), a heat stable DNA polymerase and appropriate conditions for DNA amplification. The resulting PCR product (attB-Puromycin) is then then purified and self-ligated to form a circular molecule. [0401]
  • In the second example set forth above in Section A, amplification of the GFP gene and IRES-blasticidin sequences is accomplished by combining [0402] primers 1 & 2 with DNA template pD2eGFP and primers 3 & 4 with template pIRESblasticidin under appropriate conditions to amplify the desired template. After initial amplification of the two products (attB-GFP & IRES-blasticidin) in separate reactions, a second round of amplification using both of the PCR products from the first round of amplification together with primers 1 and 4 amplifies the fusion product attB-GFP-IRES-blasticidin (FIG. 13). This technique of using complementary sequences in primer design to create a fusion product is employed in Saccharomyces cerevisiae for allele replacement (Erdeniz et al (1997) Gen Res 7:1174-1183). The amplified product is then purified from the PCR reaction mixture by standard methods and ligated to form a circular molecule.
  • C. Introduction of PCR Product onto the ACes Using a Recombinase [0403]
  • The circular PCR product is then be introduced to the platform ACes using the bacteriphage lambda integrase E174R. The introduction can be performed in vivo by transfecting the pCXLamIntR (SEQ ID NO: 112) vector encoding the lambda integrase mutant E174R together with the circularized PCR product into a cell line containing the platform ACE. [0404]
  • D. Selection for Marker Gene [0405]
  • The marker gene (in this case either puromycin, blasticidin or GFP) is used to enrich the population for cells containing the proper integration event. A proper integration event in the second example (FIG. 14) juxtaposes a promoter residing on the platform ACes 5′ to the attB-GFP-IRES-Blasticidin PCR product, allowing for transcription of both GFP and blasticidin. If enrichment is done by drug selection, blasticidin is added to the medium on the transfected cells 24-48 hours post-transfection. Selection is maintained until colonies are formed on the plates. If enrichment is done by cell sorting, cells are sorted 2-4 days post-transfection to enrich for cells expressing the fluorescent marker (GFP in this case). [0406]
  • E. Analysis of Clones [0407]
  • Clonal isolates are analyzed by PCR, FISH and sequence analysis to confirm proper integration events. [0408]
    • EXAMPLE 9
  • Construction of a Human Platform ACes “ACE 0.1”[0409]
  • A. Construction of the Targeting Vector pPACrDNA [0410]
  • Genome Systems (IncyteGenomics) was supplied with the primers 5′HETS (GGGCCGAAACGATCTCAACCTATT; SEQ ID NO:78), and 3′HETS (CGCAGCGGCCCTCCTACTC; SEQ ID NO:79), which were used to amplify a 538 bp PCR product homologous to nt 9680-10218 of the human rDNA sequences (GenBank Accession No. U13369) and used as a probe to screen a human genomic PlAC (P1 Artificial Chromosome) library constructed in the vector pCYPAC2 (loannou et al. (1994) [0411] Nat. Genet. 6(1): 84-89). Genome Systems clone #18720 was isolated in this screen and contains three repeats of human rDNA as assessed by restriction analysis. GS clone #18720, was digested with PmeI, a restriction enzyme unique to a single repeat of the human rDNA (45 Kbp), and then religated to form pPACrDNA (FIG. 15). The insert in pPACrDNA was analyzed by restriction digests and sequence analysis of the 5′ and 3′ termini. The pPACrDNA, rDNA sequences are homologous to Genbank Accession #U13369, containing an insert of about 45 kB comprising a single repeat beginning from the end of one repeat at ˜33980 (relative to the Genbank sequence) through the beginning of the next repeat up to approximately 35120 (the repeat offset from that listed in the GenBank file). Thus, the rDNA sequence is just over 1 copy of the repeat extending from 33980 (+/−10 bp) to the end of the first repeat (43 Kbp) and continuing into the second repeat to bp 35120 (+/−10 bp).
  • B. Transfection and ACes Formation. [0412]
  • Five hundred thousand MSU1.1 cells (Morgan et al., 1991, Exp. Cell Res., Nov;197(1):125-136; provided by Dr. Justin McCormick at Michigan State University) were plated per 6 cm plate (3 plates total) and allowed to grow overnight. The cells were 70-80% confluent the following day. One plate was transfected with 15 μg pPACrDNA (linearized with Pme I) and 2 μg pSV40attPsensePuro (linearized with Sca I; see Example 3). The remaining plates were controls and were transfected with either 20 μg pBS (Stratagene) or 20 μg pSV40attBsensePuro (linearized with Sca I). All three plates were transfected using a CaPO[0413] 4 protocol.
  • C. Selection of Puromycin Resistant Colonies [0414]
  • One day post-transfection the cells were “glycerol shocked” by the addition of PBS medium containing 10% glycerol for 30 seconds. Subsequently, the glycerol was removed and replaced with fresh DMEM. Four days post-transfection selective medium was added. Selective medium contains lug/ml puromycin. The transfection plates were maintained at 37° C. with 5% CO[0415] 2 in selective medium for 2 weeks at which point colonies could be seen on the plate transfected with pPACrDNA and pSV40attPsensePuro. The colonies were ring-cloned from the plate on day 17 post-selection and expanded in selective medium for analysis. Only two colonies (M2-2d & M2-2b) were able to proliferate in the selective medium after cloning. No colonies were seen on the control plates after 37 days in selective medium.
  • D. Analysis of Clones [0416]
  • FISH analysis was performed on the candidate clones to detect ACes formation. Metaphase spreads from the candidate clones were probed in multiple probe combinations. In one experiment, the probes used were biotin-labeled human alphoid DNA (pPACrDNA) and digoxigenin-labeled mouse major DNA (pFK161) as a negative control. Candidate M2-2d was single cell subcloned by flow sorting and the candidate subclones were reanalyzed by FISH. Subclone 1B1 of M2-2d was determined to be a platform ACes and is also designated human Platform ACE 0.1. [0417]
    • EXAMPLE 10
  • Site-Specific Integration of a Marker Gene onto a Human Platform ACE 0.1 [0418]
  • The promoterless delivery method was used to deliver a promoterless blasticidin marker gene onto the human platform ACes with excellent results. The human ACes platform with a promoterless blasticidin marker gene resulted in 21 of 38 blasticidin resistant clones displaying a PCR product of the expected size from the population co-transfected with pLIT38attBBSRpolyA10 and pCXLamintR (FIG. 8; SEQ ID NOs. 111 and 112). Whereas, the population transfected with pBlueScript resulted in 0 blasticidin resistant colonies. [0419]
  • A. Construction of pLIT38attB-BSRpolyA10 & pLIT38attB-BSRpolyA2. [0420]
  • The vector pLITMUS 38 (New England Biolabs; U.S. Pat. No. 5,691,140; SEQ ID NO: 119) was digested with EcoRV and ligated to two annealed oligomers, which form an attB site (attB15′-TGAAGCCTGCTTTTTTATACTAACTTGAGCGAA-3′ (SEQ ID NO:8); attB2 5′-TTCGCTCAAGTTAGTATAAAAAAGCAGGCTTCA-3′; SEQ ID NO:9). This ligation reaction resulted in the vector pLIT38attB (SEQ ID NO: 120). The blasticidin resistance gene and SV40 polyA site was PCR amplified with primers: 5BSD (ACCATGAAAACATTTAACATTTCTCAACA; SEQ ID NO:80) and SV40polyA (TTTATTTGTGAAATTTGTGATGCTATTGC; SEQ ID NO:81) using pPAC4 (Frengen, E., et al. (2000) Genomics 68 (2), 118-126; GenBank Accession No. U75992) as template. The blasticidin-SV 40polyA PCR product was then ligated into pLIT38attB at the BamHI site, which was Klenow treated following digestion with BamHI. pLIT38attB-BSDpolyA10 (SEQ ID NO: 111) and pLIT38attB-BSDpolyA2 (SEQ ID NO: 121) are the two resulting orientations of the PCR product ligated into the vector. [0421]
  • B. Transfection of MSU1.1 Cells Containing Human Platform Ace 0.1. [0422]
  • MSU1.1 cells containing human platform ACE 0.1 (see Example 9) was expanded and plated to five 10 cm dishes with 1.3×10[0423] 6 cells per dish. The cells were incubated overnight in DMEM with 10% fetal bovine serum, at 37° C. and 5% CO2. The following day the cells were transfected with 5 μg of each plasmid as set forth in Table 3, for a total of 10 μg of DNA per plate of cells transfected (see Table 3) using ExGen 500 in vitro transfection reagent (MBI fermentas, cat. no. R0511). The transfection was performed according to the manufacturers protocol.
  • Cells were incubated at 37° C. with 5% CO[0424] 2 in DMEM with 10% fetal bovine serum following the transfection.
    TABLE 3
    Plate # Plasmid 1 Plasmid 2 No. BsdR Colonies
    1 pBS None 0
    2 pCXLamInt pLIT38attB- 16
    BSRpolyA10
    3 pCXLamIntR pLIT38attB- 40
    BSRpolyA10
    4 pCXLamInt pLIT38attB- 28
    BSRpolyA2
    5 pCXLamIntR pLIT38attB- 36
    BSRpolyA2
  • C. Selection of Blasticidin Resistant Clones. [0425]
  • Three days following the transfection the cells were split from a 10 cm dish to two 15 cm dishes. The cells were maintained in DMEM with 10% fetal bovine serum for 4 days in the 15 cm dishes. Seven days post-transfection blasticidin was introduced into the medium. Stably transfected cells were selected with 1 μg/ml blasticidin. The number of colonies formed on each plate is listed in Table 3. These colonies were ring-cloned and expanded for PCR analysis. Upon expansion in blasticidin containing medium some clones failed to live and therefore do not have corresponding PCR data. [0426]
  • D. PCR analysis [0427]
  • Thirty-eight of the 40 clones from [0428] plate 3 grew after ring-cloning. Genomic DNA was isolated from these clones with the Promega Wizard Genomic cDNA purification kit, digested with EcoRI and used as template in a PCR reaction with the following primers: 3BSP-TTAATTTCGGG TATATTTGAGTGGA (SEQ ID NO:82); 5PacSV40-CTGTTAATTAACTGTGGAA TGTGTGTCAGTTAGGGTG (SEQ ID NO:76). The PCR conditions were as follows. 10 ng of genomic DNA was amplified with 0.5 ul Herculase polymerase (Stratagene) in a 50 ul reaction that contained 12.5 pmole of each primer, 2.5 mM of each dNTP, and 1× Herculase buffer (Stratagene). The reactions were placed in a PerkinElmer thermocycler programmed as follows: Initial denaturation at 95° C. for 10 minutes; 35 cycles of 94° C. for 1 minute, 53° C. for 1 minute, 72° C. for 1 minute, and 72° C. for 1 minute; Final extension for 10 minutes at 72° C.; and 4° C. hold. If pLIT38attB-BSRpolyA10 integrates onto the human platform ACE 0.1 correctly, PCR amplification with the above primers should yield an 804 bp product. Twenty-one of the 38 clones from plate 3 produced a PCR product of the expected 804 bp size.
    • EXAMPLE 11
  • Delivery of a Vector Comprising a Promoterless Marker Gene and a Gene Encoding a Therapeutic Product to a Platform ACes [0429]
  • Platform ACes containing pSV40attPsensePURO (FIG. 4) were constructed as set forth in Examples 3 and 4. [0430]
  • A. Construction of Delivery Vectors [0431]
  • 1. Erythropoietin cDNA Vector, p18EPOcDNA. [0432]
  • The erythropoietin cDNA was PCR amplified from a human cDNA library (E. Perkins et al., 1999[0433] , Proc. Natl. Acad. Sci. USA 96(5): 2204-2209) using the following primers: EPO5XBA-TATCTAGAATGGGGGTGC ACGAATGTCCTGCC (SEQ ID NO: 83); EPO3BSI-TACGTACGTCATC TGTCCCCTGTCCTGCAGGC (SEQ ID NO: 84). The cDNA was amplified through two successive rounds of PCR using the following conditions: heat denaturation at 95° C. for 3 minutes; 35 cycles of a 30 second denaturation (95° C.), 30 seconds of annealing (60° C.), and 1 minute extension (72° C.); the last cycle is followed by a 7 minute extension at 72° C. BIO-X-ACT (BIOLINE) was used to amplify the erythropoietin cDNA from 2.5 ng of the human cDNA library in the first round of amplification. Five μl of the first amplification product was used as template for the second round of amplification. Two PCR products were produced from the second amplification with Taq polymerase (Eppendorf), each product was cloned into pCR2.1-Topo (Invitrogen) and sequenced. The larger PCR product contained the expected cDNA sequence for erythropoietin. The erythropoietin cDNA was moved from pTopoEPO into p18attBZeo(6XHS4)2eGFP (SEQ ID NO: 110). pTopoEPO was digested with BsiWI and XbaI to release a 588 bp EPO cDNA. BsrGI and BsiWI create compatable ends. The eGFP gene was removed from p18attBZeo(6XHS4)2eGFP by digestion with BsiWI and XbaI, the 8.3 Kbp vector backbone was gel purified and ligated to the 588 bp EPO cDNA to create p18EPOcDNA (SEQ ID NO: 124).
  • 2. Genomic Erythropoietin Vector, p18genEPO. [0434]
  • The erythropoietin genomic clone was PCR amplified from a human genomic library (Clontech) using the following primers: GENEPO3BSI-CGTACGTCATCTGTCCCCT GTCCTGCA (SEQ ID NO: 85); GENEPO 5XBA-TCTAGAATGGGGGT GCACGGTGAGTACT (SEQ ID NO: 86). The reaction conditions for the amplification were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C. per cycle to 50° C.), and 3 minutes extension (72° C.); 15 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (50° C.), and 3 minute extension (72° C.); the last cycle is followed by a 7 minute extension at 72° C. The erythropoietin genomic PCR product (2147 bp) was gel purified and cloned into pCR2.1Topo to create pTopogenEPO. Sequence analysis revealed 2 bp substitutions and insertions in the intronic sequences of the genomic clone of erythropoietin. A partial digest with XbaI and complete digest with BsiWI excised the erythropoietin genomic insert from pTopogenEPO. The resulting 2158 bp genomic erythropoietin fragment was ligated into the 8.3 Kbp fragment resulting from the digestion of p18attBZeo(6XHS4)2eGFP (SEQ ID NO: 110) with XbaI and BsrGI to create p18genEPO (SEQ ID NO: 125). [0435]
  • B. Transfection and Selection with Drug [0436]
  • The erythropoietin genomic and cDNA genes were each moved onto the platform ACes B19-38 (constructed as set forth in Example 3) by co-transfecting with pCXLamIntR. Control transfections were also performed using pCXLamInt (SEQ ID NO: 127) together with either p18EPOcDNA (SEQ ID NO: 124) or p18genEPO (SEQ ID NO: 125). Lipofectamine Plus was used to transfect the DNA's into B19-38 cells according to the manufacturer's protocol. The cells were placed in selective medium (DMEM with 10% FBS and Zeocin @500 ug/ml) 48 hours post-transfection and maintained in selective medium for 13 days. Clones were isolated 15 days post-transfection. [0437]
  • C. Analysis of Clones (ELISA, PCR) [0438]
  • 1. ELISA Assays [0439]
  • Thirty clones were tested for erythropoietin production by an ELISA assay using a monoclonal anti-human erythropoietin antibody (R&D Systems, Catalogue # MAB287), a polyclonal anti-human erythropoietin antibody (R & D Systems, Catalogue # AB-286-NA) and alkaline phosphotase conjugated goat-anti-rabbit IgG (heavy and light chains) (Jackson ImmunoResearch Laboratories, Inc., Catalogue # 111-055-144). The negative control was a Zeocin resistant clone isolated from B19-38 cells transfected with p18attBZeo(6XHS4) (SEQ ID NO: 117; no insert control vector) and pCXLamIntR (SEQ ID NO: 112). The preliminary ELISA assay was executed as follows: 1) Nunc-Immuno Plates (MaxiSorb 96-well, Catalogue # 439454) were coated with 75 ul of a 1/200 dilution (in Phosphate buffered Saline, pH 7.4 (PBS), Sigma Catalogue # P-3813) of monoclonal anti-human erythropoietin antibody overnight at 4° C. 2) The following day the plates were washed 3 times with 300 ul PBS containing 0.15% Tween 20 (Sigma, Catalogue # P-9416). 3) The plates were then blocked with 300 ul of 1% Bovine Serum Albumin (BSA; Sigma Catalogue # A-7030) in PBS for 1 hour at 37° C. 4) Repeat the washes as in [0440] step 2. 5) The clonal supernatants (75 ul per clone per well of 96-well plate) were then added to the plate and incubated for 1 hour at 37° C. The clonal supernatant analyzed in the ELISA assay had been maintained on the cells 7 days prior to analysis. 6) Repeat the washes of step 2. 7) Add 75 ul of polyclonal anti-human erythropoietin antibody (1/250 dilution in dilution buffer (0.5% BSA, 0.01% Tween 20, 1× PBS, pH 7.4) and incubate 1 hour at 37° C. 8) Repeat washes of step 2. 9) Add 75 ul of goat anti-rabbit conjugated alkaline phosphatase diluted 1/4000 in dilution buffer and incubate 1 hour at 37° C. 10) Repeat washes of step 2. 11) Add 75 ul substrate, p-nitrophenyl phosphate (Sigma N2640), diluted to 1 mg/ml in substrate buffer (0.1 Ethanolamine-HCl (Sigma, Catalogue #E-6133), 5 mM MgCl2 (Sigma, Catalogue # M-2393), pH 9.8). Incubate the plates in the dark for 1 hour at room temperature (22° C.). 12) Read the absorption at 405 nm (reference wavelength 495 nm) on an Universal Microplate Reader (Bio-Tek Instruments, Inc., model # ELX800 UV). The erythropoietin standard curve was derived from readings of diluted human recombinant Erythropoietin (Roche, catalogue # 1-120-166; dilution range 125-7.8 mUnits/ml). From this preliminary assay the 21 clones displaying the highest expression of erythropoietin were analyzed a second time in the same manner using medium supernatants that had been on the clones for 24 hours and a 1:3 dilution therof.
  • 2. PCR Analysis [0441]
  • Genomic DNA was isolated from the 21 clones with the best expression (as assessed by the initial ELISA assay above) as well as the B19-38 cell line and used for PCR analysis. Genomic DNA was isolated using the Wizard genomic DNA purification kit (Promega) according to the manufacturers protocol. Amplification was performed on 10 ng of genomic DNA as template with MasterTaq DNA Polymerase (Eppendorf) and the primer set 5PacSV40-CTGTTAATTAACTGTGGAATGTGTG TCAGTTAGGGTG (SEQ ID NO: 76) and Antisense Zeo-TGAACAGGGTCACGTCGTCC (SEQ ID NO:77). The amplification conditions were as follows: heat denaturation for 3 minutes (95° C.); 30 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (from 65° C. decreasing 0.5° C. per cycle to 50° C.), and 1 minutes extension (72° C.); 15 cycles of a 30 second denaturation (95° C.), 30 seconds annealing (50° C.), and 1 minute extension (72° C.); the last cycle is followed by a 10 minute extension at 72° C. PCR products were size separated by gel electrophoresis. Of the 21 clones analyzed 19 produced a PCR product of 650 bp as expected for a site-specific integration event. All nineteen clones were the result of transformations with p19EPOcDNA (5) or p18genEPO (14) and pCXLamintR (i.e. mutant integrase). The remaining two clones, both of which were the result of transformation with p18genEPO (SEQ ID NO: 125) and pCXLamInt (i.e. wildtype integrase; SEQ ID NO: 127), produced a 400 bp PCR product. [0442]
    • EXAMPLE 12
  • Preparation of a Transformation Vector Useful for the Induction of Plant Artificial Chromosome Formation [0443]
  • Plant artificial chromosomes (PACs) can be generated by introducing nucleic acid, such as DNA, which can include a targeting DNA, for example rDNA or lambda DNA, into a plant cell, allowing the cell to grow, and then identifying from among the resulting cells those that include a chromosome with a structure that is distinct from that of any chromosome that existed in the cell prior to introduction of the nucleic acid. The structure of a PAC reflects amplification of chromosomal DNA, for example, segmented, repeat region-containing and heterochromatic structures. It is also possible to select cells that contain structures that are precursors to PACs, for example, chromosomes containing more than one centromere and/or fragments thereof, and culture and/or manipulate them to ultimately generate a PAC within the cell. [0444]
  • In the method of generating PACs, the nucleic acid can be introduced into a variety of plant cells. The nucleic acid can include targeting DNA and/or a plant expressable DNA encoding one or multiple selectable markers (e.g., DNA encoding bialophos (bar) resistance) or scorable markers (e.g., DNA encoding GFP). Examples of targeting DNA include, but are not limited to, [0445] N. tabacum rDNA intergenic spacer sequence (IGS) and Arabidopsis rDNA such as the 18S, 5.8S, 26S rDNA and/or the intergenic spacer sequence. The DNA can be introduced using a variety of methods, including, but not limited to Agrobacterium-mediated methods, PEG-mediated DNA uptake and electroporation using, for example, standard procedures according to Hartmann et al [(1998) Plant Molecular Biology 36:741]. The cell into which such DNA is introduced can be grown under selective conditions and can initially be grown under non-selective conditions and then transferred to selective media. The cells or protoplasts can be placed on plates containing a selection agent to grow, for example, individual calli. Resistant calli can be scored for scorable marker expression. Metaphase spreads of resistance cultures can be prepared, and the metaphase chromosomes examined by FISH analysis using specific probes in order to detect amplification of regions of the chromosomes. Cells that have artificial chromosomes with functioning centromeres or artificial chromosomal intermediate structures, including, but not limited to, dicentric chromosomes, formerly dicentric chromosomes, minichromosomes, heterochromatin structures (e.g. sausage chromosomes), and stable self-replicating artificial chromosomal intermediates as described herein, are identified and cultured. In particular, the cells containing self-replicating artificial chromosomes are identified.
  • The DNA introduced into a plant cell for the generation of PACs can be in any form, including in the form of a vector. An exemplary vector for use in methods of generating PACs can be prepared as follows. [0446]
  • For the production of artificial chromosomes, plant transformation vectors, as exemplified by pAgIIa and pAgIIb, containing a selectable marker, a targeting sequence, and a scorable marker were constructed using procedures well known in the art to combine the various fragments. [0447]
  • The vectors can be prepared using vector pAg1 as a base vector and inserting the following DNA fragments into pAg1: DNA encoding β-glucoronidase under the control of the nopaline synthase (NOS) promoter fragment and flanked at the 3′ end by the NOS terminator fragment, a fragment of mouse satellite DNA and an [0448] N. tabacum rDNA intergenic spacer sequence (IGS). In constructing plant transformation vectors, vector pAg2 can also be used as the base vector.
  • 1. Construction of pAG1 [0449]
  • Vector pAg1 (SEQ. ID. NO: 89) is a derivative of the CAMBIA vector named pCambia 3300 (Center for the Application of Molecular Biology to International Agriculture, i.e., CAMBIA, Canberra, Australia; www.cambia.org), which is a modified version of vector pCambia 1300 to which has been added DNA from the bar gene confering resistance to phosphinothricin. The nucleotide sequence of pCambia 3300 is provided in SEQ. ID. NO: 90. pCambia 3300 also contains a lacZ alpha sequence containing a polylinker region. [0450]
  • pAg1 was constructed by inserting two new functional DNA fragments into the polylinker of pCambia 3300: one sequence containing an attB site and a promoterless zeomycin resistance-encoding DNA flanked at the 3′ end by a SV40 polyA signal sequence, and a second sequence containing DNA from the hygromycin resistance gene (hygromycin phosphotransferase) confering resistance to hygromycin for selection in plants. Although the zeomycin-SV40 polyA signal fusion is not expected to function in plant cells, it can be activated in mammalian cells by insertion of a functional promoter element into the attB site by site-specific recombination catalyzed by the Lambda att integrase. Thus, the inclusion of the attB-zeomycin sequences allows for evaluation of functionality of plant artificial chromosomes in mammalian cells by activation of the zeomycin resistance-encoding DNA, and provides an att site for further insertion of new DNA sequences into plant artificial chromosomes formed as a result of using pAg1 for plant transformation. The second functional DNA fragment allows for selection of plant cells with hygromycin. Thus, pAg1 contains DNA from the bar gene confering resisance to phosphinothricin, DNA from the hygromycin resistance gene, both resistance-encoding DNAs under the control of a separate cauliflower mosaic virus (CaMV) 35S promoter, and the attB-promoterless zeomycin resistance-encoding DNA. [0451]
  • pAg1 is a binary vector containing Agrobacterium right and left T-DNA border sequences for use in Agrobacterium-mediated transformation of plant cells or protoplasts with the DNA located between the border sequences. pAg1 also contains the pBR322 Ori for replication in [0452] E. coli. pAg1 was constructed by ligating HindIII/PstI-digested p3300attBZeo with HindIII/PstI-digested pBSCaMV35SHyg as follows.
  • a. Generation of p3300attBZeo [0453]
  • Plasmid pCambia 3300 was digested with PstI/Ecl136 II and ligated with PstI/StuI-digested pLITattBZeo (the nucleotide sequence of pLITattBZeo is provided in SEQ. ID. NO: 91. (containing DNA encoding the zeocin resistance gene and an attB Integrase recognition sequence) to generate p3300attBZeo which contains an attB site, a promoterless zeomycin resistance-encoding DNA flanked at the 3′ end by a SV40 polyA signal, and a reconstructed PstI site. [0454]
  • b. Generation of pBSCaMV35SHyg [0455]
  • A DNA fragment containing DNA encoding hygromycin phosphotransferase flanked by the CaMV 35S promoter and the CaMV 35S polyA signal sequence was obtained by PCR amplification of plasmid pCambia 1302 (GenBank Accession No. AF234298 and SEQ. ID. NO: 92). The primers used in the amplification reaction were as follows: [0456]
    CaMV35SpolyA: SEQ. ID. NO:93
    5′-CTGAATTAACGCCGAATTAATTCGGGGGATCTG-3′
    CaMV35Spr: SEQ. ID. NO:94
    5′-CTAGAGCAGCTTGCCAACATGGTGGAGCA-3′
  • The 2100-bp PCR fragment was ligated with EcoRV-digested pBluescript 11 SK+ (Stratagene, La Jolla, Calif., U.S.A.) to generate pBSCaMV35SHyg. [0457]
  • C. Generation of pAg1 [0458]
  • To generate pAg1, pBSCaMV35SHyg was digested with HindIII/PstI and ligated with HindIII/PstI-digested p3300attBZeo. Thus, pAg1 contains the pCambia 3300 backbone with DNA conferring resistance to phophinothricin and hygromycin under the control of separate CaMV 35S promoters, an attB-promoterless zeomycin resistance-encoding DNA recombination cassette and unique sites for adding additional markers, e.g., DNA encoding GFP. The attB site can be used as decribed hereing for the addition of new DNA sequences to plant artificial chromosomes, including PACs formed as a result of using the pAg1 vector, or derivatives thereof, in the production of PACs. The attB site provides a convenient site for recombinase-mediated insertion of DNAs containing a homologous att site. [0459]
  • 2. pAG2 [0460]
  • The vector pAg2 (SEQ. ID. NO: 95) is a derivative of vector pAg1 formed by adding DNA encoding a green fluorescent protein (GFP), under the control of a NOS promoter and flanked at the 3′ end by a NOS polyA signal, to pAg1. pAg2 was constructed as follows. A DNA fragment containing the NOS promoter was obtained by digestion of pGEM-T-NOS, or pGEMEasyNOS (SEQ. ID. NO: 96), containing the NOS promoter in the cloning vector pGEM-T-Easy (Promega Biotech, Madison, Wis., U.S.A.), with XbaI/NcoI and was ligated to an XbaI/NcoI fragment of pCambia 1302 containing DNA encoding GFP (without the CaMV 35S promoter) to generate p1302NOS (SEQ. ID. NO: 97) containing GFP-encoding DNA in operable association with the NOS promoter. Plasmid p1302NOS was digested with SmaI/BsiWI to yield a fragment containing the NOS promoter and GFP-encoding DNA. The fragment was ligated with PmeI/BsiWI-digested pAg1 to generate pAg2. Thus, pAg2 contains DNA from the bar gene confering resistance to phosphinothricin, DNA conferring resistance to hygromycin, both resistance-encoding DNAs under the control of a cauliflower mosaic virus 35S promoter, DNA encoding kanamycin resistance, a GFP gene under the control of a NOS promoter and the attB-zeomycin resistance-encoding DNA. One of skill in the art will appreciate that other fragments can be used to generate the pAg1 and pAg2 derivatives and that other heterlogous DNA can be incorporated into pAg1 and pAg2 derivatives using methods well known in the art. [0461]
  • 3. pAgIIa and pAgIIb transformation vectors Vectors pAgIIa and pAgIIb were constructed by inserting the following DNA fragments into pAgI: DNA encoding 8-glucoronidase, the nopaline synthase terminator fragment, the nopaline synthase (NOS) promoter fragment, a fragment of mouse satellite DNA and an [0462] N. tabacum rDNA intergenic spacer sequence (IGS). The construction of pAgIIa and pAgIIb was as follows.
  • An [0463] N. tabacum rDNA intergenic spacer (IGS) sequence (SEQ. ID. NO: 98; see also GenBank Accession No. Y08422; see also Borysyuk et al. (2000) Nature Biotechnology 18:1303-1306; Borysyuk et al. (1997) Plant Mol. Biol. 35:655-660; U.S. Pat. Nos. 6,100,092 and 6,355,860) was obtained by PCR amplification of tobacco genomic DNA. The IGS can be used as a targeting sequence by virtue of its homology to tobacco rDNA genes; the sequence is also an amplification promoter sequence in plants. This fragment was amplified using standard PCR conditions (e.g., as described by Promega Biotech, Madison, Wis., U.S.A.) from tobacco genomic DNA using the primers shown below:
    NTIGS-FI (SEQ ID No.99)
    5′-GTG CTA GCC AAT GTT TAA CAA GAT G-3′
    and
    NTIGS-RI (SEQ ID No.100)
    5′-ATG TCT TAA AAA AAA AAA CCC AAG TGA C-3′
  • Following amplification, the fragment was cloned into pGEM-T Easy to give pIGS-I A fragment of mouse satellite DNA (Msatl fragment; GenBank Accession No. V00846; and SEQ ID No. 101) was amplified via PCR from pSAT-1 using the following primers: [0464]
    MSAT-F1 (SEQ ID No.102)
    5′-AAT ACC GCG GAA GCT TGA CCT GGA ATA TCG C-3′
    and
    MSAT-Ri (SEQ ID No.103)
    5′-ATA ACC GCG GAG TCC TTC AGT GTG CA T-3′
  • This amplification added a SacII and a HindIII site at the 5′end and a SacII site at the 3′ end of the PCR fragment. This fragment was then cloned into the SacII site in pIGS-1 to give pMIGS-1, providing a eukaryotic centromere-specific DNA and a convenient DNA sequence for detection via FISH. [0465]
  • A functional marker gene containing a NOS-promoter:GUS:NOS terminator fusion was then constructed containing the NOS promoter (GenBank Accession No. U09365; SEQ ID No. 104), [0466] E. coli β-glucuronidase coding sequence (from the GUS gene; GenBank Accession No. S69414; and SEQ ID No. 105), and the nopaline synthase terminator sequence (GenBank Accession No. U09365; SEQ ID No. 107). The NOS promoter in pGEM-T-NOS was added to a promoterless GUS gene in pBlueScript (Stratagene, La Jolla, Calif., U.S.A.) using NotI/SpeI to form pNGN-1, which has the NOS promoter in the opposite orientation relative to the GUS gene.
  • pMIGS-1 was digested with NotI/SpeI to yield a fragment containing the mouse major satellite DNA and the tobacco IGS which was then added to NotI-digested pNGN-1 to yield pNGN-2. The NOS promoter was then re-oriented to provide a functional GUS gene, yielding pNGN-3, by digestion and religation with SpeI. Plasmid pNGN-3 was then digested with HindIII, and the HindIII fragment containing the β-glucuronidase coding sequence and the rDNA intergenic spacer, along with the Msat sequence, was added to pAG-1 to form pAgIIa (SEQ ID NO: 108), using the unique HindIII site in pAgI located near the right T-DNA border of pAgI, within the T-DNA region. [0467]
  • Another plasmid vector, referred to as pAgIIb, was also recovered, which contained the inserted HindIII fragment (SEQ ID NO: 108) in the opposite orientation relative to that observed in pAgIIa. Thus, pAgIIa and pAgIIb differ only in the orientation of the HindIII fragment containing the mouse major satellite sequence, the GUS DNA sequence and the IGS sequence. The nucleotide sequences of pAgIIa is provided in SEQ. ID. NOS: 109. [0468]
  • Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims. [0469]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 129
    <210> SEQ ID NO 1
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer: attPUP
    <400> SEQUENCE: 1
    ccttgcgcta atgctctgtt acagg 25
    <210> SEQ ID NO 2
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer: attPDWN
    <400> SEQUENCE: 2
    cagaggcagg gagtgggaca aaattg 26
    <210> SEQ ID NO 3
    <211> LENGTH: 35
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer: Lamint 1
    <400> SEQUENCE: 3
    ttcgaattca tgggaagaag gcgaagtcat gagcg 35
    <210> SEQ ID NO 4
    <211> LENGTH: 34
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer: Lamint 2
    <400> SEQUENCE: 4
    ttcgaattct tatttgattt caattttgtc ccac 34
    <210> SEQ ID NO 5
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer
    <400> SEQUENCE: 5
    cggacaatgc ggttgtgcgt 20
    <210> SEQ ID NO 6
    <211> LENGTH: 46
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: primer
    <400> SEQUENCE: 6
    cgcgcagcaa aatctagagt aaggagatca agacttacgg ctgacg 46
    <210> SEQ ID NO 7
    <211> LENGTH: 46
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: LambdaINTER174rev
    <400> SEQUENCE: 7
    cgtcagccgt aagtcttgat ctccttactc tagattttgc tgcgcg 46
    <210> SEQ ID NO 8
    <211> LENGTH: 33
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB1
    <400> SEQUENCE: 8
    tgaagcctgc ttttttatac taacttgagc gaa 33
    <210> SEQ ID NO 9
    <211> LENGTH: 33
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB2
    <400> SEQUENCE: 9
    ttcgctcaag ttagtataaa aaagcaggct tca 33
    <210> SEQ ID NO 10
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer: attPdwn2
    <400> SEQUENCE: 10
    tcttctcggg cataagtcgg acacc 25
    <210> SEQ ID NO 11
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:CMVen
    <400> SEQUENCE: 11
    ctcacgggga tttccaagtc tccac 25
    <210> SEQ ID NO 12
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:attPdwn
    <400> SEQUENCE: 12
    cagaggcagg gagtgggaca aaattg 26
    <210> SEQ ID NO 13
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:CMVEN2
    <400> SEQUENCE: 13
    caactccgcc ccattgacgc aaatg 25
    <210> SEQ ID NO 14
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:L1
    <400> SEQUENCE: 14
    agtatcgccg aacgattagc tcttca 26
    <210> SEQ ID NO 15
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:F1 rev
    <400> SEQUENCE: 15
    gccgatttcg gcctattggt taaa 24
    <210> SEQ ID NO 16
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:RED
    <400> SEQUENCE: 16
    ccgccgacat ccccgactac aagaa 25
    <210> SEQ ID NO 17
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer:L2rev
    <400> SEQUENCE: 17
    ttccttcgaa ggggatccgc ctacc 25
    <210> SEQ ID NO 18
    <211> LENGTH: 22118
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank X82564
    <309> DATABASE ENTRY DATE: 1996-04-09
    <400> SEQUENCE: 18
    gaattcccct atccctaatc cagattggtg gaataacttg gtatagatgt ttgtgcatta 60
    aaaaccctgt aggatcttca ctctaggtca ctgttcagca ctggaacctg aattgtggcc 120
    ctgagtgata ggtcctggga catatgcagt tctgcacaga cagacagaca gacagacaga 180
    cagacagaca gacagacgtt acaaacaaac acgttgagcc gtgtgccaac acacacacaa 240
    acaccactct ggccataatt attgaggacg ttgatttatt attctgtgtt tgtgagtctg 300
    tctgtctgtc tgtctgtctg tctgtctgtc tatcaaacca aaagaaacca aacaattatg 360
    cctgcctgcc tgcctgcctg cctacacaga gaaatgattt cttcaatcaa tctaaaacga 420
    cctcctaagt ttgccttttt tctctttctt tatctttttc ttttttcttt tcttcttcct 480
    tccttccttc cttccttcct tccttccttt ctttctttct ttctttcttt cttactttct 540
    ttctttcctt cttacattta ttcttttcat acatagtttc ttagtgtaag catccctgac 600
    tgtcttgaag acactttgta ggcctcaatc ctgtaagagc cttcctctgc ttttcaaatg 660
    ctggcatgaa tgttgtacct cactatgacc agcttagtct tcaagtctga gttactggaa 720
    aggagttcca agaagactgg ttatattttt catttattat tgcattttaa ttaaaattta 780
    atttcaccaa aagaatttag actgaccaat tcagagtctg ccgtttaaaa gcataaggaa 840
    aaagtaggag aaaaacgtga ggctgtctgt ggatggtcga ggctgcttta gggagcctcg 900
    tcaccattct gcacttgcaa accgggccac tagaacccgg tgaagggaga aaccaaagcg 960
    acctggaaac aataggtcac atgaaggcca gccacctcca tcttgttgtg cgggagttca 1020
    gttagcagac aagatggctg ccatgcacat gttgtctttc agcttggtga ggtcaaagta 1080
    caaccgagtc acagaacaag gaagtataca cagtgagttc caggtcagcc agagtttaca 1140
    cagagaaacc acatcttgaa aaaaacaaaa aaataaatta aataaatata atttaaaaat 1200
    ttaaaaatag ccgggagtga tggcgcatgt ctttaatccc agctctcttc aggcagagat 1260
    gggaggattt ctgagtttga ggccagcctg gtctgcaaag tgagttccag gacagtcagg 1320
    gctatacaga gaaaccctgt cttgaaaact aaactaaatt aaactaaact aaactaaaaa 1380
    aatataaaat aaaaatttta aagaatttta aaaaactaca gaaatcaaac ataagcccac 1440
    gagatggcaa gtaactgcaa tcatagcaga aatattatac acacacacac acacagactc 1500
    tgtcataaaa tccaatgtgc cttcatgatg atcaaatttc gatagtcagt aatactagaa 1560
    gaatcatatg tctgaaaata aaagccagaa ccttttctgc ttttgttttc ttttgcccca 1620
    agatagggtt tctctcagtg tatccctggc atccctgcct ggaacttcct ttgtaggttt 1680
    ggtagcctca aactcagaga ggtcctctct gcctgcctgc ctgcctgcct gcctgcctgc 1740
    ctgcctgcct gcctgcctca cttcttctgc cacccacaca accgagtcga acctaggatc 1800
    tttatttctt tctctttctc tcttctttct ttctttcttt ctttctttct ttctttcttt 1860
    ctttctttct ttcttattca attagttttc aatgtaagtg tgtgtttgtg ctctatctgc 1920
    tgcctatagg cctgcttgcc aggagagggc aacagaacct aggagaaacc accatgcagc 1980
    tcctgagaat aagtgaaaaa acaacaaaaa aaggaaattc taatcacata gaatgtagat 2040
    atatgccgag gctgtcagag tgctttttaa ggcttagtgt aagtaatgaa aattgttgtg 2100
    tgtcttttat ccaaacacag aagagaggtg gctcggcctg catgtctgtt gtctgcatgt 2160
    agaccaggct ggccttgaac acattaatct gtctgcctct gcttccctaa tgctgcgatt 2220
    aaaggcatgt gccaccactg cccggactga tttcttcttt tttttttttt tggaaaatac 2280
    ctttctttct ttttctctct ctctttcttc cttccttcct ttctttctat tctttttttc 2340
    tttctttttt cttttttttt ttttttttaa aatttgccta aggttaaagg tgtgctccac 2400
    aattgcctca gctctgctct aattctcttt aaaaaaaaac aaacaaaaaa aaaaccaaaa 2460
    cagtatgtat gtatgtatat ttagaagaaa tactaatcca ttaataactc ttttttccta 2520
    aaattcatgt cattcttgtt ccacaaagtg agttccagga cttaccagag aaaccctgtg 2580
    ttcaaatttc tgtgttcaag gtcaccctgg cttacaaagt gagttccaag tccgataggg 2640
    ctacacagaa aaaccatatc tcagaaaaaa aaaaagttcc aaacacacac acacacacac 2700
    acacacacac acacacacac acacacacac acacacacag cgcgccgcgg cgatgagggg 2760
    aagtcgtgcc taaaataaat atttttctgg ccaaagtgaa agcaaatcac tatgaagagg 2820
    tactcctaga aaaaataaat acaaacgggc tttttaatca ttccagcact gttttaattt 2880
    aactctgaat ttagtcttgg aaaagggggc gggtgtgggt gagtgagggc gagcgagcag 2940
    acgggcgggc gggcgggtga gtggccggcg gcggtggcag cgagcaccag aaaacaacaa 3000
    accccaagcg gtagagtgtt ttaaaaatga gacctaaatg tggtggaacg gaggtcgccg 3060
    ccaccctcct cttccactgc ttagatgctc ccttcccctt actgtgctcc cttcccctaa 3120
    ctgtgcctaa ctgtgcctgt tccctcaccc cgctgattcg ccagcgacgt actttgactt 3180
    caagaacgat tttgcctgtt ttcaccgctc cctgtcatac tttcgttttt gggtgcccga 3240
    gtctagcccg ttcgctatgt tcgggcggga cgatggggac cgtttgtgcc actcgggaga 3300
    agtggtgggt gggtacgctg ctccgtcgtg cgtgcgtgag tgccggaacc tgagctcggg 3360
    agaccctccg gagagacaga atgagtgagt gaatgtggcg gcgcgtgacg gatctgtatt 3420
    ggtttgtatg gttgatcgag accattgtcg ggcgacacct agtggtgaca agtttcggga 3480
    acgctccagg cctctcaggt tggtgacaca ggagagggaa gtgcctgtgg tgaggcgacc 3540
    agggtgacag gaggccgggc aagcaggcgg gagcgtctcg gagatggtgt cgtgtttaag 3600
    gacggtctct aacaaggagg tcgtacaggg agatggccaa agcagaccga gttgctgtac 3660
    gcccttttgg gaaaaatgct agggttggtg gcaacgttac taggtcgacc agaaggctta 3720
    agtcctaccc ccccccccct tttttttttt tttcctccag aagccctctc ttgtccccgt 3780
    caccgggggc accgtacatc tgaggccgag aggacgcgat gggcccggct tccaagccgg 3840
    tgtggctcgg ccagctggcg cttcgggtct tttttttttt tttttttttt ttttcctcca 3900
    gaagccttgt ctgtcgctgt caccgggggc gctgtacttc tgaggccgag aggacgcgat 3960
    gggccccggc ttccaagccg gtgtggctcg gccagctgga gcttcgggtc tttttttttt 4020
    tttttttttt tttttttctc cagaagcctt gtctgtcgct gtcaccgggg gcgctgtact 4080
    tctgaggccg agaggacgcg atgggtcggc ttccaagccg atgtggcggg gccagctgga 4140
    gcttcgggtt tttttttttc ctccagaagc cctctcttgt ccccgtcacc gggggcgctg 4200
    tacttctgag gccgagagga cgtgatgggc ccgggttcca ggcggatgtc gcccggtcag 4260
    ctggagcttt ggatcttttt tttttttttt cctccagaag ccctctcttg tccccgtcac 4320
    cgggggcacc ttacatctga gggcgagagg acgtgatggg tccggcttcc aagccgatgt 4380
    ggcggggcca gctggagctt cgggtttttt ttttttcctc cagaagccct ctcttgtccc 4440
    cgtcaccggg ggcgctgtac ttctgaggcc gagaggacgt gatgggcccg ggttccaggc 4500
    ggatgtcgcc cggtcagctg gagctttgga tcattttttt ttttccctcc agaagccctc 4560
    tcttgtcccc gtcaccgggg gcaccgtaca tctgaggccg agaggacacg atgggcctgt 4620
    cttccaagcc gatgtggccc ggccagctgg agcttcgggt cttttttttt ttttttcctc 4680
    cagaagcctt gtctgtcgct gtcacccggg gcgctgtact tctgaggccg agaggacgcg 4740
    atgggcccgg cttccaagcc ggtgtggctc ggccagctgg agcttcgggt cttttttttt 4800
    tttttttttt ttcctccaga aaccttgtct gtcgctgtca cccggggcgc ttgtacttct 4860
    gatgccgaga ggacgcgatg ggcccgtctt ccaggccgat gtggcccggt cagctggagc 4920
    tttggatctt tttttttttt ttttcctcca gaagccctct cttgtccccg tcaccggggg 4980
    caccttacat ctgaggccta gaggacacga tgggcccggg ttccaggccg atgtggcccg 5040
    gtcagctgga gctttggatc tttttttttt ttttcttcca gaagccctct tgtccccgtc 5100
    accggtggca ctgtacatct gaggcggaga ggacattatg ggcccggctt ccaatccgat 5160
    gtggcccggt cagctggagc tttggatctt attttttttt taattttttc ttccagaagc 5220
    cctcttgtcc ctgtcaccgg tggcacggta catctgaggc cgagaggaca ttatgggccc 5280
    ggcttccagg ccgatgtggc ccggtcagct ggagctttgg atcttttttt ttttttttct 5340
    tttttcctcc agaagccctc tctgtccctg tcaccggggg ccctgtacgt ctgaggccga 5400
    gggaaagcta tgggcgcggt tttctttcat tgacctgtcg gtcttatcag ttctccgggt 5460
    tgtcagggtc gaccagttgt tcctttgagg tccggttctt ttcgttatgg ggtcattttt 5520
    gggccacctc cccaggtatg acttccaggc gtcgttgctc gcctgtcact ttcctccctg 5580
    tctcttttat gcttgtgatc ttttctatct gttcctattg gacctggaga taggtactga 5640
    cacgctgtcc tttccctatt aacactaaag gacactataa agagaccctt tcgatttaag 5700
    gctgttttgc ttgtccagcc tattcttttt actggcttgg gtctgtcgcg gtgcctgaag 5760
    ctgtccccga gccacgcttc ctgctttccc gggcttgctg cttgcgtgtg cttgctgtgg 5820
    gcagcttgtg acaactgggc gctgtgactt tgctgcgtgt cagacgtttt tcccgatttc 5880
    cccgaggtgt cgttgtcaca cctgtcccgg ttggaatggt ggagccagct gtggttgagg 5940
    gccaccttat ttcggctcac tttttttttt tttttttctc ttggagtccc gaacctccgc 6000
    tcttttctct tcccggtctt tcttccacat gcctcccgag tgcatttctt tttgtttttt 6060
    ttcttttttt tttttttttt ttggggaggt ggagagtccc gagtacttca ctcctgtctg 6120
    tggtgtccaa gtgttcatgc cacgtgcctc ccgagtgcac ttttttttgt ggcagtcgct 6180
    cgttgtgttc tcttgttctg tgtctgcccg tatcagtaac tgtcttgccc cgcgtgtaag 6240
    acattcctat ctcgcttgtt tctcccgatt gcgcgtcgtt gctcactctt agatcgatgt 6300
    ggtgctccgg agttctcttc gggccagggc caagccgcgc caggcgaggg acggacattc 6360
    atggcgaatg gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420
    cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480
    cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540
    tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600
    ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660
    gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720
    gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780
    tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840
    tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900
    ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960
    gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020
    aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080
    ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140
    gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200
    ggaaagggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260
    ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320
    ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380
    ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440
    gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggacggggtg 7500
    tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560
    tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620
    ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680
    ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740
    cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800
    gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860
    gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920
    cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980
    ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040
    tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100
    gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160
    ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220
    acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280
    cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340
    gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400
    gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460
    agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520
    ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580
    accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640
    gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700
    cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760
    catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820
    tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880
    ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940
    gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000
    gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtcagttgg gccctggcgt 9060
    tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120
    gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180
    tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240
    tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300
    cggcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360
    gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420
    gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480
    tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540
    cctcccgcga ggctctccgc cgccgccgcc tcctcctcct ctctcgcgct ctctgtcccg 9600
    cctggtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660
    ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720
    agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780
    ctacttggat aactgtggta attctagagc taatacatgc cgacgggcgc tgacccccct 9840
    tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900
    ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960
    acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020
    tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080
    gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140
    cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200
    ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260
    cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320
    tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380
    ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440
    gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500
    aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560
    taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620
    gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680
    tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740
    cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800
    ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860
    ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920
    cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980
    ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040
    ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100
    gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160
    tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220
    aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280
    gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340
    accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400
    ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460
    agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520
    ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgggagg 11580
    cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640
    gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700
    tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760
    gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820
    ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880
    cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940
    ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000
    gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060
    ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120
    cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180
    cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240
    cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300
    gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360
    cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420
    cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480
    cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540
    tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600
    catcgacact tcgaacgcac ttgcggcccc gggttcctcc cggggctacg cctgtctgag 12660
    cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720
    ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780
    gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840
    cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900
    gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960
    tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcg cgtccgcatc tgctctggtc 13020
    gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080
    ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140
    gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200
    ggcgccgcgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260
    gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320
    cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380
    cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440
    ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500
    gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560
    ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccacccc cgccgccctc 13620
    ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680
    gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740
    tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800
    attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860
    gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgt gggaaatgtg gcgtacggaa 13920
    gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980
    tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcgggtcttc ccggagtcgg 14040
    gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100
    cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160
    tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220
    gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280
    ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340
    gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcg cgcgggcggg gccgggggtg 14400
    gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460
    ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggc ccggtgggga aggtggctcg 14520
    gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580
    ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640
    ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg ggccgggccg cccctcccac 14700
    ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760
    cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820
    gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880
    cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940
    gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000
    cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060
    aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120
    cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180
    cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240
    tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300
    aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360
    agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420
    ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480
    gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540
    gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600
    aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660
    ggacgggagc ggccgcgggt gcgcgtctct cggggtcggg ggtgcgtggc gggggcccgt 15720
    cccccgcctc ccctccgcgc gccgggttcg cccccgcggc gtcgggcccc gcggagccta 15780
    cgccgcgacg agtaggaggg ccgctgcggt gagccttgaa gcctagggcg cgggcccggg 15840
    tggagccgcc gcaggtgcag atcttggtgg tagtagcaaa tattcaaacg agaactttga 15900
    aggccgaagt ggagaagggt tccatgtgaa cagcagttga acatgggtca gtcggtcctg 15960
    agagatgggc gagtgccgtt ccgaagggac gggcgatggc ctccgttgcc ctcggccgat 16020
    cgaaagggag tcgggttcag atccccgaat ccggagtggc ggagatgggc gccgcgaggc 16080
    cagtgcggta acgcgaccga tcccggagaa gccggcggga ggcctcgggg agagttctct 16140
    tttctttgtg aagggcaggg cgccctggaa tgggttcgcc ccgagagagg ggcccgtgcc 16200
    ttggaaagcg tcgcggttcc ggcggcgtcc ggtgagctct cgctggccct tgaaaatccg 16260
    ggggagaggg tgtaaatctc gcgccgggcc gtacccatat ccgcagcagg tctccaaggt 16320
    gaacagcctc tggcatgttg gaacaatgta ggtaagggaa gtcggcaagc cggatccgta 16380
    acttcgggat aaggattggc tctaagggct gggtcggtcg ggctggggcg cgaagcgggg 16440
    ctgggcgcgc gccgcggctg gacgaggcgc cgccgccctc tcccacgtcc ggggagaccc 16500
    cccgtccttt ccgcccgggc ccgccctccc ctcttccccg cggggccccg tcgtcccccg 16560
    cgtcgtcgcc acctctcttc ccccctcctt cttcccgtcg gggggcgggt cgggggtcgg 16620
    cgcgcggcgc gggctccggg gcggcgggtc caaccccgcg ggggttccgg agcgggagga 16680
    accagcggtc cccggtgggg cggggggccc ggacactcgg ggggccggcg gcggcggcga 16740
    ctctggacgc gagccgggcc cttcccgtgg atcgcctcag ctgcggcggg cgtcgcggcc 16800
    gctcccgggg agcccggcgg gtgccggcgc gggtcccctc cccgcggggc ctcgctccac 16860
    ccccccatcg cctctcccga ggtgcgtggc gggggcgggc gggcgtgtcc cgcgcgtgtg 16920
    gggggaacct ccgcgtcggt gttcccccgc cgggtccgcc ccccgggccg cggttttccg 16980
    cgcggcgccc ccgcctcggc cggcgcctag cagccgactt agaactggtg cggaccaggg 17040
    gaatccgact gtttaattaa aacaaagcat cgcgaaggcc cgcggcgggt gttgacgcga 17100
    tgtgatttct gcccagtgct ctgaatgtca aagtgaagaa attcaatgaa gcgcgggtaa 17160
    acggcgggag taactatgac tctcttaagg tagccaaatg cctcgtcatc taattagtga 17220
    cgcgcatgaa tggatgaacg agattcccac tgtccctacc tactatccag cgaaaccaca 17280
    gccaagggaa cgggcttggc ggaatcagcg gggaaagaag accctgttga gcttgactct 17340
    agtctggcac ggtgaagaga catgagaggt gtagaataag tgggaggccc ccggcgcccg 17400
    gccccgtcct cgcgtcgggg tcggggcacg ccggcctcgc gggccgccgg tgaaatacca 17460
    ctactctcat cgttttttca ctgacccggt gaggcggggg ggcgagcccc gaggggctct 17520
    cgcttctggc gccaagcgtc cgtcccgcgc gtgcgggcgg gcgcgacccg ctccggggac 17580
    agtgccaggt ggggagtttg actggggcgg tacacctgtc aaacggtaac gcaggtgtcc 17640
    taaggcgagc tcagggagga cagaaacctc ccgtggagca gaagggcaaa agctcgcttg 17700
    atcttgattt tcagtacgaa tacagaccgt gaaagcgggg cctcacgatc cttctgacct 17760
    tttgggtttt aagcaggagg tgtcagaaaa gttaccacag ggataactgg cttgtggcgg 17820
    ccaagcgttc atagcgacgt cgctttttga tccttcgatg tcggctcttc ctatcattgt 17880
    gaagcagaat tcaccaagcg ttggattgtt cacccactaa tagggaacgt gagctgggtt 17940
    tagaccgtcg tgagacaggt tagttttacc ctactgatga tgtgttgttg ccatggtaat 18000
    cctgctcagt acgagaggaa ccgcaggttc agacatttgg tgtatgtgct tggctgagga 18060
    gccaatgggg cgaagctacc atctgtggga ttatgactga acgcctctaa gtcagaatcc 18120
    gcccaagcgg aacgatacgg cagcgccgaa ggagcctcgg ttggccccgg atagccgggt 18180
    ccccgtccgt cccgctcggc ggggtccccg cgtcgccccg cggcggcgcg gggtctcccc 18240
    ccgccgggcg tcgggaccgg ggtccggtgc ggagagccgt tcgtcttggg aaacggggtg 18300
    cggccggaaa gggggccgcc ctctcgcccg tcacgttgaa cgcacgttcg tgtggaacct 18360
    ggcgctaaac cattcgtaga cgacctgctt ctgggtcggg gtttcgtacg tagcagagca 18420
    gctccctcgc tgcgatctat tgaaagtcag ccctcgacac aagggtttgt ctctgcgggc 18480
    tttcccgtcg cacgcccgct cgctcgcacg cgaccgtgtc gccgcccggg cgtcacgggg 18540
    gcggtcgcct cggcccccgc gcggttgccc gaacgaccgt gtggtggttg ggggggggat 18600
    cgtcttctcc tccgtctccc gaggacggtt cgtttctctt tccccttccg tcgctctcct 18660
    tgggtgtggg agcctcgtgc cgtcgcgacc gcggcctgcc gtcgcctgcc gccgcagccc 18720
    cttgccctcc ggccttggcc aagccggagg gcggaggagg gggatcggcg gcggcggcga 18780
    ccgcggcgcg gtgacgcacg gtgggatccc catcctcggc gcgtccgtcg gggacggccg 18840
    gttggagggg cgggaggggt ttttcccgtg aacgccgcgt tcggcgccag gcctctggcg 18900
    gccggggggg cgctctctcc gcccgagcat ccccactccc gcccctcctc ttcgcgcgcc 18960
    gcggcggcga cgtgcgtacg aggggaggat gtcgcggtgt ggaggcggag agggtccggc 19020
    gcggcgcctc ttccattttt tcccccccaa cttcggaggt cgaccagtac tccgggcgac 19080
    actttgtttt ttttttttcc cccgatgctg gaggtcgacc agatgtccga aagtgtcccc 19140
    cccccccccc ccccccggcg cggagcggcg gggccactct ggactctttt tttttttttt 19200
    tttttttttt ttaaattcct ggaaccttta ggtcgaccag ttgtccgtct tttactcctt 19260
    catataggtc gaccagtact ccgggtggta ctttgtcttt ttctgaaaat cccagaggtc 19320
    gaccagatat ccgaaagtcc tctctttccc tttactcttc cccacagcga ttctcttttt 19380
    tttttttttt tttggtgtgc ctctttttga cttatataca tgtaaatagt gtgtacgttt 19440
    atatacttat aggaggaggt cgaccagtac tccgggcgac actttgtttt tttttttttt 19500
    tccaccgatg atggaggtcg accagatgtc cgaaagtgtc ccgtcccccc cctccccccc 19560
    ccgcgacgcg gcgggctcac tctggactct tttttttttt tttttttttt tttaaatttc 19620
    tggaacctta aggtcgacca gttgtccgtc tttcactcat tcatataggt cgaccggtgg 19680
    tactttgtct ttttctgaaa atcgcagagg tcgaccagat gtcagaaagt ctggtggtcg 19740
    ataaattatc tgatctagat ttgtttttct gtttttcagt tttgtgttgt tttgtgttgt 19800
    tttgtgttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttgttttgt 19860
    tttgtgttgt gttgtgttgt gttgtgttgg gttgggttgg gttgggttgg gttgggttgg 19920
    gttgggttgg gttgggttgt gttgtttggt tttgtgttgt ttggtgttgt tggttttgtt 19980
    ttgtttgctg ttgttttgtg ttttgcgggt cgaacagttg tccctaaccg agtttttttg 20040
    tacacaaaca tgcacttttt ttaaaataaa tttttaaaat aaatgcgaaa atcgaccaat 20100
    tatccctttc cttctctctc ttttttaaaa attttctttg tgtgtgtgtg tgtgtgtgtg 20160
    tgtgtgtgtg tgcgtgtgtg tgtgtgtgtg cgtgcagcgt gcgcgcgctc gttttataaa 20220
    tacttataat aataggtcgc cgggtggtgg tagcttcccg gactccagag gcagaggcag 20280
    gcagacttct gagttcgagg ccagcctggt ctacagagga accctgtctc gaaaaatgaa 20340
    aataaataca tacatacata catacataca tacatacata catacataca tacatatgag 20400
    gttgaccagt tgtcaatcct ttagaatttt gtttttaatt aatgtgatag agagatagat 20460
    aatagataga tggatagagt gatacaaata taggtttttt tttcagtaaa tatgaggttg 20520
    attaaccact tttccctttt taggtttttt tttttttccc ctgtccatgt ggttgctggg 20580
    atttgaactc aggaccctgg caggtcaact ggaaaacgtg ttttctatat atataaatag 20640
    tggtctgtct gctgtttgtt tgtttgcttg cttgcttgct tgcttgcttg cttgcttgct 20700
    tgcttttttt tttcttctga gacagtattt ctctgtgtaa cctggtgccc tgaaactcac 20760
    tctgtagacc agcctggcct caatcgaact cagaaatcct cctgcctctt gtctacctcc 20820
    caattttgga gtaaaggtgt gctacaccac tgcctggcat tattatcatt atcattatta 20880
    attttattat tagacagaac gaaatcaact agttggtcct gtttcgttaa ttcatttgaa 20940
    attagttgga ccaattagtt ggctggtttg ggaggtttct tttgtttccg atttgggtgt 21000
    ttgtggggct ggggatcagg tatctcaacg gaatgcatga aggttaaggt gagatggctc 21060
    gatttttgta aagattactt ttcttagtct gaggaaaaaa taaaataata ttgggctacg 21120
    tttcattgct tcatttctat ttctctttct ttctttcttt ctttcagata aggaggtcgg 21180
    ccagttcctc ctgccttctg gaagatgtag gcattgcatt gggaaaagca ttgtttgaga 21240
    gatgtgctag tgaaccagag agtttggatg tcaagccgta taatgtttat tacaatatag 21300
    aaaagttcta acaaagtgat ctttaacttt tttttttttt tttctccttc tacttctact 21360
    tgttctcact ctgccaccaa cgcgctttgt acattgaatg tgagctttgt tttgcttaac 21420
    agacatatat tttttctttt ggttttgctt gacatggttt ccctttctat ccgtgcaggg 21480
    ttcccagacg gccttttgag aataaaatgg gaggccagaa ccaaagtctt ttgaataaag 21540
    caccacaact ctaacctgtt tggctgtttt ccttcccaag gcacagatct ttcccagcat 21600
    ggaaaagcat gtagcagttg taggacacac tagacgagag caccagatct cattgtgggt 21660
    ggttgtgaac cacccaccat gtggttgcct gggatttgaa ctcaggatct tcagaagacg 21720
    agtcagggct ctaaaccgat gagccatctc tccagccctc ctacattcct tcttaaggca 21780
    tgaatgatcc cagcatggga agacagtctg ccctctttgt ggtatatcac catatactca 21840
    ataaaataat gaaatgaatg aagtctccac gtatttattt cttcgagcta tctaaattct 21900
    ctcacagcac ctccccctcc cccacactgc ctttctccct atgtttgggt ggggctgggg 21960
    gaggggtggg gtgggggcag ggatctgcat gtcttcttgc aggtctgtga actatttgcg 22020
    atggcctggt tctctgaact gttgagcctt gtctatccag aggctgactg gctagttttc 22080
    tacctgaagt ccctgagtga tgatttccct gtgaattc 22118
    <210> SEQ ID NO 19
    <211> LENGTH: 175
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 19
    ctcccgcgcg gcccccgtgt tcgccgttcc cgtggcgcgg acaatgcggt tgtgcgtcca 60
    cgtgtgcgtg tccgtgcagt gccgttgtgg agtgcctcgc tctcctcctc ctccccggca 120
    gcgttcccac ggttggggac caccggtgac ctcgccctct tcgggcctgg atccg 175
    <210> SEQ ID NO 20
    <211> LENGTH: 755
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 20
    ggtctggtgg gaattgttga cctcgctctc gggtgcggcc tttggggaac ggcggggtcg 60
    gtcgtgcccg gcgccggacg tgtgtcgggg cccacttccc gctcgagggt ggcggtggcg 120
    gcggcgttgg tagtctcccg tgttgcgtct tcccgggctc ttgggggggg tgccgtcgtt 180
    ttcggggccg gcgttgcttg gcttacgcag gcttggtttg ggactgcctc aggagtcgtg 240
    ggcggtgtga ttcccgccgg ttttgcctcg cgtctgcctg ctttgcctcg ggtttgcttg 300
    gttcgtgtct cgggagcggt ggtttttttt tttttcgggt cccggggaga ggggtttttc 360
    cgggggacgt tcccgtcgcc ccctgccgcc ggtgggtttt cgtttcgggc tgtgttcgtt 420
    tccccttccc cgtttcgccg tcggttctcc ccggtcggtc ggccctctcc ccggtcggtc 480
    gcccggccgt gctgccggac ccccccttct gggggggatg cccgggcacg cacgcgtccg 540
    ggcggccact gtggtccggg agctgctcgg caggcgggtg agccagttgg aggggcgtca 600
    tgcccccgcg ggctcccgtg gccgacgcgg cgtgttcttt gggggggcct gtgcgtgcgg 660
    gaaggctgcg cacgttgtcg gtccttgcga gggaaagagg cttttttttt ttagggggtc 720
    gtccttcgtc gtcccgtcgg cggtggatcc ggcct 755
    <210> SEQ ID NO 21
    <211> LENGTH: 463
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 21
    ggccgaggtg cgtctgcggg ttggggctcg tccggccccg tcgtcctccg ggaaggcgtt 60
    tagcgggtac cgtcgccgcg ccgaggtggg cgcacgtcgg tgagataacc ccgagcgtgt 120
    ttctggttgt tggcggcggg ggctccggtc gatgtcttcc cctccccctc tccccgaggc 180
    caggtcagcc tccgcctgtg ggcttcgtcg gccgtctccc cccccctcac gtccctcgcg 240
    agcgagcccg tccgttcgac cttccttccg ccttcccccc atctttccgc gctccgttgg 300
    ccccggggtt ttcacggcgc cccccacgct cctccgcctc tccgcccgtg gtttggacgc 360
    ctggttccgg tctccccgcc aaaccccggt tgggttggtc tccggccccg gcttgctctt 420
    cgggtctccc aacccccggc cggaagggtt cgggggttcc ggg 463
    <210> SEQ ID NO 22
    <211> LENGTH: 378
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 22
    ggattcttca ggattgaaac ccaaaccggt tcagtttcct ttccggctcc ggccgggggg 60
    ggcggccccg ggcggtttgg tgagttagat aacctcgggc cgatcgcacg ccccccgtgg 120
    cggcgacgac ccattcgaac gtctgcccta tcaactttcg atggtagtcg atgtgcctac 180
    catggtgacc acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 240
    ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga cccggggagg 300
    tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtccac 360
    tttaaatcct ttaagcag 378
    <210> SEQ ID NO 23
    <211> LENGTH: 378
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 23
    gatccattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 60
    tattaaagtt gctgcagtta aaaagctcgt agttggatct tgggagcggg cgggcggtcc 120
    gccgcgaggc gagtcaccgc ccgtccccgc cccttgcctc tcggcgcccc ctcgatgctc 180
    ttagctgagt tgtcccgcgg ggcccgaagc gtttactttg aaaaaattag agttgtttca 240
    aagcaggccc gagccgcctg gataccgcca gctaggaaat aatggaatag gaccgcggtt 300
    cctattttgt ttggttttcg gaactgagcc catgattaag ggaaacggcc gggggcattc 360
    ccttattgcg ccccccta 378
    <210> SEQ ID NO 24
    <211> LENGTH: 719
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 24
    ggatctttcc cgctccccgt tcctcccggc ccctccaccc gcgcgtctcc ccccttcttt 60
    tcccctctcc ggaggggggg gaggtggggg cgcgtgggcg gggtcggggg tggggtcggc 120
    gggggaccgc ccccggccgg caaaaggccg ccgccgggcg cacttcaacc gtagcggtgc 180
    gccgcgaccg gctacgagac ggctgggaag gcccgacggg gaatgtggct cggggggggc 240
    ggcgcgtctc agggcgcgcc gaaccacctc accccgagtg ttacagccct ccggccgcgc 300
    tttcgcggaa tcccggggcc gaggggaagc ccgatacccg tcgccgcgct tttcccctcc 360
    ccccgtccgc ctcccgggcg ggcgtggggg tgggggccgg gccgcccctc ccacgcccgt 420
    ggtttctctc tctcccggtc tcggccggtt tggggggggg agcccggttg ggggcggggc 480
    ggactgtcct cagtgcgccc cgggcgtcgt cgcgccgtcg ggcccggggg gttctctcgg 540
    tcacgccgcc cccgacgaag ccgagcgcac ggggtcggcg gcgatgtcgg ctacccaccc 600
    gacccgtctt gaaacacgga ccaaggagtc taacgcgtgc gcgagtcagg ggctcgcacg 660
    aaagccgccg tggcgcaatg aaggtgaagg gccccgtccg ggggcccgag gtgggatcc 719
    <210> SEQ ID NO 25
    <211> LENGTH: 685
    <212> TYPE: DNA
    <213> ORGANISM: Mus musculus
    <400> SEQUENCE: 25
    cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgtcgggg 60
    aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ctgggcaggg 120
    cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 180
    cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 240
    tttccctcag gatagctggc gctctcgcaa ccttcggaag cagttttatc cgggtaaagg 300
    cggaatggat taggaggtct tggggccgga aacgatctca aactatttct caaactttaa 360
    atgggtaagg aagcccggct cgctggcgtg gagccgggcg tggaatgcga gtgcctagtg 420
    ggccactttt ggtaagcaga actggcgctg cgggatgaac cgaacgccgg gttaaggcgc 480
    ccgatgccga cgctcatcag accccagaaa aggtgttggt tgatatagac agcaggacgg 540
    tggccatgga agtcggaatc cgctaaggag tgtgtaacaa ctcacctgcc gaatcaacta 600
    gccctgaaaa tggatggcgc tggagcgtcg ggcccatacc cggccgtcgc cggcagtcgg 660
    aacgggacgg gacgggagcg gccgc 685
    <210> SEQ ID NO 26
    <211> LENGTH: 5162
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Chimeric bacterial plasmid
    <400> SEQUENCE: 26
    gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
    ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
    cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
    ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
    gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
    tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
    cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
    attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
    atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
    atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
    tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
    actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
    aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
    gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
    ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900
    gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccac tagtaacggc 960
    cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc 1020
    ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat 1080
    ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa 1140
    aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact 1200
    tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 1260
    tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa 1320
    cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc 1380
    caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1440
    gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 1500
    gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1560
    caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1620
    agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1680
    aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1740
    accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1800
    gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1860
    acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtg agcaagggcg 1920
    aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1980
    acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2040
    agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 2100
    cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2160
    agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 2220
    actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2280
    tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2340
    acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2400
    tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2460
    acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2520
    ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2580
    ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagagctc 2640
    gctgatcagc ctcgactgtg cctctagttg ccagccatct gttgtttgcc cctcccccgt 2700
    gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2760
    tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2820
    caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2880
    ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac 2940
    tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 3000
    catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 3060
    gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 3120
    ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3180
    gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3240
    tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3300
    cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3360
    gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3420
    gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3480
    gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3540
    ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3600
    aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3660
    tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3720
    ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3780
    gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3840
    ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3900
    ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3960
    agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4020
    ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4080
    aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4140
    tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4200
    cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4260
    tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4320
    cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4380
    ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4440
    gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4500
    gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4560
    gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4620
    gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4680
    tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4740
    aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4800
    cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4860
    caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4920
    cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4980
    ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5040
    aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5100
    tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5160
    tc 5162
    <210> SEQ ID NO 27
    <211> LENGTH: 5627
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pMG plasmid from InvivoGen; IRES sequence
    modified EMCV nucleotides 2736-3308
    <400> SEQUENCE: 27
    caccggcgaa ggaggcctag atctatcgat tgtacagcta gctcgacatg ataagataca 60
    ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 120
    tttgtgatgc tattgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 180
    tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 240
    gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtagatccat 300
    ttaaatgtta attaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 360
    ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 420
    acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 480
    tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 540
    ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 600
    ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 660
    ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 720
    actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 780
    gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 840
    tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 900
    caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 960
    atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 1020
    acgttaaggg attttggtca tggctagtta attaagctgc aataaacaat cattattttc 1080
    attggatctg tgtgttggtt ttttgtgtgg gcttggggga gggggaggcc agaatgactc 1140
    caagagctac aggaaggcag gtcagagacc ccactggaca aacagtggct ggactctgca 1200
    ccataacaca caatcaacag gggagtgagc tggatcgagc tagagtccgt tacataactt 1260
    acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 1320
    acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 1380
    ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 1440
    attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 1500
    gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 1560
    ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 1620
    caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 1680
    tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 1740
    tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt 1800
    tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt 1860
    ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc 1920
    acccccttgg cttcttatgc atgctatact gtttttggct tggggtctat acacccccgc 1980
    ttcctcatgt tataggtgat ggtatagctt agcctatagg tgtgggttat tgaccattat 2040
    tgaccactcc cctattggtg acgatacttt ccattactaa tccataacat ggctctttgc 2100
    cacaactctc tttattggct atatgccaat acactgtcct tcagagactg acacggactc 2160
    tgtattttta caggatgggg tctcatttat tatttacaaa ttcacatata caacaccacc 2220
    gtccccagtg cccgcagttt ttattaaaca taacgtggga tctccacgcg aatctcgggt 2280
    acgtgttccg gacatgggct cttctccggt agcggcggag cttctacatc cgagccctgc 2340
    tcccatgcct ccagcgactc atggtcgctc ggcagctcct tgctcctaac agtggaggcc 2400
    agacttaggc acagcacgat gcccaccacc accagtgtgc cgcacaaggc cgtggcggta 2460
    gggtatgtgt ctgaaaatga gctcggggag cgggcttgca ccgctgacgc atttggaaga 2520
    cttaaggcag cggcagaaga agatgcaggc agctgagttg ttgtgttctg ataagagtca 2580
    gaggtaactc ccgttgcggt gctgttaacg gtggagggca gtgtagtctg agcagtactc 2640
    gttgctgccg cgcgcgccac cagacataat agctgacaga ctaacagact gttcctttcc 2700
    atgggtcttt tctgcagtca cccgggggat ccttcgaacg tagctctaga ttgagtcgac 2760
    gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 2820
    accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 2880
    agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 2940
    aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 3000
    aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 3060
    gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 3120
    agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 3180
    ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg 3240
    aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 3300
    cgataatacc atgggtaagt gatatctact agttgtgacc ggcgcctagt gttgacaatt 3360
    aatcatcggc atagtatatc ggcatagtat aatacgactc actataggag ggccaccatg 3420
    tcgactacta accttcttct ctttcctaca gctgagatca ccggtaggag ggccatcatg 3480
    aaaaagcctg aactcaccgc gacgtctgtc gcgaagtttc tgatcgaaaa gttcgacagc 3540
    gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta 3600
    ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 3660
    tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg 3720
    gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 3780
    gacctgcctg aaaccgaact gcccgctgtt ctgcaacccg tcgcggagct catggatgcg 3840
    atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 3900
    ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 3960
    tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 4020
    atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 4080
    aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 4140
    ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 4200
    atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg 4260
    ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 4320
    aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 4380
    gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 4440
    gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa 4500
    tgagtcgaga attcgctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc 4560
    tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 4620
    ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 4680
    gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 4740
    aagggggagg attgggaaga caatagcagg catgcgcagg gcccaattgc tcgagcggcc 4800
    gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgtaac 4860
    taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc 4920
    ccagtgcaag tgcaggtgcc agaacatttc tctatcgaag gatctgcgat cgctccggtg 4980
    cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 5040
    gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 5100
    actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 5160
    tgaacgttct ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc 5220
    gcatctctcc ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc 5280
    gcgttctgcc gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt 5340
    taaagctcag gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca 5400
    gccggctctc cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt 5460
    ctgttctgcg ccgttacaga tccaagctgt gaccggcgcc tacgtaagtg atatctacta 5520
    gatttatcaa aaagagtgtt gacttgtgag cgctcacaat tgatacttag attcatcgag 5580
    agggacacgt cgactactaa ccttcttctc tttcctacag ctgagat 5627
    <210> SEQ ID NO 28
    <211> LENGTH: 553
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pMG plasmid from InvivoGen: EMCV IRES sequence
    <400> SEQUENCE: 28
    aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt 60
    tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg 120
    acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc 180
    gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt 240
    tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta 300
    taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg 360
    gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag 420
    gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac gtgtgtttag 480
    tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa 540
    cacgatgata ata 553
    <210> SEQ ID NO 29
    <211> LENGTH: 4692
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pDSred1-N1 plasmid from Clontech
    <400> SEQUENCE: 29
    tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
    cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
    gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
    atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
    aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
    catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
    catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
    atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
    ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
    acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600
    ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660
    gatccaccgg tcgccaccat ggtgcgctcc tccaagaacg tcatcaagga gttcatgcgc 720
    ttcaaggtgc gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc 780
    gagggccgcc cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc 840
    ctgcccttcg cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg 900
    aagcaccccg ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg 960
    gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg 1020
    caggacggct gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc 1080
    cccgtaatgc agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc 1140
    gacggcgtgc tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac 1200
    ctggtggagt tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac 1260
    tacgtggact ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag 1320
    tacgagcgca ccgagggccg ccaccacctg ttcctgtagc ggccgcgact ctagatcata 1380
    atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 1440
    ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 1500
    aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 1560
    cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt 1620
    taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1680
    ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1740
    tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1800
    aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 1860
    ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 1920
    ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 1980
    cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 2040
    taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2100
    tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 2160
    ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg 2220
    aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2280
    agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 2340
    agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 2400
    cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 2460
    ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 2520
    ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc 2580
    gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 2640
    gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 2700
    gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 2760
    tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 2820
    agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 2880
    ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 2940
    tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3000
    acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3060
    ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 3120
    gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3180
    ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3240
    tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3300
    ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 3360
    ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 3420
    cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 3480
    ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 3540
    ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga 3600
    acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt 3660
    cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat 3720
    tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa 3780
    ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca 3840
    tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3900
    ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3960
    gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 4020
    tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4080
    ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 4140
    ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 4200
    gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4260
    ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4320
    tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4380
    ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4440
    agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4500
    agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4560
    gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4620
    tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4680
    accgccatgc at 4692
    <210> SEQ ID NO 30
    <211> LENGTH: 4257
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pPur plasmid from Clontech
    <400> SEQUENCE: 30
    ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 60
    atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 120
    gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 180
    actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 240
    ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 300
    tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttgcatg cctgcaggtc 360
    ggccgccacg accggtgccg ccaccatccc ctgacccacg cccctgaccc ctcacaagga 420
    gacgaccttc catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 480
    cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 540
    tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 600
    tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga 660
    ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 720
    agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 780
    ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca 840
    agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 900
    ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 960
    ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 1020
    ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc 1080
    atggctccga ccgaagccga cccgggcggc cccgccgacc ccgcacccgc ccccgaggcc 1140
    caccgactct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 1200
    aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 1260
    aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1320
    aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1380
    tatcatgtct ggatccccag gaagctcctc tgtgtcctca taaaccctaa cctcctctac 1440
    ttgagaggac attccaatca taggctgccc atccaccctc tgtgtcctcc tgttaattag 1500
    gtcacttaac aaaaaggaaa ttgggtaggg gtttttcaca gaccgctttc taagggtaat 1560
    tttaaaatat ctgggaagtc ccttccactg ctgtgttcca gaagtgttgg taaacagccc 1620
    acaaatgtca acagcagaaa catacaagct gtcagctttg cacaagggcc caacaccctg 1680
    ctcatcaaga agcactgtgg ttgctgtgtt agtaatgtgc aaaacaggag gcacattttc 1740
    cccacctgtg taggttccaa aatatctagt gttttcattt ttacttggat caggaaccca 1800
    gcactccact ggataagcat tatccttatc caaaacagcc ttgtggtcag tgttcatctg 1860
    ctgactgtca actgtagcat tttttggggt tacagtttga gcaggatatt tggtcctgta 1920
    gtttgctaac acaccctgca gctccaaagg ttccccacca acagcaaaaa aatgaaaatt 1980
    tgacccttga atgggttttc cagcaccatt ttcatgagtt ttttgtgtcc ctgaatgcaa 2040
    gtttaacata gcagttaccc caataacctc agttttaaca gtaacagctt cccacatcaa 2100
    aatatttcca caggttaagt cctcatttaa attaggcaaa ggaattcttg aagacgaaag 2160
    ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 2220
    tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 2280
    cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 2340
    aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 2400
    ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 2460
    cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 2520
    agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 2580
    gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 2640
    cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2700
    gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2760
    ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 2820
    gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 2880
    gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta 2940
    cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3000
    ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3060
    gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3120
    gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 3180
    gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 3240
    ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3300
    gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3360
    gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3420
    caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3480
    ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 3540
    tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3600
    ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3660
    tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3720
    cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3780
    gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3840
    ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3900
    gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3960
    agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 4020
    tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 4080
    tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 4140
    gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4200
    caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccag 4257
    <210> SEQ ID NO 31
    <211> LENGTH: 8136
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pWE15 cosmid vector
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank X65279
    <309> DATABASE ENTRY DATE: 1995-04-14
    <400> SEQUENCE: 31
    ctatagtgag tcgtattatg cggccgcgaa ttcttgaaga cgaaagggcc tcgtgatacg 60
    cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 120
    tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 180
    tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 240
    gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gcttcctgtt 300
    tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 360
    gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 420
    gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 480
    gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 540
    gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 600
    agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 660
    ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 720
    cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 780
    gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 840
    cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 900
    gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 960
    ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 1020
    acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 1080
    ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 1140
    aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 1200
    aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 1260
    ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 1320
    ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 1380
    actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 1440
    caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 1500
    gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 1560
    ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 1620
    cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 1680
    ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 1740
    cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg gtttcgccac 1800
    ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 1860
    gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 1920
    tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 1980
    accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 2040
    cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 2100
    tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 2160
    ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 2220
    cacgatcatg cgcacccgtc agatccagac atgataagat acattgatga gtttggacaa 2280
    accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2340
    ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2400
    atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2460
    tgtggtatgg ctgattatga tctctagtca aggcactata catcaaatat tccttattaa 2520
    cccctttaca aattaaaaag ctaaaggtac acaatttttg agcatagtta ttaatagcag 2580
    acactctatg cctgtgtgga gtaagaaaaa acagtatgtt atgattataa ctgttatgcc 2640
    tacttataaa ggttacagaa tatttttcca taattttctt gtatagcagt gcagcttttt 2700
    cctttgtggt gtaaatagca aagcaagcaa gagttctatt actaaacaca gcatgactca 2760
    aaaaacttag caattctgaa ggaaagtcct tggggtcttc tacctttctc ttcttttttg 2820
    gaggagtaga atgttgagag tcagcagtag cctcatcatc actagatggc atttcttctg 2880
    agcaaaacag gttttcctca ttaaaggcat tccaccactg ctcccattca tcagttccat 2940
    aggttggaat ctaaaataca caaacaatta gaatcagtag tttaacacat tatacactta 3000
    aaaattttat atttacctta gagctttaaa tctctgtagg tagtttgtcc aattatgtca 3060
    caccacagaa gtaaggttcc ttcacaaaga tccggaccaa agcggccatc gtgcctcccc 3120
    actcctgcag ttcgggggca tggatgcgcg gatagccgct gctggtttcc tggatgccga 3180
    cggatttgca ctgccggtag aactcgcgag gtcgtccagc ctcaggcagc agctgaacca 3240
    actcgcgagg ggatcgagcc cggggtgggc gaagaactcc agcatgagat ccccgcgctg 3300
    gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc 3360
    ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa 3420
    ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 3480
    tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 3540
    tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 3600
    ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 3660
    tcgccatggg tcacgacgag atcctcgccg tcgggatgcg cgccttgagc ctggcgaaca 3720
    gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 3780
    cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 3840
    tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 3900
    caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 3960
    cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 4020
    gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct 4080
    tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 4140
    cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 4200
    ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 4260
    atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 4320
    agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 4380
    ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 4440
    ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc 4500
    accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg 4560
    agtgcttgcg gcagcgtgaa agctttttgc aaaagcctag gcctccaaaa aagcctcctc 4620
    actacttctg gaatagctca gaggccgagg cggcctaaat aaaaaaaatt agtcagccat 4680
    ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg 4740
    ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc 4800
    ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc 4860
    ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agccggatct 4920
    gcaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat 4980
    ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc 5040
    tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 5100
    gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 5160
    cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cgcataaatc gccgtgacga 5220
    tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc 5280
    cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcggca tcccgatgcc 5340
    gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag 5400
    caagacgtag cccagcgcgt cgggccgcca tgccggcgat aatggcctgc ttctcgccga 5460
    aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 5520
    ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 5580
    cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 5640
    cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 5700
    agggcatcgg tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 5760
    ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 5820
    acagtccccc ggccacgggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 5880
    gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 5940
    acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatct tggcagtcac 6000
    agcatgcgca tatccatgct tcgaccatgc gctcacaaag taggtgaatg cgcaatgtag 6060
    tacccacatc gtcatcgctt tccactgctc tcgcgaataa agatggaaaa tcaatctcat 6120
    ggtaatagtc catgaaaatc cttgtattca taaatcctcc aggtagctat atgcaaattg 6180
    aaacaaaaga gatggtgatc tttctaagag atgatggaat ctcccttcag tatcccgatg 6240
    gtcaatgcgc tggatatggg atagatggga atatgctgat ttttatggga cagagttgcg 6300
    aactgttccc aactaaaatc attttgcacg atcagcgcac tacgaacttt acccacaaat 6360
    agtcaggtaa tgaatcctga tataaagaca ggttgataaa tcagtcttct acgcgcatcg 6420
    cacgcgcaca ccgtagaaag tctttcagtt gtgagcctgg gcaaaccgtt aactttcggc 6480
    ggctttgctg tgcgacaggc tcacgtctaa aaggaaataa atcatgggtc ataaaattat 6540
    cacgttgtcc ggcgcggcga cggatgttct gtatgcgctg tttttccgtg gcgcgttgct 6600
    gtctggtgat ctgccttcta aatctggcac agccgaattg cgcgagcttg gttttgctga 6660
    aaccagacac acagcaactg aataccagaa agaaaatcac tttacctttc tgacatcaga 6720
    agggcagaaa tttgccgttg aacacctggt caatacgcgt tttggtgagc agcaatattg 6780
    cgcttcgatg acgcttggcg ttgagattga tacctctgct gcacaaaagg caatcgacga 6840
    gctggaccag cgcattcgtg acaccgtctc cttcgaactt attcgcaatg gagtgtcatt 6900
    catcaaggac gccgctatcg caaatggtgc tatccacgca gcggcaatcg aaacacctca 6960
    gccggtgacc aatatctaca acatcagcct tggtatccag cgtgatgagc cagcgcagaa 7020
    caaggtaacc gtcagtgccg ataagttcaa agttaaacct ggtgttgata ccaacattga 7080
    aacgttgatc gaaaacgcgc tgaaaaacgc tgctgaatgt gcggcgctgg atgtcacaaa 7140
    gcaaatggca gcagacaaga aagcgatgga tgaactggct tcctatgtcc gcacggccat 7200
    catgatggaa tgtttccccg gtggtgttat ctggcagcag tgccgtcgat agtatgcaat 7260
    tgataattat tatcatttgc gggtcctttc cggcgatccg ccttgttacg gggcggcgac 7320
    ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg ttcttcttcg 7380
    tcataactta atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg acaggtgctg 7440
    aaagcgagct ttttggcctc tgtcgtttcc tttctctgtt tttgtccgtg gaatgaacaa 7500
    tggaagtcaa caaaaagcag ctggctgaca ttttcggtgc gagtatccgt accattcaga 7560
    actggcagga acagggaatg cccgttctgc gaggcggtgg caagggtaat gaggtgcttt 7620
    atgactctgc cgccgtcata aaatggtatg ccgaaaggga tgctgaaatt gagaacgaaa 7680
    agctgcgccg ggaggttgaa gaactgcggc aggccagcga ggcagatcca caggacgggt 7740
    gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg 7800
    cggcggcaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg 7860
    catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc 7920
    gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg 7980
    tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc 8040
    aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagcggt caaacatgag 8100
    aattcgcggc cgcaattaac cctcactaaa ggatcc 8136
    <210> SEQ ID NO 32
    <211> LENGTH: 2713
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pNEB193 plasmid
    <400> SEQUENCE: 32
    tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
    cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
    ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
    accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
    attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
    tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
    tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgggggc 420
    gcgccggatc cttaattaag tctagagtcg actgtttaaa cctgcaggca tgcaagcttg 480
    gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 540
    aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 600
    acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 660
    cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 720
    tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 780
    tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 840
    gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 900
    aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 960
    ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1020
    gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1080
    ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1140
    ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 1200
    cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 1260
    attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 1320
    ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 1380
    aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 1440
    gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 1500
    tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1560
    ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 1620
    taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 1680
    atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 1740
    actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 1800
    cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 1860
    agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 1920
    gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 1980
    gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 2040
    gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 2100
    gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 2160
    cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 2220
    ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 2280
    accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 2340
    aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 2400
    aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 2460
    caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 2520
    ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 2580
    gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 2640
    cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 2700
    aggccctttc gtc 2713
    <210> SEQ ID NO 33
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attP
    <400> SEQUENCE: 33
    cagctttttt atactaagtt g 21
    <210> SEQ ID NO 34
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB
    <400> SEQUENCE: 34
    ctgctttttt atactaactt g 21
    <210> SEQ ID NO 35
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attL
    <400> SEQUENCE: 35
    ctgctttttt atactaagtt g 21
    <210> SEQ ID NO 36
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attR
    <400> SEQUENCE: 36
    cagctttttt atactaactt g 21
    <210> SEQ ID NO 37
    <211> LENGTH: 1071
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Integrase E174R
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(1071)
    <223> OTHER INFORMATION: Nucleotide sequence encoding Integrase E147R
    <400> SEQUENCE: 37
    atg gga aga agg cga agt cat gag cgc cgg gat tta ccc cct aac ctt 48
    Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu
    1 5 10 15
    tat ata aga aac aat gga tat tac tgc tac agg gac cca agg acg ggt 96
    Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly
    20 25 30
    aaa gag ttt gga tta ggc aga gac agg cga atc gca atc act gaa gct 144
    Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala
    35 40 45
    ata cag gcc aac att gag tta ttt tca gga cac aaa cac aag cct ctg 192
    Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu
    50 55 60
    aca gcg aga atc aac agt gat aat tcc gtt acg tta cat tca tgg ctt 240
    Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu
    65 70 75 80
    gat cgc tac gaa aaa atc ctg gcc agc aga gga atc aag cag aag aca 288
    Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr
    85 90 95
    ctc ata aat tac atg agc aaa att aaa gca ata agg agg ggt ctg cct 336
    Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro
    100 105 110
    gat gct cca ctt gaa gac atc acc aca aaa gaa att gcg gca atg ctc 384
    Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu
    115 120 125
    aat gga tac ata gac gag ggc aag gcg gcg tca gcc aag tta atc aga 432
    Asn Gly Tyr Ile Asp Glu Gly Lys Ala Ala Ser Ala Lys Leu Ile Arg
    130 135 140
    tca aca ctg agc gat gca ttc cga gag gca ata gct gaa ggc cat ata 480
    Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile
    145 150 155 160
    aca aca aac cat gtc gct gcc act cgc gca gca aaa tct aga gta agg 528
    Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg
    165 170 175
    aga tca aga ctt acg gct gac gaa tac ctg aaa att tat caa gca gca 576
    Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala
    180 185 190
    gaa tca tca cca tgt tgg ctc aga ctt gca atg gaa ctg gct gtt gtt 624
    Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val
    195 200 205
    acc ggg caa cga gtt ggt gat tta tgc gaa atg aag tgg tct gat atc 672
    Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile
    210 215 220
    gta gat gga tat ctt tat gtc gag caa agc aaa aca ggc gta aaa att 720
    Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile
    225 230 235 240
    gcc atc cca aca gca ttg cat att gat gct ctc gga ata tca atg aag 768
    Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys
    245 250 255
    gaa aca ctt gat aaa tgc aaa gag att ctt ggc gga gaa acc ata att 816
    Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile
    260 265 270
    gca tct act cgt cgc gaa ccg ctt tca tcc ggc aca gta tca agg tat 864
    Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr
    275 280 285
    ttt atg cgc gca cga aaa gca tca ggt ctt tcc ttc gaa ggg gat ccg 912
    Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro
    290 295 300
    cct acc ttt cac gag ttg cgc agt ttg tct gca aga ctc tat gag aag 960
    Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys
    305 310 315 320
    cag ata agc gat aag ttt gct caa cat ctt ctc ggg cat aag tcg gac 1008
    Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp
    325 330 335
    acc atg gca tca cag tat cgt gat gac aga ggc agg gag tgg gac aaa 1056
    Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys
    340 345 350
    att gaa atc aaa taa 1071
    Ile Glu Ile Lys *
    355
    <210> SEQ ID NO 38
    <211> LENGTH: 356
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Integrase E147R
    <400> SEQUENCE: 38
    Met Gly Arg Arg Arg Ser His Glu Arg Arg Asp Leu Pro Pro Asn Leu
    1 5 10 15
    Tyr Ile Arg Asn Asn Gly Tyr Tyr Cys Tyr Arg Asp Pro Arg Thr Gly
    20 25 30
    Lys Glu Phe Gly Leu Gly Arg Asp Arg Arg Ile Ala Ile Thr Glu Ala
    35 40 45
    Ile Gln Ala Asn Ile Glu Leu Phe Ser Gly His Lys His Lys Pro Leu
    50 55 60
    Thr Ala Arg Ile Asn Ser Asp Asn Ser Val Thr Leu His Ser Trp Leu
    65 70 75 80
    Asp Arg Tyr Glu Lys Ile Leu Ala Ser Arg Gly Ile Lys Gln Lys Thr
    85 90 95
    Leu Ile Asn Tyr Met Ser Lys Ile Lys Ala Ile Arg Arg Gly Leu Pro
    100 105 110
    Asp Ala Pro Leu Glu Asp Ile Thr Thr Lys Glu Ile Ala Ala Met Leu
    115 120 125
    Asn Gly Tyr Ile Asp Glu Gly Lys Ala Ala Ser Ala Lys Leu Ile Arg
    130 135 140
    Ser Thr Leu Ser Asp Ala Phe Arg Glu Ala Ile Ala Glu Gly His Ile
    145 150 155 160
    Thr Thr Asn His Val Ala Ala Thr Arg Ala Ala Lys Ser Arg Val Arg
    165 170 175
    Arg Ser Arg Leu Thr Ala Asp Glu Tyr Leu Lys Ile Tyr Gln Ala Ala
    180 185 190
    Glu Ser Ser Pro Cys Trp Leu Arg Leu Ala Met Glu Leu Ala Val Val
    195 200 205
    Thr Gly Gln Arg Val Gly Asp Leu Cys Glu Met Lys Trp Ser Asp Ile
    210 215 220
    Val Asp Gly Tyr Leu Tyr Val Glu Gln Ser Lys Thr Gly Val Lys Ile
    225 230 235 240
    Ala Ile Pro Thr Ala Leu His Ile Asp Ala Leu Gly Ile Ser Met Lys
    245 250 255
    Glu Thr Leu Asp Lys Cys Lys Glu Ile Leu Gly Gly Glu Thr Ile Ile
    260 265 270
    Ala Ser Thr Arg Arg Glu Pro Leu Ser Ser Gly Thr Val Ser Arg Tyr
    275 280 285
    Phe Met Arg Ala Arg Lys Ala Ser Gly Leu Ser Phe Glu Gly Asp Pro
    290 295 300
    Pro Thr Phe His Glu Leu Arg Ser Leu Ser Ala Arg Leu Tyr Glu Lys
    305 310 315 320
    Gln Ile Ser Asp Lys Phe Ala Gln His Leu Leu Gly His Lys Ser Asp
    325 330 335
    Thr Met Ala Ser Gln Tyr Arg Asp Asp Arg Gly Arg Glu Trp Asp Lys
    340 345 350
    Ile Glu Ile Lys
    355
    <210> SEQ ID NO 39
    <211> LENGTH: 876
    <212> TYPE: DNA
    <213> ORGANISM: Discosoma species
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (45)...(737)
    <223> OTHER INFORMATION: Nucleotide sequence encoding red flourescent
    protein (FP593)
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AF272711
    <309> DATABASE ENTRY DATE: 2000-09-26
    <400> SEQUENCE: 39
    agtttcagcc agtgacaggg tgagctgcca ggtattctaa caag atg agt tgt tcc 56
    Met Ser Cys Ser
    1
    aag aat gtg atc aag gag ttc atg agg ttc aag gtt cgt atg gaa gga 104
    Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly
    5 10 15 20
    acg gtc aat ggg cac gag ttt gaa ata aaa ggc gaa ggt gaa ggg agg 152
    Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu Gly Glu Gly Arg
    25 30 35
    cct tac gaa ggt cac tgt tcc gta aag ctt atg gta acc aag ggt gga 200
    Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val Thr Lys Gly Gly
    40 45 50
    cct ttg cca ttt gct ttt gat att ttg tca cca caa ttt cag tat gga 248
    Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly
    55 60 65
    agc aag gta tat gtc aaa cac cct gcc gac ata cca gac tat aaa aag 296
    Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys
    70 75 80
    ctg tca ttt cct gag gga ttt aaa tgg gaa agg gtc atg aac ttt gaa 344
    Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu
    85 90 95 100
    gac ggt ggc gtg gtt act gta tcc caa gat tcc agt ttg aaa gac ggc 392
    Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser Leu Lys Asp Gly
    105 110 115
    tgt ttc atc tac gag gtc aag ttc att ggg gtg aac ttt cct tct gat 440
    Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp
    120 125 130
    gga cct gtt atg cag agg agg aca cgg ggc tgg gaa gcc agc tct gag 488
    Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu Ala Ser Ser Glu
    135 140 145
    cgt ttg tat cct cgt gat ggg gtg ctg aaa gga gac atc cat atg gct 536
    Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp Ile His Met Ala
    150 155 160
    ctg agg ctg gaa gga ggc ggc cat tac ctc gtt gaa ttc aaa agt att 584
    Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu Phe Lys Ser Ile
    165 170 175 180
    tac atg gta aag aag cct tca gtg cag ttg cca ggc tac tat tat gtt 632
    Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly Tyr Tyr Tyr Val
    185 190 195
    gac tcc aaa ctg gat atg acg agc cac aac gaa gat tac aca gtc gtt 680
    Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp Tyr Thr Val Val
    200 205 210
    gag cag tat gaa aaa acc cag gga cgc cac cat ccg ttc att aag cct 728
    Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro Phe Ile Lys Pro
    215 220 225
    ctg cag tga actcggctca gtcatggatt agcggtaatg gccacaaaag 777
    Leu Gln *
    230
    gcacgatgat cgttttttag gaatgcagcc aaaaattgaa ggttatgaca gtagaaatac 837
    aagcaacagg ctttgcttat taaacatgta attgaaaac 876
    <210> SEQ ID NO 40
    <211> LENGTH: 230
    <212> TYPE: PRT
    <213> ORGANISM: Discosoma species
    <400> SEQUENCE: 40
    Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val
    1 5 10 15
    Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Lys Gly Glu
    20 25 30
    Gly Glu Gly Arg Pro Tyr Glu Gly His Cys Ser Val Lys Leu Met Val
    35 40 45
    Thr Lys Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln
    50 55 60
    Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro
    65 70 75 80
    Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val
    85 90 95
    Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser Ser
    100 105 110
    Leu Lys Asp Gly Cys Phe Ile Tyr Glu Val Lys Phe Ile Gly Val Asn
    115 120 125
    Phe Pro Ser Asp Gly Pro Val Met Gln Arg Arg Thr Arg Gly Trp Glu
    130 135 140
    Ala Ser Ser Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Asp
    145 150 155 160
    Ile His Met Ala Leu Arg Leu Glu Gly Gly Gly His Tyr Leu Val Glu
    165 170 175
    Phe Lys Ser Ile Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly
    180 185 190
    Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp
    195 200 205
    Tyr Thr Val Val Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro
    210 215 220
    Phe Ile Lys Pro Leu Gln
    225 230
    <210> SEQ ID NO 41
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: m-att;
    <220> FEATURE:
    <221> NAME/KEY: misc_difference
    <222> LOCATION: 18
    <223> OTHER INFORMATION: n is a or g or c or t/u
    <400> SEQUENCE: 41
    rkycwgcttt yktrtacnaa stsgb 25
    <210> SEQ ID NO 42
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: m-attB;
    <220> FEATURE:
    <221> NAME/KEY: misc_difference
    <222> LOCATION: 18
    <223> OTHER INFORMATION: n is a or g or c or t/u
    <400> SEQUENCE: 42
    agccwgcttt yktrtacnaa ctsgb 25
    <210> SEQ ID NO 43
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: m-attR
    <220> FEATURE:
    <221> NAME/KEY: misc_difference
    <222> LOCATION: 18
    <223> OTHER INFORMATION: n is a or g or c or t/u
    <400> SEQUENCE: 43
    gttcagcttt cktrtacnaa ctsgb 25
    <210> SEQ ID NO 44
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: m-attL
    <220> FEATURE:
    <221> NAME/KEY: misc_difference
    <222> LOCATION: 18
    <223> OTHER INFORMATION: n is a or g or c or t/u
    <400> SEQUENCE: 44
    agccwgcttt cktrtacnaa gtsgb 25
    <210> SEQ ID NO 45
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: m-attP1
    <220> FEATURE:
    <221> NAME/KEY: misc_difference
    <222> LOCATION: 18
    <223> OTHER INFORMATION: n is a or g or c or t/u
    <400> SEQUENCE: 45
    gttcagcttt yktrtacnaa gtsgb 25
    <210> SEQ ID NO 46
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB1
    <400> SEQUENCE: 46
    agcctgcttt tttgtacaaa cttgt 25
    <210> SEQ ID NO 47
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB2
    <400> SEQUENCE: 47
    agcctgcttt cttgtacaaa cttgt 25
    <210> SEQ ID NO 48
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attB3
    <400> SEQUENCE: 48
    acccagcttt cttgtacaaa cttgt 25
    <210> SEQ ID NO 49
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attR1
    <400> SEQUENCE: 49
    gttcagcttt tttgtacaaa cttgt 25
    <210> SEQ ID NO 50
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attR2
    <400> SEQUENCE: 50
    gttcagcttt cttgtacaaa cttgt 25
    <210> SEQ ID NO 51
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attR3
    <400> SEQUENCE: 51
    gttcagcttt cttgtacaaa gttgg 25
    <210> SEQ ID NO 52
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attL1
    <400> SEQUENCE: 52
    agcctgcttt tttgtacaaa gttgg 25
    <210> SEQ ID NO 53
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attL2
    <400> SEQUENCE: 53
    agcctgcttt cttgtacaaa gttgg 25
    <210> SEQ ID NO 54
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attL3
    <400> SEQUENCE: 54
    acccagcttt cttgtacaaa gttgg 25
    <210> SEQ ID NO 55
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attP1
    <400> SEQUENCE: 55
    gttcagcttt tttgtacaaa gttgg 25
    <210> SEQ ID NO 56
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attP2,P3
    <400> SEQUENCE: 56
    gttcagcttt cttgtacaaa gttgg 25
    <210> SEQ ID NO 57
    <211> LENGTH: 34
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Lox P site
    <400> SEQUENCE: 57
    ataacttcgt ataatgtatg ctatacgaag ttat 34
    <210> SEQ ID NO 58
    <211> LENGTH: 1032
    <212> TYPE: DNA
    <213> ORGANISM: Escherichia coli
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(1032)
    <223> OTHER INFORMATION: nucleotide sequence encoding Cre recombinase
    <400> SEQUENCE: 58
    atg tcc aat tta ctg acc gta cac caa aat ttg cct gca tta ccg gtc 48
    Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val
    1 5 10 15
    gat gca acg agt gat gag gtt cgc aag aac ctg atg gac atg ttc agg 96
    Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg
    20 25 30
    gat cgc cag gcg ttt tct gag cat acc tgg aaa atg ctt ctg tcc gtt 144
    Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val
    35 40 45
    tgc cgg tcg tgg gcg gca tgg tgc aag ttg aat aac cgg aaa tgg ttt 192
    Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe
    50 55 60
    ccc gca gaa cct gaa gat gtt cgc gat tat ctt cta tat ctt cag gcg 240
    Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala
    65 70 75 80
    cgc ggt ctg gca gta aaa act atc cag caa cat ttg ggc cag cta aac 288
    Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn
    85 90 95
    atg ctt cat cgt cgg tcc ggg ctg cca cga cca agt gac agc aat gct 336
    Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala
    100 105 110
    gtt tca ctg gtt atg cgg cgg atc cga aaa gaa aac gtt gat gcc ggt 384
    Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly
    115 120 125
    gaa cgt gca aaa cag gct cta gcg ttc gaa cgc act gat ttc gac cag 432
    Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln
    130 135 140
    gtt cgt tca ctc atg gaa aat agc gat cgc tgc cag gat ata cgt aat 480
    Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn
    145 150 155 160
    ctg gca ttt ctg ggg att gct tat aac acc ctg tta cgt ata gcc gaa 528
    Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu
    165 170 175
    att gcc agg atc agg gtt aaa gat atc tca cgt act gac ggt ggg aga 576
    Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg
    180 185 190
    atg tta atc cat att ggc aga acg aaa acg ctg gtt agc acc gca ggt 624
    Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly
    195 200 205
    gta gag aag gca ctt agc ctg ggg gta act aaa ctg gtc gag cga tgg 672
    Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp
    210 215 220
    att tcc gtc tct ggt gta gct gat gat ccg aat aac tac ctg ttt tgc 720
    Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys
    225 230 235 240
    cgg gtc aga aaa aat ggt gtt gcc gcg cca tct gcc acc agc cag cta 768
    Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu
    245 250 255
    tca act cgc gcc ctg gaa ggg att ttt gaa gca act cat cga ttg att 816
    Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile
    260 265 270
    tac ggc gct aag gat gac tct ggt cag aga tac ctg gcc tgg tct gga 864
    Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly
    275 280 285
    cac agt gcc cgt gtc gga gcc gcg cga gat atg gcc cgc gct gga gtt 912
    His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val
    290 295 300
    tca ata ccg gag atc atg caa gct ggt ggc tgg acc aat gta aat att 960
    Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile
    305 310 315 320
    gtc atg aac tat atc cgt aac ctg gat agt gaa aca ggg gca atg gtg 1008
    Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val
    325 330 335
    cgc ctg ctg gaa gat ggc gat tag 1032
    Arg Leu Leu Glu Asp Gly Asp *
    340
    <210> SEQ ID NO 59
    <211> LENGTH: 343
    <212> TYPE: PRT
    <213> ORGANISM: Escherichia coli
    <400> SEQUENCE: 59
    Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val
    1 5 10 15
    Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg
    20 25 30
    Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val
    35 40 45
    Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe
    50 55 60
    Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala
    65 70 75 80
    Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn
    85 90 95
    Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala
    100 105 110
    Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly
    115 120 125
    Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln
    130 135 140
    Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn
    145 150 155 160
    Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu
    165 170 175
    Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg
    180 185 190
    Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly
    195 200 205
    Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp
    210 215 220
    Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys
    225 230 235 240
    Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu
    245 250 255
    Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile
    260 265 270
    Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly
    275 280 285
    His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val
    290 295 300
    Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile
    305 310 315 320
    Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val
    325 330 335
    Arg Leu Leu Glu Asp Gly Asp
    340
    <210> SEQ ID NO 60
    <211> LENGTH: 1272
    <212> TYPE: DNA
    <213> ORGANISM: Saccharomyces cerevisiae
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(1272)
    <223> OTHER INFORMATION: nucleotide sequence encoding Flip recombinase
    <400> SEQUENCE: 60
    atg cca caa ttt ggt ata tta tgt aaa aca cca cct aag gtg ctt gtt 48
    Met Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val
    1 5 10 15
    cgt cag ttt gtg gaa agg ttt gaa aga cct tca ggt gag aaa ata gca 96
    Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala
    20 25 30
    tta tgt gct gct gaa cta acc tat tta tgt tgg atg att aca cat aac 144
    Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn
    35 40 45
    gga aca gca atc aag aga gcc aca ttc atg agc tat aat act atc ata 192
    Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile
    50 55 60
    agc aat tcg ctg agt ttc gat att gtc aat aaa tca ctc cag ttt aaa 240
    Ser Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys
    65 70 75 80
    tac aag acg caa aaa gca aca att ctg gaa gcc tca tta aag aaa ttg 288
    Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu
    85 90 95
    att cct gct tgg gaa ttt aca att att cct tac tat gga caa aaa cat 336
    Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His
    100 105 110
    caa tct gat atc act gat att gta agt agt ttg caa tta cag ttc gaa 384
    Gln Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu
    115 120 125
    tca tcg gaa gaa gca gat aag gga aat agc cac agt aaa aaa atg ctt 432
    Ser Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu
    130 135 140
    aaa gca ctt cta agt gag ggt gaa agc atc tgg gag atc act gag aaa 480
    Lys Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys
    145 150 155 160
    ata cta aat tcg ttt gag tat act tcg aga ttt aca aaa aca aaa act 528
    Ile Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr
    165 170 175
    tta tac caa ttc ctc ttc cta gct act ttc atc aat tgt gga aga ttc 576
    Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe
    180 185 190
    agc gat att aag aac gtt gat ccg aaa tca ttt aaa tta gtc caa aat 624
    Ser Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn
    195 200 205
    aag tat ctg gga gta ata atc cag tgt tta gtg aca gag aca aag aca 672
    Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr
    210 215 220
    agc gtt agt agg cac ata tac ttc ttt agc gca agg ggt agg atc gat 720
    Ser Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp
    225 230 235 240
    cca ctt gta tat ttg gat gaa ttt ttg agg aat tct gaa cca gtc cta 768
    Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu
    245 250 255
    aaa cga gta aat agg acc ggc aat tct tca agc aat aaa cag gaa tac 816
    Lys Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gln Glu Tyr
    260 265 270
    caa tta tta aaa gat aac tta gtc aga tcg tac aat aaa gct ttg aag 864
    Gln Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys
    275 280 285
    aaa aat gcg cct tat tca atc ttt gct ata aaa aat ggc cca aaa tct 912
    Lys Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser
    290 295 300
    cac att gga aga cat ttg atg acc tca ttt ctt tca atg aag ggc cta 960
    His Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu
    305 310 315 320
    acg gag ttg act aat gtt gtg gga aat tgg agc gat aag cgt gct tct 1008
    Thr Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser
    325 330 335
    gcc gtg gcc agg aca acg tat act cat cag ata aca gca ata cct gat 1056
    Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp
    340 345 350
    cac tac ttc gca cta gtt tct cgg tac tat gca tat gat cca ata tca 1104
    His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser
    355 360 365
    aag gaa atg ata gca ttg aag gat gag act aat cca att gag gag tgg 1152
    Lys Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp
    370 375 380
    cag cat ata gaa cag cta aag ggt agt gct gaa gga agc ata cga tac 1200
    Gln His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr
    385 390 395 400
    ccc gca tgg aat ggg ata ata tca cag gag gta cta gac tac ctt tca 1248
    Pro Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser
    405 410 415
    tcc tac ata aat aga cgc ata taa 1272
    Ser Tyr Ile Asn Arg Arg Ile *
    420
    <210> SEQ ID NO 61
    <211> LENGTH: 422
    <212> TYPE: PRT
    <213> ORGANISM: Saccharomyces cerevisiae
    <400> SEQUENCE: 61
    Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val Arg
    1 5 10 15
    Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala Leu
    20 25 30
    Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His Asn Gly
    35 40 45
    Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile Ser
    50 55 60
    Asn Ser Leu Ser Phe Asp Ile Val Asn Lys Ser Leu Gln Phe Lys Tyr
    65 70 75 80
    Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu Ile
    85 90 95
    Pro Ala Trp Glu Phe Thr Ile Ile Pro Tyr Tyr Gly Gln Lys His Gln
    100 105 110
    Ser Asp Ile Thr Asp Ile Val Ser Ser Leu Gln Leu Gln Phe Glu Ser
    115 120 125
    Ser Glu Glu Ala Asp Lys Gly Asn Ser His Ser Lys Lys Met Leu Lys
    130 135 140
    Ala Leu Leu Ser Glu Gly Glu Ser Ile Trp Glu Ile Thr Glu Lys Ile
    145 150 155 160
    Leu Asn Ser Phe Glu Tyr Thr Ser Arg Phe Thr Lys Thr Lys Thr Leu
    165 170 175
    Tyr Gln Phe Leu Phe Leu Ala Thr Phe Ile Asn Cys Gly Arg Phe Ser
    180 185 190
    Asp Ile Lys Asn Val Asp Pro Lys Ser Phe Lys Leu Val Gln Asn Lys
    195 200 205
    Tyr Leu Gly Val Ile Ile Gln Cys Leu Val Thr Glu Thr Lys Thr Ser
    210 215 220
    Val Ser Arg His Ile Tyr Phe Phe Ser Ala Arg Gly Arg Ile Asp Pro
    225 230 235 240
    Leu Val Tyr Leu Asp Glu Phe Leu Arg Asn Ser Glu Pro Val Leu Lys
    245 250 255
    Arg Val Asn Arg Thr Gly Asn Ser Ser Ser Asn Lys Gln Glu Tyr Gln
    260 265 270
    Leu Leu Lys Asp Asn Leu Val Arg Ser Tyr Asn Lys Ala Leu Lys Lys
    275 280 285
    Asn Ala Pro Tyr Ser Ile Phe Ala Ile Lys Asn Gly Pro Lys Ser His
    290 295 300
    Ile Gly Arg His Leu Met Thr Ser Phe Leu Ser Met Lys Gly Leu Thr
    305 310 315 320
    Glu Leu Thr Asn Val Val Gly Asn Trp Ser Asp Lys Arg Ala Ser Ala
    325 330 335
    Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp His
    340 345 350
    Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Ser Lys
    355 360 365
    Glu Met Ile Ala Leu Lys Asp Glu Thr Asn Pro Ile Glu Glu Trp Gln
    370 375 380
    His Ile Glu Gln Leu Lys Gly Ser Ala Glu Gly Ser Ile Arg Tyr Pro
    385 390 395 400
    Ala Trp Asn Gly Ile Ile Ser Gln Glu Val Leu Asp Tyr Leu Ser Ser
    405 410 415
    Tyr Ile Asn Arg Arg Ile
    420
    <210> SEQ ID NO 62
    <211> LENGTH: 48
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: IR2
    <400> SEQUENCE: 62
    gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 48
    <210> SEQ ID NO 63
    <211> LENGTH: 48
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: IR1
    <400> SEQUENCE: 63
    gaagttccta tactttctag agaataggaa cttcggaata ggaacttc 48
    <210> SEQ ID NO 64
    <211> LENGTH: 66
    <212> TYPE: DNA
    <213> ORGANISM: Bacteriophage mu
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(66)
    <223> OTHER INFORMATION: nucleotide sequence encoding GIN recombinase
    <400> SEQUENCE: 64
    tca act ctg tat aaa aaa cac ccc gcg aaa cga gcg cat ata gaa aac 48
    Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn
    1 5 10 15
    gac gat cga atc aat taa 66
    Asp Asp Arg Ile Asn *
    20
    <210> SEQ ID NO 65
    <211> LENGTH: 21
    <212> TYPE: PRT
    <213> ORGANISM: bacteriophage mu
    <400> SEQUENCE: 65
    Ser Thr Leu Tyr Lys Lys His Pro Ala Lys Arg Ala His Ile Glu Asn
    1 5 10 15
    Asp Asp Arg Ile Asn
    20
    <210> SEQ ID NO 66
    <211> LENGTH: 69
    <212> TYPE: DNA
    <213> ORGANISM: Bacteriophage mu
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(69)
    <223> OTHER INFORMATION: nucleotide sequence encoding Gin recombinase
    <400> SEQUENCE: 66
    tat aaa aaa cat ccc gcg aaa cga acg cat ata gaa aac gac gat cga 48
    Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg
    1 5 10 15
    atc aat caa atc gat cgg taa 69
    Ile Asn Gln Ile Asp Arg *
    20
    <210> SEQ ID NO 67
    <211> LENGTH: 22
    <212> TYPE: PRT
    <213> ORGANISM: bacteriophage mu
    <220> FEATURE:
    <223> OTHER INFORMATION: Gin recombinase of bacteriophage mu
    <400> SEQUENCE: 67
    Tyr Lys Lys His Pro Ala Lys Arg Thr His Ile Glu Asn Asp Asp Arg
    1 5 10 15
    Ile Asn Gln Ile Asp Arg
    20
    <210> SEQ ID NO 68
    <211> LENGTH: 555
    <212> TYPE: DNA
    <213> ORGANISM: Escherichia coli
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(555)
    <223> OTHER INFORMATION: nucleotide sequence encoding PIN recombinase
    <400> SEQUENCE: 68
    atg ctt att ggc tat gta cgc gta tca aca aat gac cag aac aca gat 48
    Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asn Asp Gln Asn Thr Asp
    1 5 10 15
    cta caa cgt aat gcg ctg aac tgt gca gga tgc gag ctg att ttt gaa 96
    Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu
    20 25 30
    gac aag ata agc ggc aca aag tcc gaa agg ccg gga ctg aaa aaa ctg 144
    Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu
    35 40 45
    ctc agg aca tta tcg gca ggt gac act ctg gtt gtc tgg aag ctg gat 192
    Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp
    50 55 60
    cgg ctg ggg cgt agt atg cgg cat ctt gtc gtg ctg gtg gag gag ttg 240
    Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu
    65 70 75 80
    cgc gaa cga ggc atc aac ttt cgt agt ctg acg gat tca att gat acc 288
    Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr
    85 90 95
    agc aca cca atg gga cgc ttt ttc ttt cat gtg atg ggt gcc ctg gct 336
    Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala
    100 105 110
    gaa atg gag cgt gaa ctg att gtt gaa cga aca aaa gct gga ctg gaa 384
    Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu
    115 120 125
    act gct cgt gca cag gga cga att ggt gga cgt cgt ccc aaa ctt aca 432
    Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr
    130 135 140
    cca gaa caa tgg gca caa gct gga cga tta att gca gca gga act cct 480
    Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro
    145 150 155 160
    cgc cag aag gtg gcg att atc tat gat gtt ggt gtg tca act ttg tat 528
    Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr
    165 170 175
    aag agg ttt cct gca ggg gat aaa taa 555
    Lys Arg Phe Pro Ala Gly Asp Lys *
    180
    <210> SEQ ID NO 69
    <211> LENGTH: 184
    <212> TYPE: PRT
    <213> ORGANISM: Escherichia coli
    <400> SEQUENCE: 69
    Met Leu Ile Gly Tyr Val Arg Val Ser Thr Asn Asp Gln Asn Thr Asp
    1 5 10 15
    Leu Gln Arg Asn Ala Leu Asn Cys Ala Gly Cys Glu Leu Ile Phe Glu
    20 25 30
    Asp Lys Ile Ser Gly Thr Lys Ser Glu Arg Pro Gly Leu Lys Lys Leu
    35 40 45
    Leu Arg Thr Leu Ser Ala Gly Asp Thr Leu Val Val Trp Lys Leu Asp
    50 55 60
    Arg Leu Gly Arg Ser Met Arg His Leu Val Val Leu Val Glu Glu Leu
    65 70 75 80
    Arg Glu Arg Gly Ile Asn Phe Arg Ser Leu Thr Asp Ser Ile Asp Thr
    85 90 95
    Ser Thr Pro Met Gly Arg Phe Phe Phe His Val Met Gly Ala Leu Ala
    100 105 110
    Glu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Lys Ala Gly Leu Glu
    115 120 125
    Thr Ala Arg Ala Gln Gly Arg Ile Gly Gly Arg Arg Pro Lys Leu Thr
    130 135 140
    Pro Glu Gln Trp Ala Gln Ala Gly Arg Leu Ile Ala Ala Gly Thr Pro
    145 150 155 160
    Arg Gln Lys Val Ala Ile Ile Tyr Asp Val Gly Val Ser Thr Leu Tyr
    165 170 175
    Lys Arg Phe Pro Ala Gly Asp Lys
    180
    <210> SEQ ID NO 70
    <211> LENGTH: 4778
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pcx plasmid
    <400> SEQUENCE: 70
    gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
    gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
    ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
    ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
    atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
    cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
    tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
    atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
    gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
    gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
    tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
    gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
    ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
    gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
    ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
    tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
    cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
    ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
    tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
    cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
    gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
    ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
    gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
    gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
    tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
    cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
    acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
    gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
    acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcactcct caggtgcagg 1740
    ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag 1800
    atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt 1860
    ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 1920
    cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt 1980
    agagtttggc aacatatgcc atatgctggc tgccatgaac aaaggtggct ataaagaggt 2040
    catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact 2100
    tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa 2160
    ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata 2220
    gctgtccctc ttctcttatg aagatccctc gacctgcagc ccaagcttgg cgtaatcatg 2280
    gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 2340
    cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 2400
    gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc 2460
    aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 2520
    agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 2580
    gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 2640
    ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 2700
    tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 2760
    tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg 2820
    gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2880
    ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2940
    agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 3000
    ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 3060
    caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 3120
    gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 3180
    cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 3240
    tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 3300
    gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 3360
    cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3420
    tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 3480
    tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3540
    caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3600
    aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3660
    cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3720
    ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3780
    tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 3840
    atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3900
    tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3960
    aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 4020
    catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 4080
    gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 4140
    ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 4200
    aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 4260
    atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 4320
    cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 4380
    gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 4440
    agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4500
    gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4560
    caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4620
    ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4680
    tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4740
    aggggttccg cgcacatttc cccgaaaagt gccacctg 4778
    <210> SEQ ID NO 71
    <211> LENGTH: 5510
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCXeGFP plasmid
    <400> SEQUENCE: 71
    gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
    gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
    ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
    ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
    atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
    cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
    tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
    atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
    gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
    gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
    tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
    gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
    ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
    gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
    ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
    tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
    cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
    ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
    tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
    cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
    gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
    ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
    gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
    gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
    tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
    cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
    acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
    gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
    acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740
    agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800
    acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860
    ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1920
    ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980
    tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2040
    acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100
    tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160
    acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220
    tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280
    agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2340
    cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400
    tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460
    ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520
    aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580
    agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640
    ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 2700
    agtatttggt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760
    ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820
    aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880
    acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 2940
    ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000
    ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060
    caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120
    cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180
    gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240
    ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300
    gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360
    ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat 3420
    aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3480
    gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg 3540
    ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3600
    ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3660
    acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3720
    aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3780
    tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3840
    aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3900
    gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 3960
    acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4020
    accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4080
    ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4140
    gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4200
    gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4260
    ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4320
    gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380
    cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440
    cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500
    gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560
    tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620
    gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680
    agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740
    tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800
    agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860
    gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920
    catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980
    ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040
    atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100
    tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5160
    cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220
    cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280
    atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340
    aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400
    ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460
    aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5510
    <210> SEQ ID NO 72
    <211> LENGTH: 282
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: attp
    <400> SEQUENCE: 72
    ccttgcgcta atgctctgtt acaggtcact aataccatct aagtagttga ttcatagtga 60
    ctgcatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa tctaatttaa 120
    tatattgata tttatatcat tttacgtttc tcgttcagct tttttatact aagttggcat 180
    tataaaaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa 240
    aatcattatt tgatttcaat tttgtcccac tccctgcctc tg 282
    <210> SEQ ID NO 73
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer
    <400> SEQUENCE: 73
    ggccccgtaa tgcagaagaa 20
    <210> SEQ ID NO 74
    <211> LENGTH: 32
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer
    <400> SEQUENCE: 74
    ggtttaaagt gcgctcctcc aagaacgtca tc 32
    <210> SEQ ID NO 75
    <211> LENGTH: 40
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer
    <400> SEQUENCE: 75
    agatctagag ccgccgctac aggaacaggt ggtggcggcc 40
    <210> SEQ ID NO 76
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer 5PacSV40
    <400> SEQUENCE: 76
    ctgttaatta actgtggaat gtgtgtcagt tagggtg 37
    <210> SEQ ID NO 77
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer Antisense Zeo
    <400> SEQUENCE: 77
    tgaacagggt cacgtcgtcc 20
    <210> SEQ ID NO 78
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer 5′ HETS
    <400> SEQUENCE: 78
    gggccgaaac gatctcaacc tatt 24
    <210> SEQ ID NO 79
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer 3′ HETS
    <400> SEQUENCE: 79
    cgcagcggcc ctcctactc 19
    <210> SEQ ID NO 80
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer 5BSD
    <400> SEQUENCE: 80
    accatgaaaa catttaacat ttctcaaca 29
    <210> SEQ ID NO 81
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer SV40polyA
    <400> SEQUENCE: 81
    tttatttgtg aaatttgtga tgctattgc 29
    <210> SEQ ID NO 82
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer 3BSP
    <400> SEQUENCE: 82
    ttaatttcgg gtatatttga gtgga 25
    <210> SEQ ID NO 83
    <211> LENGTH: 32
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer EPO5XBA
    <400> SEQUENCE: 83
    tatctagaat gggggtgcac gaatgtcctg cc 32
    <210> SEQ ID NO 84
    <211> LENGTH: 32
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer EPO3SBI
    <400> SEQUENCE: 84
    tacgtacgtc atctgtcccc tgtcctgcag gc 32
    <210> SEQ ID NO 85
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer GENEPO3BSI
    <400> SEQUENCE: 85
    cgtacgtcat ctgtcccctg tcctgca 27
    <210> SEQ ID NO 86
    <211> LENGTH: 28
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Primer GENEPO5XBA
    <400> SEQUENCE: 86
    tctagaatgg gggtgcacgg tgagtact 28
    <210> SEQ ID NO 87
    <211> LENGTH: 4862
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pD2eGFP-1N plasmid from Clontech
    <400> SEQUENCE: 87
    tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
    cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
    gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
    atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
    aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
    catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
    catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
    atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
    ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
    acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600
    ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 660
    gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 720
    atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 780
    gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 840
    cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 900
    taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 960
    caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1020
    ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1080
    ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1140
    gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 1200
    ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1260
    ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1320
    aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 1380
    gacgagctgt acaagaagct tagccatggc ttcccgccgg aggtggagga gcaggatgat 1440
    ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg accgtcaccc tgcagcctgt 1500
    gcttctgcta ggatcaatgt gtagatgcgc ggccgcgact ctagatcata atcagccata 1560
    ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 1620
    aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 1680
    aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 1740
    gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg 1800
    ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 1860
    ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 1920
    tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 1980
    tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 2040
    tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 2100
    aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 2160
    ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 2220
    ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2280
    tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 2340
    caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc 2400
    agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 2460
    tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520
    aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 2580
    ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 2640
    atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 2700
    ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg 2760
    attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 2820
    tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 2880
    caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 2940
    gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3000
    gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3060
    ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3120
    cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3180
    gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3240
    catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 3300
    gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 3360
    cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 3420
    gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 3480
    gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 3540
    gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 3600
    catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 3660
    tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 3720
    accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta 3780
    tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg 3840
    gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat 3900
    acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc 3960
    tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt 4020
    agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 4080
    atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 4140
    aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 4200
    caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 4260
    ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 4320
    cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 4380
    tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 4440
    gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 4500
    ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 4560
    gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 4620
    caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 4680
    ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 4740
    tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 4800
    ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc 4860
    at 4862
    <210> SEQ ID NO 88
    <211> LENGTH: 5192
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pIRESpuro2 plasmid from Clontech
    <400> SEQUENCE: 88
    gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
    ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
    cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
    ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
    gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
    tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
    cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
    attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
    atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
    atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
    tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
    actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
    aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
    gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
    ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900
    gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt 960
    ctccggattc gaattcggat ccgcggccgc atagataact gatccagtgt gctggaatta 1020
    attcgctgtc tgcgagggcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga 1080
    cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg 1140
    cggtgatgcc tttgagggtg gccgcgtcca tctggtcaga aaagacaatc tttttgttgt 1200
    caagcttgag gtgtggcagg cttgagatct ggccatacac ttgagtgaca atgacatcca 1260
    ctttgccttt ctctccacag gtgtccactc ccaggtccaa ctgcaggtcg agcatgcatc 1320
    tagggcggcc aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc 1380
    ttggaataag gccggtgtgc gtttgtctat atgtgatttt ccaccatatt gccgtctttt 1440
    ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt 1500
    tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 1560
    gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 1620
    cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg 1680
    gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 1740
    tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct 1800
    gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 1860
    ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac 1920
    aacccacaag gagacgacct tccatgaccg agtacaagcc cacggtgcgc ctcgccaccc 1980
    gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 2040
    cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg caagaactct 2100
    tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 2160
    tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 2220
    cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 2280
    tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 2340
    accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 2400
    gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 2460
    ggctcggctt caccgtcacc gccgacgtcg agtgcccgaa ggaccgcgcg acctggtgca 2520
    tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga 2580
    gcgcacgacc ccatggctcc gaccgaagcc gacccgggcg gccccgccga ccccgcaccc 2640
    gcccccgagg cccaccgact ctagagctcg ctgatcagcc tcgactgtgc cttctagttg 2700
    ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 2760
    cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 2820
    tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2880
    gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2940
    gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 3000
    cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 3060
    gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3120
    gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3180
    cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3240
    tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3300
    tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3360
    ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3420
    ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3480
    gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3540
    gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3600
    ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 3660
    tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3720
    gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3780
    tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3840
    tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3900
    tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3960
    ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4020
    ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4080
    gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4140
    aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4200
    aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4260
    cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4320
    ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4380
    cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4440
    ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4500
    ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4560
    ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4620
    gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4680
    ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4740
    ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4800
    gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4860
    ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4920
    cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4980
    ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5040
    aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5100
    gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5160
    gcacatttcc ccgaaaagtg ccacctgacg tc 5192
    <210> SEQ ID NO 89
    <211> LENGTH: 11182
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pAg1 Plasmid
    <400> SEQUENCE: 89
    catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60
    atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120
    agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180
    gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240
    agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300
    ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360
    ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420
    acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480
    ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540
    acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600
    agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660
    tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720
    tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780
    ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840
    gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900
    gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960
    cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020
    ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080
    gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140
    tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200
    aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260
    aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320
    ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380
    ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440
    cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500
    atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560
    accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620
    gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680
    gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740
    ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800
    cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860
    aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920
    gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980
    agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040
    ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100
    atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160
    accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220
    tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280
    cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340
    gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400
    tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460
    cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520
    gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580
    tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640
    cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700
    gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760
    gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820
    tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880
    tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940
    agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000
    gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060
    gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120
    ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180
    cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240
    aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300
    catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360
    gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420
    tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480
    cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540
    aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600
    ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660
    gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720
    aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780
    actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840
    aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900
    ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960
    tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020
    gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080
    ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
    gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
    ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
    ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
    acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
    tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
    ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500
    ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
    ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
    actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
    gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
    tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
    caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
    atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
    acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980
    atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040
    ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100
    gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160
    gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220
    ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280
    gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340
    taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400
    cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460
    gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520
    gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580
    atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640
    tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700
    tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760
    tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820
    aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880
    ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940
    caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000
    gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060
    tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120
    cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180
    tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240
    taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300
    gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360
    ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420
    ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480
    ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540
    ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600
    atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660
    gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720
    cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780
    gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840
    cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900
    aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960
    gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020
    gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080
    ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140
    agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200
    cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260
    aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320
    tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380
    taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440
    cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500
    agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560
    gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620
    tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680
    atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740
    gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800
    gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860
    taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920
    aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980
    atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040
    tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100
    gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160
    gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220
    gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280
    tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 8340
    gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 8400
    ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460
    cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 8520
    ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580
    gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640
    ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700
    caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760
    gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820
    accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880
    attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940
    aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000
    ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 9060
    cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 9120
    agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180
    taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240
    cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9300
    ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360
    tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420
    ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480
    aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 9540
    tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600
    cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660
    tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 9720
    tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9780
    catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggagtttagc gagagcctga 9840
    cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9900
    tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960
    gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020
    gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080
    acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140
    gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200
    atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 10260
    aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320
    acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380
    gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440
    gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500
    aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560
    gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620
    atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680
    ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740
    gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800
    agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttggcact 10860
    ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 10920
    tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 10980
    ttcccaacag ttgcgcagcc tgaatggcga atgctagagc agcttgagct tggatcagat 11040
    tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 11100
    cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 11160
    tccgttcgtc catttgtatg tg 11182
    <210> SEQ ID NO 90
    <211> LENGTH: 8428
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCambia3300 Plasmid
    <400> SEQUENCE: 90
    catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60
    atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120
    agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180
    gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240
    agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300
    ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360
    ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420
    acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480
    ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540
    acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600
    agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660
    tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720
    tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780
    ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840
    gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900
    gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960
    cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020
    ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080
    gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140
    tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200
    aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260
    aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320
    ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380
    ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440
    cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500
    atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560
    accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620
    gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680
    gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740
    ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800
    cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860
    aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920
    gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980
    agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040
    ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100
    atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160
    accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220
    tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280
    cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340
    gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400
    tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460
    cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520
    gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580
    tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640
    cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700
    gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760
    gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820
    tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880
    tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940
    agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000
    gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060
    gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120
    ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180
    cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240
    aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300
    catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360
    gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420
    tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480
    cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540
    aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600
    ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660
    gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720
    aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780
    actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840
    aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900
    ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960
    tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020
    gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080
    ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
    gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
    ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
    ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
    acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
    tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
    ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500
    ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
    ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
    actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
    gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
    tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
    caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
    atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
    acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980
    atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040
    ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100
    gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160
    gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220
    ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280
    gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340
    taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400
    cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460
    gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520
    gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580
    atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640
    tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700
    tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760
    tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820
    aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880
    ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940
    caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000
    gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060
    tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120
    cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180
    tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240
    taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300
    gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360
    ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420
    ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480
    ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540
    ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600
    atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660
    gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720
    cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780
    gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840
    cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900
    aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960
    gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020
    gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080
    ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140
    agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200
    cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260
    aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320
    tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380
    taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440
    cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500
    agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560
    gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620
    tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680
    atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740
    gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800
    gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860
    taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920
    aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980
    atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040
    tacgaattcg agctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctt 8100
    ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 8160
    tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 8220
    tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct tgagcttgga 8280
    tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg 8340
    ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa 8400
    ggtttatccg ttcgtccatt tgtatgtg 8428
    <210> SEQ ID NO 91
    <211> LENGTH: 3438
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pLIT38attBZeo Plasmid
    <400> SEQUENCE: 91
    tcgaccctct agtcaaggcc ttaagtgagt cgtattacgg actggccgtc gttttacaac 60
    gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 120
    tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 180
    gcctgaatgg cgaatggcgc ttcgcttggt aataaagccc gcttcggcgg gctttttttt 240
    gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 300
    tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 360
    ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 420
    ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 480
    tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 540
    gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 600
    gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 660
    acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 720
    tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 780
    caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 840
    gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 900
    cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 960
    tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 1020
    agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 1080
    tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 1140
    ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 1200
    acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1260
    ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1320
    aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1380
    aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1440
    aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1500
    ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1560
    ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1620
    aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1680
    gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1740
    cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1800
    atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1860
    ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1920
    ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1980
    ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2040
    ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2100
    ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2160
    tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2220
    tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2280
    tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2340
    tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2400
    gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2460
    tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2520
    ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2580
    accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2640
    acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2700
    ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2760
    tgctttttta tactaacttg agcgaaatct ggatccatgg ccaagttgac cagtgccgtt 2820
    ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 2880
    ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 2940
    ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 3000
    cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 3060
    gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 3120
    cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta 3180
    cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg 3240
    gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc 3300
    aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 3360
    aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 3420
    tatcatgtct gtataccg 3438
    <210> SEQ ID NO 92
    <211> LENGTH: 10549
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCambia1302 Plasmid
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #AF234398
    <309> DATABASE ENTRY DATE: 2000-04-24
    <400> SEQUENCE: 92
    catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60
    tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120
    tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180
    gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240
    tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300
    gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360
    agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420
    cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480
    gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag acggcggcgt 540
    gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600
    agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 660
    ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 720
    atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780
    ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840
    cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 900
    gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960
    acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020
    ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080
    cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140
    tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200
    ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260
    tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320
    tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380
    cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 1440
    agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500
    aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560
    cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 1620
    agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 1680
    agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 1740
    ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800
    gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 1860
    atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 1920
    ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 1980
    cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040
    ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100
    aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160
    tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220
    ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280
    gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340
    tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400
    taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 2460
    actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 2520
    ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580
    ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 2640
    ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700
    aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760
    ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820
    catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 2880
    tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940
    agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 3000
    actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 3060
    ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120
    gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180
    gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240
    gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300
    ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360
    cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420
    caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 3480
    cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540
    tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600
    aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660
    agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720
    tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780
    gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 3840
    tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 3900
    accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960
    tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020
    ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080
    tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140
    ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200
    aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260
    tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320
    tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380
    ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 4440
    ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500
    ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560
    gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 4620
    accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680
    tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740
    aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 4800
    tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860
    ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 4920
    gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980
    gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040
    gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100
    ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160
    ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5220
    cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5280
    actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340
    tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400
    aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460
    ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520
    aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580
    cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640
    cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700
    aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760
    cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5820
    ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880
    ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940
    gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000
    agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060
    acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120
    acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180
    agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240
    agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300
    ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360
    gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420
    gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480
    ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540
    agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600
    cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 6660
    catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720
    tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780
    ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840
    ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900
    ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960
    taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020
    gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080
    gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140
    taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 7200
    agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260
    cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320
    tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380
    aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440
    tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500
    acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 7560
    ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620
    gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680
    gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740
    tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800
    tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860
    gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920
    cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa 7980
    gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040
    tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100
    ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160
    cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220
    gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280
    cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340
    tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400
    ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg 8460
    gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520
    gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580
    ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640
    ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc cgggatctgc 8700
    gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760
    aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820
    atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880
    gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940
    agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000
    ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060
    gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120
    atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180
    cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240
    gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300
    gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 9360
    gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420
    aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480
    gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540
    tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600
    cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660
    cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9720
    tatgaccatg attacgaatt cgagctcggt acccggggat cctctagagt cgacctgcag 9780
    gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 9840
    tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 9900
    ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gctagagcag 9960
    cttgagcttg gatcagattg tcgtttcccg ccttcagttt agcttcatgg agtcaaagat 10020
    tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 10080
    cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 10140
    ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 10200
    acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 10260
    tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 10320
    ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 10380
    gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 10440
    tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 10500
    tatataagga agttcatttc atttggagag aacacggggg actcttgac 10549
    <210> SEQ ID NO 93
    <211> LENGTH: 33
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: CaMV35SpolyA Primer
    <400> SEQUENCE: 93
    ctgaattaac gccgaattaa ttcgggggat ctg 33
    <210> SEQ ID NO 94
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: CaMV35Spr Primer
    <400> SEQUENCE: 94
    ctagagcagc ttgccaacat ggtggagca 29
    <210> SEQ ID NO 95
    <211> LENGTH: 12592
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pAg2 Plasmid
    <400> SEQUENCE: 95
    gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta 60
    gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag 120
    ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc 180
    ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg 240
    ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg 300
    ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc 360
    cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc 420
    gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc 480
    aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca 540
    ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt 600
    acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt 660
    ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc tgtgcataac 720
    tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct 780
    ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg 840
    gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc 900
    ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 960
    cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 1020
    gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 1080
    cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 1140
    gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 1200
    ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1260
    cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1320
    gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1380
    tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1440
    agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1500
    tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1560
    cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1620
    ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1680
    ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1740
    ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1800
    ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1860
    cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1920
    gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1980
    atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2040
    ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa tattttattt 2100
    tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc 2160
    cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc 2220
    cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt 2280
    ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat 2340
    catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat 2400
    cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg 2460
    tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct 2520
    cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg 2580
    cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa 2640
    caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg 2700
    tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct 2760
    tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca 2820
    gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt cctcttttct 2880
    acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt 2940
    ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt 3000
    ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac 3060
    gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc 3120
    ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt 3180
    acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat 3240
    tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt 3300
    gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga 3360
    attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg ttttaggaat 3420
    tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg gtttcttata 3480
    tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg gaactactca 3540
    cacattatta tggagaaact cgagtcaaat ctcggtgacg ggcaggaccg gacggggcgg 3600
    taccggcagg ctgaagtcca gctgccagaa acccacgtca tgccagttcc cgtgcttgaa 3660
    gccggccgcc cgcagcatgc cgcggggggc atatccgagc gcctcgtgca tgcgcacgct 3720
    cgggtcgttg ggcagcccga tgacagcgac cacgctcttg aagccctgtg cctccaggga 3780
    cttcagcagg tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc ggggggagac 3840
    gtacacggtc gactcggccg tccagtcgta ggcgttgcgt gccttccagg ggcccgcgta 3900
    ggcgatgccg gcgacctcgc cgtccacctc ggcgacgagc cagggatagc gctcccgcag 3960
    acggacgagg tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga agttgaccgt 4020
    gcttgtctcg atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg cctcggtggc 4080
    acggcggatg tcggccgggc gtcgttctgg gctcatggta gactcgagag agatagattt 4140
    gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata 4200
    gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 4260
    cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 4320
    tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 4380
    ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 4440
    tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 4500
    gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 4560
    cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa gacgtggttg 4620
    gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 4680
    cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 4740
    caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 4800
    ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 4860
    ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct 4920
    gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 4980
    gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 5040
    gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 5100
    aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattcga 5160
    gccttgacta gagggtcgac ggtatacaga catgataaga tacattgatg agtttggaca 5220
    aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc 5280
    tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca 5340
    tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca 5400
    acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag tcctgctcct 5460
    cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc cgcccccacg 5520
    gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc gtggacacga 5580
    cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag gccagggtgt 5640
    tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg tcccggacca 5700
    caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg gtccagaact 5760
    cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc aacttggcca 5820
    tggatccaga tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg caggaattcg 5880
    atcgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 5940
    attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 6000
    atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 6060
    tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 6120
    cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 6180
    gtggattgat gtgataacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 6240
    gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 6300
    aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 6360
    gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 6420
    tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 6480
    gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 6540
    gatgacgcac aatcccacta tccttcgcaa gaccttcctc tatataagga agttcatttc 6600
    atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct ctctcgagct 6660
    ttcgcagatc cgggggggca atgagatatg aaaaagcctg aactcaccgc gacgtctgtc 6720
    gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 6780
    gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 6840
    agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 6900
    ctcccgattc cggaagtgct tgacattggg gagtttagcg agagcctgac ctattgcatc 6960
    tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 7020
    ctacaaccgg tcgcggaggc tatggatgcg atcgctgcgg ccgatcttag ccagacgagc 7080
    gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 7140
    tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 7200
    gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 7260
    cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 7320
    acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 7380
    atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 7440
    aggcatccgg agcttgcagg atcgccacga ctccgggcgt atatgctccg cattggtctt 7500
    gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 7560
    cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 7620
    agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 7680
    cgccccagca ctcgtccgag ggcaaagaaa tagagtagat gccgaccgga tctgtcgatc 7740
    gacaagctcg agtttctcca taataatgtg tgagtagttc ccagataagg gaattagggt 7800
    tcctataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt 7860
    tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat 7920
    ccagatcccc cgaattaatt cggcgttaat tcagatcaag cttggcactg gccgtcgttt 7980
    tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 8040
    cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 8100
    tgcgcagcct gaatggcgaa tgctagagca gcttgagctt ggatcagatt gtcgtttccc 8160
    gccttcagtt tggggatcct ctagactgaa ggcgggaaac gacaatctga tcatgagcgg 8220
    agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc gttttacgtt 8280
    tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc tggagtttaa 8340
    tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc taaggtcact 8400
    atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa attcccctcg 8460
    gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg gatctcgaga 8520
    atcgaattcc cgcggccgcc atggtagatc tgactagtaa aggagaagaa cttttcactg 8580
    gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca 8640
    gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta 8700
    ctggaaaact acctgttccg tggccaacac ttgtcactac tttctcttat ggtgttcaat 8760
    gcttttcaag atacccagat catatgaagc ggcacgactt cttcaagagc gccatgcctg 8820
    agggatacgt gcaggagagg accatcttct tcaaggacga cgggaactac aagacacgtg 8880
    ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat cgagcttaag ggaatcgatt 8940
    tcaaggagga cggaaacatc ctcggccaca agttggaata caactacaac tcccacaacg 9000
    tatacatcat ggccgacaag caaaagaacg gcatcaaagc caacttcaag acccgccaca 9060
    acatcgaaga cggcggcgtg caactcgctg atcattatca acaaaatact ccaattggcg 9120
    atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag 9180
    atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta 9240
    cacatggcat ggatgaacta tacaaagcta gccaccacca ccaccaccac gtgtgaattg 9300
    gtgaccagct cgaatttccc cgatcgttca aacatttggc aataaagttt cttaagattg 9360
    aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat 9420
    gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc 9480
    ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa 9540
    ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat taaactatca gtgtttgaca 9600
    ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac ggatatttaa 9660
    aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc 9720
    ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg 9780
    acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca 9840
    ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc gcataaagta 9900
    gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct 9960
    gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact 10020
    gcacgcggcc ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg 10080
    cccggagctg gccaggatgc ttgaccacct acgccctggc gacgttgtga cagtgaccag 10140
    gctagaccgc ctggcccgca gcacccgcga cctactggac attgccgagc gcatccagga 10200
    ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc gacaccacca cgccggccgg 10260
    ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc taatcatcga 10320
    ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc 10380
    taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac 10440
    cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg accctgtacc gcgcacttga 10500
    gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc 10560
    attgaccgag gccgacgccc tggcggccgc cgagaatgaa cgccaagagg aacaagcatg 10620
    aaaccgcacc aggacggcca ggacgaaccg tttttcatta ccgaagagat cgaggcggag 10680
    atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat 10740
    gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct ggccggccag cttggccgct 10800
    gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt 10860
    catgcggtcg ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa ggggaacgca 10920
    tgaaggttat cgctgtactt aaccagaaag gcgggtcagg caagacgacc atcgcaaccc 10980
    atctagcccg cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat tccgatcccc 11040
    agggcagtgc ccgcgattgg gcggccgtgc gggaagatca accgctaacc gttgtcggca 11100
    tcgaccgccc gacgattgac cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga 11160
    tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg 11220
    tgctgattcc ggtgcagcca agcccttacg acatatgggc caccgccgac ctggtggagc 11280
    tggttaagca gcgcattgag gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc 11340
    gggcgatcaa aggcacgcgc atcggcggtg aggttgccga ggcgctggcc gggtacgagc 11400
    tgcccattct tgagtcccgt atcacgcagc gcgtgagcta cccaggcact gccgccgccg 11460
    gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg 11520
    ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag 11580
    cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg caacgttggc 11640
    cagcctggca gacacgccag ccatgaagcg ggtcaacttt cagttgccgg cggaggatca 11700
    caccaagctg aagatgtacg cggtacgcca aggcaagacc attaccgagc tgctatctga 11760
    atacatcgcg cagctaccag agtaaatgag caaatgaata aatgagtaga tgaattttag 11820
    cggctaaagg aggcggcatg gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc 11880
    ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc tgccggccct 11940
    gcaatggcac tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa accatccggc 12000
    ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag 12060
    gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc 12120
    gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg 12180
    aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc 12240
    acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga 12300
    cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg 12360
    ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta 12420
    accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt 12480
    ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac 12540
    gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca gc 12592
    <210> SEQ ID NO 96
    <211> LENGTH: 3357
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pGEMEasyNOS Plasmid
    <400> SEQUENCE: 96
    tatcactagt gaattcgcgg ccgcctgcag gtcgaccata tgggagagct cccaacgcgt 60
    tggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta atcatggtca 120
    tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 180
    agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 240
    cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 300
    caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 360
    tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 420
    cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 480
    aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 540
    gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 600
    agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 660
    cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 720
    cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 780
    ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 840
    gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 900
    tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 960
    acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 1020
    tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 1080
    attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 1140
    gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 1200
    ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 1260
    taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 1320
    ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 1380
    ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 1440
    gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 1500
    ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 1560
    gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 1620
    tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 1680
    atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 1740
    gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 1800
    tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 1860
    atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 1920
    agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 1980
    ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 2040
    tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 2100
    aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 2160
    tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 2220
    aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga tgcggtgtga 2280
    aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag cgttaatatt 2340
    ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 2400
    atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 2460
    gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 2520
    gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 2580
    aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 2640
    ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 2700
    gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 2760
    ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 2820
    tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 2880
    gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattgt 2940
    aatacgactc actatagggc gaattgggcc cgacgtcgca tgctcccggc cgccatggcg 3000
    gccgcgggaa ttcgattctc gagatccggt gcagattatt tggattgaga gtgaatatga 3060
    gactctaatt ggataccgag gggaatttat ggaacgtcag tggagcattt ttgacaagaa 3120
    atatttgcta gctgatagtg accttaggcg acttttgaac gcgcaataat ggtttctgac 3180
    gtatgtgctt agctcattaa actccagaaa cccgcggctg agtggctcct tcaacgttgc 3240
    ggttctgtca gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg 3300
    tgactccctt aattctccgc tcatgatcag attgtcgttt cccgccttca gtctaga 3357
    <210> SEQ ID NO 97
    <211> LENGTH: 10122
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p1302NOS Plasmid
    <400> SEQUENCE: 97
    catggtagat ctgactagta aaggagaaga acttttcact ggagttgtcc caattcttgt 60
    tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 120
    tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 180
    gtggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 240
    tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 300
    gaccatcttc ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 360
    agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 420
    cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca tggccgacaa 480
    gcaaaagaac ggcatcaaag ccaacttcaa gacccgccac aacatcgaag acggcggcgt 540
    gcaactcgct gatcattatc aacaaaatac tccaattggc gatggccctg tccttttacc 600
    agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 660
    ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 720
    atacaaagct agccaccacc accaccacca cgtgtgaatt ggtgaccagc tcgaatttcc 780
    ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 840
    cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 900
    gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 960
    acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 1020
    ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 1080
    cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 1140
    tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 1200
    ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 1260
    tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 1320
    tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 1380
    cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 1440
    agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 1500
    aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 1560
    cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 1620
    agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 1680
    agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 1740
    ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 1800
    gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 1860
    atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 1920
    ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 1980
    cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 2040
    ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 2100
    aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 2160
    tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 2220
    ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 2280
    gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 2340
    tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 2400
    taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 2460
    actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 2520
    ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 2580
    ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 2640
    ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 2700
    aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 2760
    ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 2820
    catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 2880
    tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 2940
    agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 3000
    actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 3060
    ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 3120
    gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 3180
    gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 3240
    gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 3300
    ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 3360
    cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 3420
    caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 3480
    cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 3540
    tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 3600
    aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 3660
    agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 3720
    tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 3780
    gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 3840
    tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 3900
    accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 3960
    tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 4020
    ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 4080
    tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 4140
    ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 4200
    aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 4260
    tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 4320
    tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 4380
    ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 4440
    ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 4500
    ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 4560
    gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 4620
    accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 4680
    tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 4740
    aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 4800
    tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 4860
    ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 4920
    gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 4980
    gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 5040
    gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 5100
    ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 5160
    ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 5220
    cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 5280
    actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5340
    tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5400
    aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5460
    ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5520
    aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5580
    cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5640
    cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700
    aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5760
    cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5820
    ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5880
    ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5940
    gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000
    agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6060
    acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 6120
    acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 6180
    agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 6240
    agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 6300
    ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 6360
    gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 6420
    gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 6480
    ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 6540
    agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 6600
    cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 6660
    catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 6720
    tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 6780
    ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 6840
    ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 6900
    ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 6960
    taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 7020
    gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 7080
    gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 7140
    taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 7200
    agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 7260
    cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 7320
    tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 7380
    aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcgggggatc 7440
    tggattttag tactggattt tggttttagg aattagaaat tttattgata gaagtatttt 7500
    acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg aaaccctata 7560
    ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa actcgagctt 7620
    gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac gagtgctggg 7680
    gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct 7740
    tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca 7800
    tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg 7860
    gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa gctccggatg 7920
    cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa 7980
    gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc 8040
    tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg 8100
    ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga 8160
    cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc 8220
    gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa 8280
    cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggttg 8340
    tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg 8400
    ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt ccggaatcgg 8460
    gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca 8520
    gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc 8580
    ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt 8640
    ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc ccggatctgc 8700
    gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt gtcctctcca 8760
    aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg attgtgcgtc 8820
    atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac 8880
    gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc 8940
    agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt aggtgccacc 9000
    ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga atccgaggag 9060
    gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt 9120
    atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat cacatcaatc 9180
    cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg 9240
    gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc 9300
    gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca 9360
    gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc 9420
    aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc 9480
    gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct ccccgcgcgt 9540
    tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 9600
    cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 9660
    cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 9720
    tatgaccatg attacgaatt cgagctcggt acccggggat cctctagact gaaggcggga 9780
    aacgacaatc tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga 9840
    cgcgggacaa gccgttttac gtttggaact gacagaaccg caacgttgaa ggagccactc 9900
    agccgcgggt ttctggagtt taatgagcta agcacatacg tcagaaacca ttattgcgcg 9960
    ttcaaaagtc gcctaaggtc actatcagct agcaaatatt tcttgtcaaa aatgctccac 10020
    tgacgttcca taaattcccc tcggtatcca attagagtct catattcact ctcaatccaa 10080
    ataatctgca ccggatctcg agaatcgaat tcccgcggcc gc 10122
    <210> SEQ ID NO 98
    <211> LENGTH: 621
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: N. tabacum rDNA intergnic spacer (IGS) sequence
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #Y08422
    <309> DATABASE ENTRY DATE: 1997-10-31
    <400> SEQUENCE: 98
    gtgctagcca atgtttaaca agatgtcaag cacaatgaat gttggtggtt ggtggtcgtg 60
    gctggcggtg gtggaaaatt gcggtggttc gagcggtagt gatcggcgat ggttggtgtt 120
    tgcagcggtg tttgatatcg gaatcactta tggtggttgt cacaatggag gtgcgtcatg 180
    gttattggtg gttggtcatc tatatatttt tataataata ttaagtattt tacctatttt 240
    ttacatattt tttattaaat ttatgcattg tttgtatttt taaatagttt ttatcgtact 300
    tgttttataa aatattttat tattttatgt gttatattat tacttgatgt attggaaatt 360
    ttctccattg ttttttctat atttataata attttcttat ttttttttgt tttattatgt 420
    attttttcgt tttataataa atatttatta aaaaaaatat tatttttgta aaatatatca 480
    tttacaatgt ttaaaagtca tttgtgaata tattagctaa gttgtacttc tttttgtgca 540
    tttggtgttg tacatgtcta ttatgattct ctggccaaaa catgtctact cctgtcactt 600
    gggttttttt ttttaagaca t 621
    <210> SEQ ID NO 99
    <211> LENGTH: 25
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: NTIGS-F1 Primer
    <400> SEQUENCE: 99
    gtgctagcca atgtttaaca agatg 25
    <210> SEQ ID NO 100
    <211> LENGTH: 28
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: NTIGS-R1 Primer
    <400> SEQUENCE: 100
    atgtcttaaa aaaaaaaacc caagtgac 28
    <210> SEQ ID NO 101
    <211> LENGTH: 233
    <212> TYPE: DNA
    <213> ORGANISM: Mus Musculus
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #V00846
    <309> DATABASE ENTRY DATE: 1989-07-06
    <400> SEQUENCE: 101
    gacctggaat atggcgagaa aactgaaaat cacggaaaat gagaaataca cactttagga 60
    cgtgaaatat ggcgaggaaa actgaaaaag gtggaaaatt tagaaatgtc cactgtagga 120
    cgtggaatat ggcaagaaaa ctgaaaatca tggaaaatga gaaacatcca cttgacgact 180
    tgaaaaatga cgaaatcact aaaaaacgtg aaaaatgaga aatgcacact gaa 233
    <210> SEQ ID NO 102
    <211> LENGTH: 31
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MSAT-F1 Primer
    <400> SEQUENCE: 102
    aataccgcgg aagcttgacc tggaatatcg c 31
    <210> SEQ ID NO 103
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: MSAT-Ri Primer
    <400> SEQUENCE: 103
    ataaccgcgg agtccttcag tgtgcat 27
    <210> SEQ ID NO 104
    <211> LENGTH: 277
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Nopaline Synthase Promoter Sequence
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #U09365
    <309> DATABASE ENTRY DATE: 1997-10-17
    <400> SEQUENCE: 104
    gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60
    tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120
    aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180
    attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240
    gcgcgcggtg tcatctatgt tactagatcg ggaattc 277
    <210> SEQ ID NO 105
    <211> LENGTH: 1812
    <212> TYPE: DNA
    <213> ORGANISM: Escherichia coli
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)...(1812)
    <223> OTHER INFORMATION: Beta-Glucuronidase
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #S69414
    <309> DATABASE ENTRY DATE: 1994-09-23
    <400> SEQUENCE: 105
    atg tta cgt cct gta gaa acc cca acc cgt gaa atc aaa aaa ctc gac 48
    Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp
    1 5 10 15
    ggc ctg tgg gca ttc agt ctg gat cgc gaa aac tgt gga att gat cag 96
    Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln
    20 25 30
    cgt tgg tgg gaa agc gcg tta caa gaa agc cgg gca att gct gtg cca 144
    Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro
    35 40 45
    ggc agt ttt aac gat cag ttc gcc gat gca gat att cgt aat tat gcg 192
    Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala
    50 55 60
    ggc aac gtc tgg tat cag cgc gaa gtc ttt ata ccg aaa ggt tgg gca 240
    Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala
    65 70 75 80
    ggc cag cgt atc gtg ctg cgt ttc gat gcg gtc act cat tac ggc aaa 288
    Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys
    85 90 95
    gtg tgg gtc aat aat cag gaa gtg atg gag cat cag ggc ggc tat acg 336
    Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr
    100 105 110
    cca ttt gaa gcc gat gtc acg ccg tat gtt att gcc ggg aaa agt gta 384
    Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val
    115 120 125
    cgt atc acc gtt tgt gtg aac aac gaa ctg aac tgg cag act atc ccg 432
    Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro
    130 135 140
    ccg gga atg gtg att acc gac gaa aac ggc aag aaa aag cag tct tac 480
    Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr
    145 150 155 160
    ttc cat gat ttc ttt aac tat gcc gga atc cat cgc agc gta atg ctc 528
    Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu
    165 170 175
    tac acc acg ccg aac acc tgg gtg gac gat atc acc gtg gtg acg cat 576
    Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His
    180 185 190
    gtc gcg caa gac tgt aac cac gcg tct gtt gac tgg cag gtg gtg gcc 624
    Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala
    195 200 205
    aat ggt gat gtc agc gtt gaa ctg cgt gat gcg gat caa cag gtg gtt 672
    Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val
    210 215 220
    gca act gga caa ggc act agc ggg act ttg caa gtg gtg aat ccg cac 720
    Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His
    225 230 235 240
    ctc tgg caa ccg ggt gaa ggt tat ctc tat gaa ctg tgc gtc aca gcc 768
    Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala
    245 250 255
    aaa agc cag aca gag tgt gat atc tac ccg ctt cgc gtc ggc atc cgg 816
    Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg
    260 265 270
    tca gtg gca gtg aag ggc gaa cag ttc ctg att aac cac aaa ccg ttc 864
    Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe
    275 280 285
    tac ttt act ggc ttt ggt cgt cat gaa gat gcg gac ttg cgt ggc aaa 912
    Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys
    290 295 300
    gga ttc gat aac gtg ctg atg gtg cac gac cac gca tta atg gac tgg 960
    Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp
    305 310 315 320
    att ggg gcc aac tcc tac cgt acc tcg cat tac cct tac gct gaa gag 1008
    Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu
    325 330 335
    atg ctc gac tgg gca gat gaa cat ggc atc gtg gtg att gat gaa act 1056
    Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr
    340 345 350
    gct gct gtc ggc ttt aac ctc tct tta ggc att ggt ttc gaa gcg ggc 1104
    Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly
    355 360 365
    aac aag ccg aaa gaa ctg tac agc gaa gag gca gtc aac ggg gaa act 1152
    Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr
    370 375 380
    cag caa gcg cac tta cag gcg att aaa gag ctg ata gcg cgt gac aaa 1200
    Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys
    385 390 395 400
    aac cac cca agc gtg gtg atg tgg agt att gcc aac gaa ccg gat acc 1248
    Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr
    405 410 415
    cgt ccg caa ggt gca cgg gaa tat ttc gcg cca ctg gcg gaa gca acg 1296
    Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr
    420 425 430
    cgt aaa ctc gac ccg acg cgt ccg atc acc tgc gtc aat gta atg ttc 1344
    Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe
    435 440 445
    tgc gac gct cac acc gat acc atc agc gat ctc ttt gat gtg ctg tgc 1392
    Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys
    450 455 460
    ctg aac cgt tat tac gga tgg tat gtc caa agc ggc gat ttg gaa acg 1440
    Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr
    465 470 475 480
    gca gag aag gta ctg gaa aaa gaa ctt ctg gcc tgg cag gag aaa ctg 1488
    Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu
    485 490 495
    cat cag ccg att atc atc acc gaa tac ggc gtg gat acg tta gcc ggg 1536
    His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly
    500 505 510
    ctg cac tca atg tac acc gac atg tgg agt gaa gag tat cag tgt gca 1584
    Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala
    515 520 525
    tgg ctg gat atg tat cac cgc gtc ttt gat cgc gtc agc gcc gtc gtc 1632
    Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val
    530 535 540
    ggt gaa cag gta tgg aat ttc gcc gat ttt gcg acc tcg caa ggc ata 1680
    Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile
    545 550 555 560
    ttg cgc gtt ggc ggt aac aag aaa ggg atc ttc act cgc gac cgc aaa 1728
    Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys
    565 570 575
    ccg aag tcg gcg gct ttt ctg ctg caa aaa cgc tgg act ggc atg aac 1776
    Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn
    580 585 590
    ttc ggt gaa aaa ccg cag cag gga ggc aaa caa tga 1812
    Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln *
    595 600
    <210> SEQ ID NO 106
    <211> LENGTH: 603
    <212> TYPE: PRT
    <213> ORGANISM: Escherichia coli
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: Genbank #S69414
    <309> DATABASE ENTRY DATE: 1994-09-23
    <400> SEQUENCE: 106
    Met Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp
    1 5 10 15
    Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln
    20 25 30
    Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro
    35 40 45
    Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala
    50 55 60
    Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala
    65 70 75 80
    Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys
    85 90 95
    Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr
    100 105 110
    Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val
    115 120 125
    Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro
    130 135 140
    Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr
    145 150 155 160
    Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu
    165 170 175
    Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His
    180 185 190
    Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala
    195 200 205
    Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val
    210 215 220
    Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His
    225 230 235 240
    Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala
    245 250 255
    Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg
    260 265 270
    Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe
    275 280 285
    Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys
    290 295 300
    Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp
    305 310 315 320
    Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu
    325 330 335
    Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr
    340 345 350
    Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly
    355 360 365
    Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr
    370 375 380
    Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys
    385 390 395 400
    Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr
    405 410 415
    Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr
    420 425 430
    Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe
    435 440 445
    Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys
    450 455 460
    Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr
    465 470 475 480
    Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu
    485 490 495
    His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly
    500 505 510
    Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala
    515 520 525
    Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val
    530 535 540
    Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile
    545 550 555 560
    Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys
    565 570 575
    Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn
    580 585 590
    Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln
    595 600
    <210> SEQ ID NO 107
    <211> LENGTH: 277
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Nopaline Synthase Terminator Sequence
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: U09365
    <309> DATABASE ENTRY DATE: 1995-10-17
    <400> SEQUENCE: 107
    gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 60
    tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 120
    aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 180
    attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 240
    gcgcgcggtg tcatctatgt tactagatcg ggaattc 277
    <210> SEQ ID NO 108
    <211> LENGTH: 3451
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: HindIII Fragment containing the
    beta-glucuronidase coding sequence, the rDNA intergenic spacer,
    and the Mast1 sequence
    <400> SEQUENCE: 108
    aagcttgacc tggaatatcg cgagtaaact gaaaatcacg gaaaatgaga aatacacact 60
    ttaggacgtg aaatatggcg aggaaaactg aaaaaggtgg aaaatttaga aatgtccact 120
    gtaggacgtg gaatatggca agaaaactga aaatcatgga aaatgagaaa catccacttg 180
    acgacttgaa aaatgacgaa atcactaaaa aacgtgaaaa atgagaaatg cacactgaag 240
    gactccgcgg gaattcgatt gtgctagcca atgtttaaca agatgtcaag cacaatgaat 300
    gttggtggtt ggtggtcgtg gctggcggtg gtggaaaatt gcggtggttc gagcggtagt 360
    gatcggcgat ggttggtgtt tgcagcggtg tttgatatcg gaatcactta tggtggttgt 420
    cacaatggag gtgcgtcatg gttattggtg gttggtcatc tatatatttt tataataata 480
    ttaagtattt tacctatttt ttacatattt tttattaaat ttatgcattg tttgtatttt 540
    taaatagttt ttatcgtact tgttttataa aatattttat tattttatgt gttatattat 600
    tacttgatgt attggaaatt ttctccattg ttttttctat atttataata attttcttat 660
    ttttttttgt tttattatgt attttttcgt tttataataa atatttatta aaaaaaatat 720
    tatttttgta aaatatatca tttacaatgt ttaaaagtca tttgtgaata tattagctaa 780
    gttgtacttc tttttgtgca tttggtgttg tacatgtcta ttatgattct ctggccaaaa 840
    catgtctact cctgtcactt gggttttttt ttttaagaca taatcactag tgattatatc 900
    tagactgaag gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgtta 960
    tgacccccgc cgatgacgcg ggacaagccg ttttacgttt ggaactgaca gaaccgcaac 1020
    gttgaaggag ccactcagcc gcgggtttct ggagtttaat gagctaagca catacgtcag 1080
    aaaccattat tgcgcgttca aaagtcgcct aaggtcacta tcagctagca aatatttctt 1140
    gtcaaaaatg ctccactgac gttccataaa ttcccctcgg tatccaatta gagtctcata 1200
    ttcactctca atccaaataa tctgcaccgg atctcgagat cgaattcccg cggccgcgaa 1260
    ttcactagtg gatccccggg tacggtcagt cccttatgtt acgtcctgta gaaaccccaa 1320
    cccgtgaaat caaaaaactc gacggcctgt gggcattcag tctggatcgc gaaaactgtg 1380
    gaattgagca gcgttggtgg gaaagcgcgt tacaagaaag ccgggcaatt gctgtgccag 1440
    gcagttttaa cgatcagttc gccgatgcag atattcgtaa ttatgtgggc aacgtctggt 1500
    atcagcgcga agtctttata ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg 1560
    atgcggtcac tcattacggc aaagtgtggg tcaataatca ggaagtgatg gagcatcagg 1620
    gcggctatac gccatttgaa gccgatgtca cgccgtatgt tattgccggg aaaagtgtac 1680
    gtatcacagt ttgtgtgaac aacgaactga actggcagac tatcccgccg ggaatggtga 1740
    ttaccgacga aaacggcaag aaaaagcagt cttacttcca tgatttcttt aactacgccg 1800
    ggatccatcg cagcgtaatg ctctacacca cgccgaacac ctgggtggac gatatcaccg 1860
    tggtgacgca tgtcgcgcaa gactgtaacc acgcgtctgt tgactggcag gtggtggcca 1920
    atggtgatgt cagcgttgaa ctgcgtgatg cggatcaaca ggtggttgca actggacaag 1980
    gcaccagcgg gactttgcaa gtggtgaatc cgcacctctg gcaaccgggt gaaggttatc 2040
    tctatgaact gtacgtcaca gccaaaagcc agacagagtg tgatatctac ccgctgcgcg 2100
    tcggcatccg gtcagtggca gtgaagggcg aacagttcct gatcaaccac aaaccgttct 2160
    actttactgg ctttggccgt catgaagatg cggatttgcg cggcaaagga ttcgataacg 2220
    tgctgatggt gcacgatcac gcattaatgg actggattgg ggccaactcc taccgtacct 2280
    cgcattaccc ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga 2340
    ttgatgaaac tgcagctgtc ggctttaacc tctctttagg cattggtttc gaagcgggca 2400
    acaagccgaa agaactgtac agcgaagagg cagtcaacgg ggaaactcag caggcgcact 2460
    tacaggcgat taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga 2520
    gtattgccaa cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg 2580
    cggaagcaac gcgtaaactc gatccgacgc gtccgatcac ctgcgtcaat gtaatgttct 2640
    gcgacgctca caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt 2700
    acggttggta tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac 2760
    ttctggcctg gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata 2820
    cgttagccgg gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat 2880
    ggctggatat gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat 2940
    ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaagg 3000
    ggatcttcac ccgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga 3060
    ctggcatgaa cttcggtgaa aaaccgcagc agggaggcaa acaatgaatc aacaactctc 3120
    ctggcgcacc atcgtcggct acagcctcgg gaattgcgta ccgagctcga atttccccga 3180
    tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 3240
    gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 3300
    gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 3360
    gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 3420
    gttactagat cgggaattcg atatcaagct t 3451
    <210> SEQ ID NO 109
    <211> LENGTH: 14627
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pAg11a Plasmid
    <400> SEQUENCE: 109
    catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60
    atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120
    agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180
    gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240
    agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300
    ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360
    ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420
    acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480
    ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540
    acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600
    agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660
    tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720
    tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780
    ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840
    gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900
    gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960
    cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020
    ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080
    gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140
    tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200
    aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260
    aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320
    ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380
    ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440
    cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500
    atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560
    accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620
    gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680
    gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740
    ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800
    cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860
    aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920
    gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980
    agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040
    ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100
    atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160
    accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220
    tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280
    cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340
    gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400
    tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460
    cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520
    gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580
    tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640
    cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700
    gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760
    gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820
    tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880
    tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940
    agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000
    gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060
    gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120
    ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180
    cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240
    aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300
    catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360
    gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420
    tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480
    cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540
    aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600
    ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660
    gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720
    aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780
    actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840
    aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900
    ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960
    tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020
    gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080
    ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
    gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
    ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
    ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
    acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
    tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
    ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500
    ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
    ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
    actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
    gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
    tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
    caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
    atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
    acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980
    atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040
    ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100
    gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160
    gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220
    ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280
    gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340
    taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400
    cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460
    gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520
    gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580
    atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640
    tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700
    tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760
    tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820
    aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880
    ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940
    caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000
    gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060
    tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120
    cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180
    tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240
    taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300
    gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360
    ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420
    ggaactactc acacattatt atggagaaac tcgagtcaaa tctcggtgac gggcaggacc 6480
    ggacggggcg gtaccggcag gctgaagtcc agctgccaga aacccacgtc atgccagttc 6540
    ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg catatccgag cgcctcgtgc 6600
    atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga ccacgctctt gaagccctgt 6660
    gcctccaggg acttcagcag gtgggtgtag agcgtggagc ccagtcccgt ccgctggtgg 6720
    cggggggaga cgtacacggt cgactcggcc gtccagtcgt aggcgttgcg tgccttccag 6780
    gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct cggcgacgag ccagggatag 6840
    cgctcccgca gacggacgag gtcgtccgtc cactcctgcg gttcctgcgg ctcggtacgg 6900
    aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg tgcagaccgc cggcatgtcc 6960
    gcctcggtgg cacggcggat gtcggccggg cgtcgttctg ggctcatggt agactcgaga 7020
    gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 7080
    ttccttatat agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc 7140
    agtggagata tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc 7200
    cacgatgctc ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg 7260
    aacgatagcc tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac 7320
    tgtccttttg atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat 7380
    taccctttgt tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt 7440
    cttggagtag acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga 7500
    agacgtggtt ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt 7560
    gggaccactg tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca 7620
    tttgtaggtg ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca 7680
    atggaatccg aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg 7740
    gtcttctgag actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 7800
    gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 7860
    taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 7920
    aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 7980
    atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 8040
    tacgaattcg agccttgact agagggtcga cggtatacag acatgataag atacattgat 8100
    gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 8160
    gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa 8220
    gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat 8280
    tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca 8340
    gtcctgctcc tcggccacga agtgcacgca gttgccggcc gggtcgcgca gggcgaactc 8400
    ccgcccccac ggctgctcgc cgatctcggt catggccggc ccggaggcgt cccggaagtt 8460
    cgtggacacg acctccgacc actcggcgta cagctcgtcc aggccgcgca cccacaccca 8520
    ggccagggtg ttgtccggca ccacctggtc ctggaccgcg ctgatgaaca gggtcacgtc 8580
    gtcccggacc acaccggcga agtcgtcctc cacgaagtcc cgggagaacc cgagccggtc 8640
    ggtccagaac tcgaccgctc cggcgacgtc gcgcgcggtg agcaccggaa cggcactggt 8700
    caacttggcc atggatccag atttcgctca agttagtata aaaaagcagg cttcaatcct 8760
    gcaggaattc gatcgacact ctcgtctact ccaagaatat caaagataca gtctcagaag 8820
    accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc ctcggattcc 8880
    attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca 8940
    aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc 9000
    ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 9060
    cttcaaagca agtggattga tgtgataaca tggtggagca cgacactctc gtctactcca 9120
    agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg 9180
    taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga 9240
    cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg 9300
    ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 9360
    tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 9420
    ctgacgtaag ggatgacgca caatcccact atccttcgca agaccttcct ctatataagg 9480
    aagttcattt catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc 9540
    tctctcgagc tttcgcagat ccgggggggc aatgagatat gaaaaagcct gaactcaccg 9600
    cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9660
    tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 9720
    tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9780
    catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggagtttagc gagagcctga 9840
    cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9900
    tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc gatcgctgcg gccgatctta 9960
    gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 10020
    gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 10080
    acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 10140
    gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 10200
    atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 10260
    aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 10320
    acttcgagcg gaggcatccg gagcttgcag gatcgccacg actccgggcg tatatgctcc 10380
    gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10440
    gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10500
    aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10560
    gtggaaaccg acgccccagc actcgtccga gggcaaagaa atagagtaga tgccgaccgg 10620
    atctgtcgat cgacaagctc gagtttctcc ataataatgt gtgagtagtt cccagataag 10680
    ggaattaggg ttcctatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat 10740
    gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc 10800
    agtactaaaa tccagatccc ccgaattaat tcggcgttaa ttcagatcaa gcttgacctg 10860
    gaatatcgcg agtaaactga aaatcacgga aaatgagaaa tacacacttt aggacgtgaa 10920
    atatggcgag gaaaactgaa aaaggtggaa aatttagaaa tgtccactgt aggacgtgga 10980
    atatggcaag aaaactgaaa atcatggaaa atgagaaaca tccacttgac gacttgaaaa 11040
    atgacgaaat cactaaaaaa cgtgaaaaat gagaaatgca cactgaagga ctccgcggga 11100
    attcgattgt gctagccaat gtttaacaag atgtcaagca caatgaatgt tggtggttgg 11160
    tggtcgtggc tggcggtggt ggaaaattgc ggtggttcga gcggtagtga tcggcgatgg 11220
    ttggtgtttg cagcggtgtt tgatatcgga atcacttatg gtggttgtca caatggaggt 11280
    gcgtcatggt tattggtggt tggtcatcta tatattttta taataatatt aagtatttta 11340
    cctatttttt acatattttt tattaaattt atgcattgtt tgtattttta aatagttttt 11400
    atcgtacttg ttttataaaa tattttatta ttttatgtgt tatattatta cttgatgtat 11460
    tggaaatttt ctccattgtt ttttctatat ttataataat tttcttattt ttttttgttt 11520
    tattatgtat tttttcgttt tataataaat atttattaaa aaaaatatta tttttgtaaa 11580
    atatatcatt tacaatgttt aaaagtcatt tgtgaatata ttagctaagt tgtacttctt 11640
    tttgtgcatt tggtgttgta catgtctatt atgattctct ggccaaaaca tgtctactcc 11700
    tgtcacttgg gttttttttt ttaagacata atcactagtg attatatcta gactgaaggc 11760
    gggaaacgac aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg 11820
    atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc 11880
    actcagccgc gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg 11940
    cgcgttcaaa agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct 12000
    ccactgacgt tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat 12060
    ccaaataatc tgcaccggat ctcgagatcg aattcccgcg gccgcgaatt cactagtgga 12120
    tccccgggta cggtcagtcc cttatgttac gtcctgtaga aaccccaacc cgtgaaatca 12180
    aaaaactcga cggcctgtgg gcattcagtc tggatcgcga aaactgtgga attgagcagc 12240
    gttggtggga aagcgcgtta caagaaagcc gggcaattgc tgtgccaggc agttttaacg 12300
    atcagttcgc cgatgcagat attcgtaatt atgtgggcaa cgtctggtat cagcgcgaag 12360
    tctttatacc gaaaggttgg gcaggccagc gtatcgtgct gcgtttcgat gcggtcactc 12420
    attacggcaa agtgtgggtc aataatcagg aagtgatgga gcatcagggc ggctatacgc 12480
    catttgaagc cgatgtcacg ccgtatgtta ttgccgggaa aagtgtacgt atcacagttt 12540
    gtgtgaacaa cgaactgaac tggcagacta tcccgccggg aatggtgatt accgacgaaa 12600
    acggcaagaa aaagcagtct tacttccatg atttctttaa ctacgccggg atccatcgca 12660
    gcgtaatgct ctacaccacg ccgaacacct gggtggacga tatcaccgtg gtgacgcatg 12720
    tcgcgcaaga ctgtaaccac gcgtctgttg actggcaggt ggtggccaat ggtgatgtca 12780
    gcgttgaact gcgtgatgcg gatcaacagg tggttgcaac tggacaaggc accagcggga 12840
    ctttgcaagt ggtgaatccg cacctctggc aaccgggtga aggttatctc tatgaactgt 12900
    acgtcacagc caaaagccag acagagtgtg atatctaccc gctgcgcgtc ggcatccggt 12960
    cagtggcagt gaagggcgaa cagttcctga tcaaccacaa accgttctac tttactggct 13020
    ttggccgtca tgaagatgcg gatttgcgcg gcaaaggatt cgataacgtg ctgatggtgc 13080
    acgatcacgc attaatggac tggattgggg ccaactccta ccgtacctcg cattaccctt 13140
    acgctgaaga gatgctcgac tgggcagatg aacatggcat cgtggtgatt gatgaaactg 13200
    cagctgtcgg ctttaacctc tctttaggca ttggtttcga agcgggcaac aagccgaaag 13260
    aactgtacag cgaagaggca gtcaacgggg aaactcagca ggcgcactta caggcgatta 13320
    aagagctgat agcgcgtgac aaaaaccacc caagcgtggt gatgtggagt attgccaacg 13380
    aaccggatac ccgtccgcaa ggtgcacggg aatatttcgc gccactggcg gaagcaacgc 13440
    gtaaactcga tccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 13500
    ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggttggtatg 13560
    tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 13620
    aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 13680
    tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 13740
    atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 13800
    attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaagggg atcttcaccc 13860
    gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 13920
    tcggtgaaaa accgcagcag ggaggcaaac aatgaatcaa caactctcct ggcgcaccat 13980
    cgtcggctac agcctcggga attgcgtacc gagctcgaat ttccccgatc gttcaaacat 14040
    ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 14100
    atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 14160
    gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 14220
    aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 14280
    ggaattcgat atcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc 14340
    ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 14400
    gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct 14460
    agagcagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt 14520
    ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag aataacggat 14580
    atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtg 14627
    <210> SEQ ID NO 110
    <211> LENGTH: 9080
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeo(6XHS4)2eGFP Plasmid
    <400> SEQUENCE: 110
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420
    agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480
    gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540
    ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600
    gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660
    acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720
    acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780
    ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840
    acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900
    atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960
    ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020
    cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080
    gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140
    ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200
    aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260
    ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320
    tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380
    ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440
    gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500
    ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560
    ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620
    tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680
    atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740
    gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800
    aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860
    cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920
    tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980
    gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040
    cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100
    tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160
    aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220
    acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280
    actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340
    ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400
    cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460
    ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520
    gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580
    ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640
    ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700
    gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760
    gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820
    tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880
    gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940
    gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000
    ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060
    gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120
    ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180
    ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240
    gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300
    caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360
    gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420
    cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480
    tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540
    tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600
    tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660
    gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3720
    gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780
    tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840
    agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900
    tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960
    tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020
    agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080
    gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140
    accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4200
    acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260
    tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320
    aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 4380
    tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440
    agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500
    gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560
    atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620
    caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680
    ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740
    ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800
    tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860
    cccaagcttg catgcctgca ggtcgactct agtggatccc ccgccccgta tcccccaggt 4920
    gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc 4980
    cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc 5040
    ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc 5100
    cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggccccg tatcccccag 5160
    gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct 5220
    tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga 5280
    ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca 5340
    tccctggggg ctttgggggg gggctgtccc cgtgagcgga tccgcggccc cgtatccccc 5400
    aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac 5460
    cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg 5520
    gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta 5580
    catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc cccgtatccc 5640
    ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc 5700
    accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc 5760
    cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat 5820
    tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg gccccgtatc 5880
    ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg 5940
    ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc 6000
    gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta 6060
    attacatccc tgggggcttt gggggggggc tgtccccgtg agcggatccg cggccccgta 6120
    tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg 6180
    tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga 6240
    gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg 6300
    taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc cgcggggctg 6360
    caggaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 6420
    tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 6480
    ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 6540
    ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 6600
    ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 6660
    agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 6720
    catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 6780
    tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 6840
    gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 6900
    ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 6960
    cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7020
    caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7080
    ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7140
    taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7200
    taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 7260
    cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 7320
    tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7380
    gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7440
    catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7500
    atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7560
    ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7620
    gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 7680
    agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 7740
    gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 7800
    agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 7860
    catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 7920
    aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 7980
    gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8040
    taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8100
    caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 8160
    ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8220
    ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8280
    tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8340
    aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8400
    actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8460
    catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8520
    agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa gcgggcttta ttaccaagcg 8580
    aagcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 8640
    tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 8700
    ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtccgtaata cgactcactt 8760
    aaggccttga ctagagggtc gacggtatac agacatgata agatacattg atgagtttgg 8820
    acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 8880
    tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg aagaactcca 8940
    gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc 9000
    ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tagcacgtgt cagtcctgct 9060
    cctcggccac gaagtgcacg 9080
    <210> SEQ ID NO 111
    <211> LENGTH: 4223
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pLIT38attBBSRpolyA10 Plasmid
    <400> SEQUENCE: 111
    gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60
    tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120
    ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180
    ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240
    tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300
    gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360
    gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420
    acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480
    tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540
    caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600
    gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660
    cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720
    tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780
    agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840
    tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900
    ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960
    acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020
    ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080
    aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140
    aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200
    aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260
    ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320
    ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380
    aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440
    gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500
    cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560
    atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620
    ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680
    ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740
    ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800
    ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860
    ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920
    tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980
    tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040
    tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100
    tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160
    gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220
    tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280
    ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340
    accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400
    acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460
    ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520
    tgctttttta tactaacttg agcgaaatct ggatcaccat gaaaacattt aacatttctc 2580
    aacaagatct agaattagta gaagtagcga cagagaagat tacaatgctt tatgaggata 2640
    ataaacatca tgtgggagcg gcaattcgta cgaaaacagg agaaatcatt tcggcagtac 2700
    atattgaagc gtatatagga cgagtaactg tttgtgcaga agccattgcg attggtagtg 2760
    cagtttcgaa tggacaaaag gattttgaca cgattgtagc tgttagacac ccttattctg 2820
    acgaagtaga tagaagtatt cgagtggtaa gtccttgtgg tatgtgtagg gagttgattt 2880
    cagactatgc accagattgt tttgtgttaa tagaaatgaa tggcaagtta gtcaaaacta 2940
    cgattgaaga actcattcca ctcaaatata cccgaaatta aaagttttac cataccaagc 3000
    ttggctgctg cctgaggctg gacgacctcg cggagttcta ccggcagtgc aaatccgtcg 3060
    gcatccagga aaccagcagc ggctatccgc gcatccatgc ccccgaactg caggagtggg 3120
    gaggcacgat ggccgctttg gtccggatct ttgtgaagga accttacttc tgtggtgtga 3180
    cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa 3240
    gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg 3300
    gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag 3360
    aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa 3420
    aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga 3480
    gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa 3540
    aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc 3600
    ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg 3660
    ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta 3720
    ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt 3780
    gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa 3840
    atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc 3900
    aatagcatca caaatttcac aaataaagat ccacgaattc gctagcttcg gccgtgacgc 3960
    gtctccggat gtacaggcat gcgtcgaccc tctagtcaag gccttaagtg agtcgtatta 4020
    cggactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 4080
    tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 4140
    tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcttcgctt ggtaataaag 4200
    cccgcttcgg cgggcttttt ttt 4223
    <210> SEQ ID NO 112
    <211> LENGTH: 5855
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCX-LamIntR Plasmid
    <400> SEQUENCE: 112
    gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
    gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
    ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
    ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
    atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
    cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
    tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
    atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
    gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
    gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
    tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
    gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
    ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
    gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
    ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
    tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
    cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
    ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
    tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
    cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
    gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
    ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
    gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
    gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
    tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
    cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
    acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
    gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
    acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740
    gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800
    acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atcgcaatca 1860
    ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920
    cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980
    tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040
    caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaattgcgg 2100
    caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160
    cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220
    ctgccactcg cgcagcaaaa tctagagtaa ggagatcaag acttacggct gacgaatacc 2280
    tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340
    ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400
    atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460
    tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520
    ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580
    caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640
    cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700
    ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760
    gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820
    cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 2880
    tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940
    gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000
    tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060
    gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120
    cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180
    ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt 3240
    tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300
    gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360
    atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420
    aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480
    gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540
    tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600
    tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3660
    gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720
    tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780
    caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840
    tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900
    aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960
    cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020
    atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4080
    taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140
    aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200
    tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260
    gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 4320
    cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4380
    cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440
    atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500
    tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560
    ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620
    acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680
    aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740
    aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800
    tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860
    cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920
    catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980
    ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040
    aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100
    ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160
    caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220
    attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280
    agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340
    actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400
    ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460
    ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5520
    gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580
    atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640
    cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700
    gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760
    gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820
    ggttccgcgc acatttcccc gaaaagtgcc acctg 5855
    <210> SEQ ID NO 113
    <211> LENGTH: 4346
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pSV40-193AttpsensePur Plasmid
    <400> SEQUENCE: 113
    ccggtgccgc caccatcccc tgacccacgc ccctgacccc tcacaaggag acgaccttcc 60
    atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta 120
    cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac 180
    cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 240
    atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 300
    agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 360
    tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 420
    cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 480
    agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 540
    gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 600
    gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 660
    cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tggctccgac 720
    cgaagccgac ccgggcggcc ccgccgaccc cgcacccgcc cccgaggccc accgactcta 780
    gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 840
    acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat 900
    tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 960
    tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 1020
    gatccgcgcc ggatccttaa ttaagtctag agtcgactgt ttaaacctgc aggcatgcaa 1080
    gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 1140
    cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 1200
    aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 1260
    agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 1320
    ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 1380
    ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 1440
    tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 1500
    tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 1560
    gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1620
    ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 1680
    tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 1740
    agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 1800
    atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 1860
    acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1920
    actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 1980
    tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 2040
    tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 2100
    tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 2160
    tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 2220
    caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 2280
    cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 2340
    agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 2400
    acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 2460
    gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 2520
    ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 2580
    tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 2640
    ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 2700
    tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 2760
    attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 2820
    agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 2880
    ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 2940
    ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3000
    cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3060
    gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 3120
    tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 3180
    tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 3240
    tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 3300
    tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 3360
    agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 3420
    agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 3480
    agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 3540
    aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 3600
    tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 3660
    gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcg 3720
    agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3780
    gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3840
    cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3900
    taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3960
    gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 4020
    agtagtgagg aggctttttt ggaggctcgg tacccccttg cgctaatgct ctgttacagg 4080
    tcactaatac catctaagta gttgattcat agtgactgca tatgttgtgt tttacagtat 4140
    tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac 4200
    gtttctcgtt cagctttttt atactaagtt ggcattataa aaaagcattg cttatcaatt 4260
    tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgatt tcaattttgt 4320
    cccactccct gcctctgggg ggcgcg 4346
    <210> SEQ ID NO 114
    <211> LENGTH: 3166
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeo Plasmid
    <400> SEQUENCE: 114
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgcatgc ctgcaggtcg 420
    actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480
    gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540
    aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600
    gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660
    agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720
    gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780
    gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840
    cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900
    aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960
    tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020
    ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080
    ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140
    cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200
    ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260
    gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320
    atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380
    aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440
    aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500
    gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560
    cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620
    gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680
    tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740
    ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800
    ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860
    atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920
    cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980
    tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040
    aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100
    tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160
    ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220
    agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280
    gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340
    agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400
    accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460
    gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520
    cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580
    ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tagttaacaa aaaaaagccc 2640
    gccgaagcgg gctttattac caagcgaagc gccattcgcc attcaggctg cgcaactgtt 2700
    gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 2760
    ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 2820
    cggccagtcc gtaatacgac tcacttaagg ccttgactag agggtcgacg gtatacagac 2880
    atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 2940
    tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 3000
    caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc 3060
    ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa 3120
    atctcgtagc acgtgtcagt cctgctcctc ggccacgaag tgcacg 3166
    <210> SEQ ID NO 115
    <211> LENGTH: 7600
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeo3′6XHS4eGFP Plasmid
    <400> SEQUENCE: 115
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatcta gttattaata 420
    gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 480
    tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 540
    gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggacta 600
    tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 660
    tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 720
    ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgggtcgagg 780
    tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 840
    atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg 900
    ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca 960
    gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg 1020
    ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc 1080
    ccgctccgcg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag 1140
    gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 1200
    ctcgtttctt ttctgtggct gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc 1260
    gggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg 1320
    cccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 1380
    gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg 1440
    gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc 1500
    ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt 1560
    cgggtgcggg gctccgtgcg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc 1620
    ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg 1680
    ggcgcggcgg ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct 1740
    tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct ggcggagccg 1800
    aaatctggga ggcgccgccg caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg 1860
    gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc 1920
    atctccagcc tcggggctgc cgcaggggga cggctgcctt cgggggggac ggggcagggc 1980
    ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 2040
    cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt 2100
    tggcaaagaa ttcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 2160
    catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 2220
    cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 2280
    gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg 2340
    ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 2400
    ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 2460
    gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 2520
    cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 2580
    ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 2640
    cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 2700
    gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga 2760
    gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 2820
    ggacgagctg tacaagtaag aattcactcc tcaggtgcag gctgcctatc agaaggtggt 2880
    ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa 2940
    aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt 3000
    tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg 3060
    gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc 3120
    catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc 3180
    cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta 3240
    tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac 3300
    tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat 3360
    gaagatccct cgacctgcag cccaagcttg catgcctgca ggtcgactct agtggatccc 3420
    ccgccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 3480
    agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 3540
    tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 3600
    gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 3660
    cgcggccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 3720
    aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 3780
    gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 3840
    gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 3900
    tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 3960
    ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 4020
    gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 4080
    gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg 4140
    gatccgcggc cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca 4200
    gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct 4260
    cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc 4320
    tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag 4380
    cggatccgcg gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt 4440
    cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg 4500
    ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc 4560
    cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg 4620
    agcggatccg cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg 4680
    ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc 4740
    ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc 4800
    cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg 4860
    tgagcggatc cgcggggctg caggaattcg taatcatggt catagctgtt tcctgtgtga 4920
    aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 4980
    tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5040
    cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5100
    ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5160
    cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5220
    ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5280
    aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5340
    cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5400
    cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5460
    gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5520
    tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5580
    cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5640
    ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5700
    gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 5760
    gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5820
    accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5880
    ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 5940
    tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6000
    aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6060
    taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6120
    gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6180
    agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6240
    cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6300
    tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6360
    gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6420
    agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6480
    gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6540
    atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6600
    gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6660
    tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6720
    atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6780
    agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6840
    gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6900
    cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6960
    tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7020
    ccgcgcacat ttccccgaaa agtgccacct gacgtagtta acaaaaaaaa gcccgccgaa 7080
    gcgggcttta ttaccaagcg aagcgccatt cgccattcag gctgcgcaac tgttgggaag 7140
    ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 7200
    ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 7260
    gtccgtaata cgactcactt aaggccttga ctagagggtc gacggtatac agacatgata 7320
    agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 7380
    tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 7440
    ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc 7500
    ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg 7560
    tagcacgtgt cagtcctgct cctcggccac gaagtgcacg 7600
    <210> SEQ ID NO 116
    <211> LENGTH: 7631
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeo5′6XHS4eGFP Plasmid
    <400> SEQUENCE: 116
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420
    agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480
    gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540
    ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600
    gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660
    acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720
    acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780
    ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840
    acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900
    atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960
    ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020
    cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080
    gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140
    ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200
    aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260
    ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320
    tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380
    ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440
    gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500
    ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560
    ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620
    tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680
    atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740
    gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800
    aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860
    cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920
    tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980
    gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040
    cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100
    tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160
    aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220
    acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280
    actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340
    ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400
    cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460
    ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520
    gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580
    ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640
    ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700
    gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760
    gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820
    tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880
    gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940
    gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000
    ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060
    gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120
    ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180
    ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240
    gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300
    caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360
    gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420
    cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480
    tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 3540
    tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt tggcaaagaa ttcgccacca 3600
    tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3660
    gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3720
    gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3780
    tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3840
    agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 3900
    tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 3960
    tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4020
    agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4080
    gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4140
    accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4200
    acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4260
    tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag 4320
    aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 4380
    tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 4440
    agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 4500
    gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 4560
    atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa 4620
    caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta 4680
    ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt 4740
    ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc 4800
    tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag 4860
    cccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc 4920
    gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4980
    catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 5040
    attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 5100
    ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 5160
    ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5220
    aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5280
    aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5340
    gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5400
    gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5460
    tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5520
    ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5580
    ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5640
    tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5700
    tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5760
    ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5820
    aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5880
    ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5940
    tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 6000
    atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 6060
    aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 6120
    ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 6180
    tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6240
    ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6300
    tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6360
    aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6420
    gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6480
    tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6540
    cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6600
    tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6660
    ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6720
    cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6780
    actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6840
    ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6900
    aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6960
    ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 7020
    atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 7080
    tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt attaccaagc gaagcgccat 7140
    tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 7200
    cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 7260
    tcccagtcac gacgttgtaa aacgacggcc agtccgtaat acgactcact taaggccttg 7320
    actagagggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac 7380
    aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 7440
    tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat 7500
    ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt 7560
    catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca 7620
    cgaagtgcac g 7631
    <210> SEQ ID NO 117
    <211> LENGTH: 4615
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeo6XHS4 Plasmid
    <400> SEQUENCE: 117
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgcatgc ctgcaggtcg 420
    actctagtgg atcccccgcc ccgtatcccc caggtgtctg caggctcaaa gagcagcgag 480
    aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg 540
    ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct 600
    gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt 660
    ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 720
    agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 780
    cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 840
    ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct 900
    gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 960
    cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 1020
    cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 1080
    cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 1140
    ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 1200
    agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 1260
    cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 1320
    ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 1380
    ggctgtcccc gtgagcggat ccgcggcccc gtatccccca ggtgtctgca ggctcaaaga 1440
    gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc 1500
    cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc 1560
    ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 1620
    ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc caggtgtctg caggctcaaa 1680
    gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 1740
    tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 1800
    gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 1860
    ggggggctgt ccccgtgagc ggatccgcgg ggctgcagga attcgtaatc atggtcatag 1920
    ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 1980
    ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc 2040
    tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 2100
    cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 2160
    ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 2220
    ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 2280
    gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2340
    gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2400
    taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2460
    accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2520
    tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2580
    cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 2640
    agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2700
    gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 2760
    gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2820
    tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2880
    acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2940
    cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3000
    acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3060
    acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3120
    tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3180
    ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3240
    ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3300
    tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3360
    aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3420
    ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3480
    ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3540
    gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3600
    gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 3660
    cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 3720
    actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 3780
    ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 3840
    tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 3900
    ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 3960
    agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4020
    aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt agttaacaaa 4080
    aaaaagcccg ccgaagcggg ctttattacc aagcgaagcg ccattcgcca ttcaggctgc 4140
    gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 4200
    ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 4260
    gtaaaacgac ggccagtccg taatacgact cacttaaggc cttgactaga gggtcgacgg 4320
    tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga 4380
    aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc 4440
    tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat 4500
    catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt 4560
    ggaatcgaaa tctcgtagca cgtgtcagtc ctgctcctcg gccacgaagt gcacg 4615
    <210> SEQ ID NO 118
    <211> LENGTH: 17384
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pFK161 Plasmid
    <400> SEQUENCE: 118
    gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcggggtttc 60
    gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 120
    aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 180
    tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 240
    ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 300
    aagagcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat 360
    gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc 420
    ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac 480
    aggagcacga tcatgcgcac ccgtcagatc cagacatgat aagatacatt gatgagtttg 540
    gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 600
    ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 660
    attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 720
    acaaatgtgg tatggctgat tatgatctct agtcaaggca ctatacatca aatattcctt 780
    attaacccct ttacaaatta aaaagctaaa ggtacacaat ttttgagcat agttattaat 840
    agcagacact ctatgcctgt gtggagtaag aaaaaacagt atgttatgat tataactgtt 900
    atgcctactt ataaaggtta cagaatattt ttccataatt ttcttgtata gcagtgcagc 960
    tttttccttt gtggtgtaaa tagcaaagca agcaagagtt ctattactaa acacagcatg 1020
    actcaaaaaa cttagcaatt ctgaaggaaa gtccttgggg tcttctacct ttctcttctt 1080
    ttttggagga gtagaatgtt gagagtcagc agtagcctca tcatcactag atggcatttc 1140
    ttctgagcaa aacaggtttt cctcattaaa ggcattccac cactgctccc attcatcagt 1200
    tccataggtt ggaatctaaa atacacaaac aattagaatc agtagtttaa cacattatac 1260
    acttaaaaat tttatattta ccttagagct ttaaatctct gtaggtagtt tgtccaatta 1320
    tgtcacacca cagaagtaag gttccttcac aaagatccgg accaaagcgg ccatcgtgcc 1380
    tccccactcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat 1440
    gccgacggat ttgcactgcc ggtagaactc gcgaggtcgt ccagcctcag gcagcagctg 1500
    aaccaactcg cgaggggatc gagcccgggg tgggcgaaga actccagcat gagatccccg 1560
    cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 1620
    aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt 1680
    tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct 1740
    gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa 1800
    gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca 1860
    gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc 1920
    aggcatcgcc atgggtcacg acgagatcct cgccgtcggg atgcgcgcct tgagcctggc 1980
    gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2040
    accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2100
    gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2160
    ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2220
    ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2280
    ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc 2340
    ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2400
    gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2460
    agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2520
    atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2580
    tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2640
    tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2700
    tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2760
    tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg 2820
    ccctgagtgc ttgcggcagc gtgaaagctt tttgcaaaag cctaggcctc caaaaaagcc 2880
    tcctcactac ttctggaata gctcagaggc cgaggcggcc taaataaaaa aaattagtca 2940
    gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag 3000
    gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg 3060
    ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact 3120
    tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccacagccg 3180
    gatctgcagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac 3240
    gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga 3300
    ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg 3360
    tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg 3420
    cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcgcat aaatcgccgt 3480
    gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag 3540
    ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cggcatcccg 3600
    atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac 3660
    gccagcaaga cgtagcccag cgcgtcgggc cgccatgccg gcgataatgg cctgcttctc 3720
    gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 3780
    gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 3840
    aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 3900
    aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 3960
    tctcaagggc atcggtcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4020
    tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4080
    gcccaacagt cccccggcca cgggcctgcc accataccca cgccgaaaca agcgctcatg 4140
    agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca 4200
    accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcttggca 4260
    gtcacagcat gcgcatatcc atgcttcgac catgcgctca caaagtaggt gaatgcgcaa 4320
    tgtagtaccc acatcgtcat cgctttccac tgctctcgcg aataaagatg gaaaatcaat 4380
    ctcatggtaa tagtccatga aaatccttgt attcataaat cctccaggta gctatatgca 4440
    aattgaaaca aaagagatgg tgatctttct aagagatgat ggaatctccc ttcagtatcc 4500
    cgatggtcaa tgcgctggat atgggataga tgggaatatg ctgattttta tgggacagag 4560
    ttgcgaactg ttcccaacta aaatcatttt gcacgatcag cgcactacga actttaccca 4620
    caaatagtca ggtaatgaat cctgatataa agacaggttg ataaatcagt cttctacgcg 4680
    catcgcacgc gcacaccgta gaaagtcttt cagttgtgag cctgggcaaa ccgttaactt 4740
    tcggcggctt tgctgtgcga caggctcacg tctaaaagga aataaatcat gggtcataaa 4800
    attatcacgt tgtccggcgc ggcgacggat gttctgtatg cgctgttttt ccgtggcgcg 4860
    ttgctgtctg gtgatctgcc ttctaaatct ggcacagccg aattgcgcga gcttggtttt 4920
    gctgaaacca gacacacagc aactgaatac cagaaagaaa atcactttac ctttctgaca 4980
    tcagaagggc agaaatttgc cgttgaacac ctggtcaata cgcgttttgg tgagcagcaa 5040
    tattgcgctt cgatgacgct tggcgttgag attgatacct ctgctgcaca aaaggcaatc 5100
    gacgagctgg accagcgcat tcgtgacacc gtctccttcg aacttattcg caatggagtg 5160
    tcattcatca aggacgccgc tatcgcaaat ggtgctatcc acgcagcggc aatcgaaaca 5220
    cctcagccgg tgaccaatat ctacaacatc agccttggta tccagcgtga tgagccagcg 5280
    cagaacaagg taaccgtcag tgccgataag ttcaaagtta aacctggtgt tgataccaac 5340
    attgaaacgt tgatcgaaaa cgcgctgaaa aacgctgctg aatgtgcggc gctggatgtc 5400
    acaaagcaaa tggcagcaga caagaaagcg atggatgaac tggcttccta tgtccgcacg 5460
    gccatcatga tggaatgttt ccccggtggt gttatctggc agcagtgccg tcgatagtat 5520
    gcaattgata attattatca tttgcgggtc ctttccggcg atccgccttg ttacggggcg 5580
    gcgacctcgc gggttttcgc tatttatgaa aattttccgg tttaaggcgt ttccgttctt 5640
    cttcgtcata acttaatgtt tttatttaaa ataccctctg aaaagaaagg aaacgacagg 5700
    tgctgaaagc gagctttttg gcctctgtcg tttcctttct ctgtttttgt ccgtggaatg 5760
    aacaatggaa gtcaacaaaa agcagctggc tgacattttc ggtgcgagta tccgtaccat 5820
    tcagaactgg caggaacagg gaatgcccgt tctgcgaggc ggtggcaagg gtaatgaggt 5880
    gctttatgac tctgccgccg tcataaaatg gtatgccgaa agggatgctg aaattgagaa 5940
    cgaaaagctg cgccgggagg ttgaagaact gcggcaggcc agcgaggcag atccacagga 6000
    cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga 6060
    ctgggcggcg gcaaagcggt cggacagtgc tccgagaacg ggtgcgcata gaaattgcat 6120
    caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg aatggacgat 6180
    atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt 6240
    gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg 6300
    ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gcggtcaaac 6360
    atgagaattc gcggccgctc ttctcgttct gccagcgggc cctcgtctct ccaccccatc 6420
    cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctc cggcccgacg ctgccccgcg 6480
    cgcacttttc tcagtggttc gcgtggtcct tgtggatgtg tgaggcgccc ggttgtgccc 6540
    tcacgtgttt cactttggtc gtgtctcgct tgaccatgtt cccagagtcg gtggatgtgg 6600
    ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacg cgctgtttct tgtaagcgtc 6660
    gaggtgctcc tggagcgttc caggtttgtc tcctaggtgc ctgcttctga gctggtggtg 6720
    gcgctcccca ttccctggtg tgcctccggt gctccgtctg gctgtgtgcc ttcccgtttg 6780
    tgtctgagaa gcccgtgaga ggggggtcga ggagagaagg aggggcaaga ccccccttct 6840
    tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgt gcgtagggct ggtgctgagc 6900
    ggtcgcggct ggggttggaa agtttctcga gagactcatt gctttcccgt ggggagcttt 6960
    gagaggcctg gctttcgggg gggaccggtt gcagggtctc ccctgtccgc ggatgctcag 7020
    aatgcccttg gaagagaacc ttcctgttgc cgcagacccc cccgcgcggt cgcccgcgtg 7080
    ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcc tctctcggtg gccggggctc 7140
    gtcggggttt tgggtccgtc ccgccctcag tgagaaagtt tccttctcta gctatcttcc 7200
    ggaaagggtg cgggcttctt acggtctcga ggggtctctc ccgaatggtc ccctggaggg 7260
    ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctc tctcgtctac cgcggcccgc 7320
    ggcctccccg ctccgagttc ggggagggat cacgcggggc agagcctgtc tgtcgtcctg 7380
    ccgttgctgc ggagcatgtg gctcggcttg tgtggttggt ggctggggag agggctccgt 7440
    gcacaccccc gcgtgcgcgt actttcctcc cctcctgagg gccgccgtgc ggacggggtg 7500
    tgggtaggcg acggtgggct cccgggtccc cacccgtctt cccgtgcctc acccgtgcct 7560
    tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttg gccgctcccg cgacggcggc 7620
    ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctg tgtggttgtg tcgcctcgcc 7680
    ccccccttcc cgcggcagcg ttcccacggc tggcgaaatc gcgggagtcc tccttcccct 7740
    cctcggggtc gagagggtcc gtgtctggcg ttgattgatc tcgctctcgg ggacgggacc 7800
    gttctgtggg agaacggctg ttggccgcgt ccggcgcgac gtcggacgtg gggacccact 7860
    gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcgg catcggtctc tctctcgtgt 7920
    cggtgtcgcc tcctcgggct cccggggggc cgtcgtgttt cgggtcggct cggcgctgca 7980
    ggtgtggtgg gactgctcag gggagtggtg cagtgtgatt cccgccggtt ttgcctcgcg 8040
    tgccctgacc ggtccgacgc ccgagcggtc tctcggtccc ttgtgaggac ccccttccgg 8100
    gaggggcccg tttcggccgc ccttgccgtc gtcgccggcc ctcgttctgc tgtgtcgttc 8160
    ccccctcccc gctcgccgca gccggtcttt tttcctctct ccccccctct cctctgactg 8220
    acccgtggcc gtgctgtcgg accccccgca tgggggcggc cgggcacgta cgcgtccggg 8280
    cggtcaccgg ggtcttgggg gggggccgag gggtaagaaa gtcggctcgg cgggcgggag 8340
    gagctgtggt ttggagggcg tcccggcccc gcggccgtgg cggtgtcttg cgcggtcttg 8400
    gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagg gcaaagggaa agaggctagc 8460
    agtggtcatt gtcccgacgg tgtggtggtc tgttggccga ggtgcgtctg gggggctcgt 8520
    ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcct gccggagtgc cgaggtgggt 8580
    accctggcgg tgggattaac cccgcgcgcg tgtcccggtg tggcggtggg ggctccggtc 8640
    gatgtctacc tccctctccc cgaggtctca ggccttctcc gcgcgggctc tcggccctcc 8700
    cctcgttcct ccctctcgcg gggttcaagt cgctcgtcga cctcccctcc tccgtccttc 8760
    catctctcgc gcaatggcgc cgcccgagtt cacggtgggt tcgtcctccg cctccgcttc 8820
    tcgccggggg ctggccgctg tccggtctct cctgcccgac ccccgttggc gtggtcttct 8880
    ctcgccggct tcgcggactc ctggcttcgc ccggagggtc agggggcttc ccggttcccc 8940
    gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgc tgcggcctcc gcccgcccgt 9000
    gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgc ggtcagttgg gccctggcgt 9060
    tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctaga cgcgggtgtc gccgggctcc 9120
    gacgggtggc ctatccaggg ctcgcccccg ccgacccccg cctgcccgtc ccggtggtgg 9180
    tcgttggtgt ggggagtgaa tggtgctacc ggtcattccc tcccgcgtgg tttgactgtc 9240
    tcgccggtgt cgcgcttctc tttccgccaa cccccacgcc aacccaccac cctgctctcc 9300
    cggcccggtg cggtcgacgt tccggctctc ccgatgccga ggggttcggg atttgtgccg 9360
    gggacggagg ggagagcggg taagagaggt gtcggagagc tgtcccgggg cgacgctcgg 9420
    gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttg tcggaccccg acggggtcgg 9480
    tccggccgca tgcactctcc cgttccgcgc gagcgcccgc ccggctcacc cccggtttgt 9540
    cctcccgcga ggctctccgc cgccgccgcc tcctcctcct ctctcgcgct ctctgtcccg 9600
    cctggtcctg tcccaccccc gacgctccgc tcgcgcttcc ttacctggtt gatcctgcca 9660
    ggtagcatat gcttgtctca aagattaagc catgcatgtc taagtacgca cggccggtac 9720
    agtgaaactg cgaatggctc attaaatcag ttatggttcc tttggtcgct cgctcctctc 9780
    ctacttggat aactgtggta attctagagc taatacatgc cgacgggcgc tgacccccct 9840
    tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaa cccggtgagc tccctcccgg 9900
    ctccggccgg gggtcgggcg ccggcggctt ggtgactcta gataacctcg ggccgatcgc 9960
    acgccccccg tggcggcgac gacccattcg aacgtctgcc ctatcaactt tcgatggtag 10020
    tcgccgtgcc taccatggtg accacgggtg acggggaatc agggttcgat tccggagagg 10080
    gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat tacccactcc 10140
    cgacccgggg aggtagtgac gaaaaataac aatacaggac tctttcgagg ccctgtaatt 10200
    ggaatgagtc cactttaaat cctttaacga ggatccattg gagggcaagt ctggtgccag 10260
    cagccgcggt aattccagct ccaatagcgt atattaaagt tgctgcagtt aaaaagctcg 10320
    tagttggatc ttgggagcgg gcgggcggtc cgccgcgagg cgagtcaccg cccgtccccg 10380
    ccccttgcct ctcggcgccc cctcgatgct cttagctgag tgtcccgcgg ggcccgaagc 10440
    gtttactttg aaaaaattag agtgttcaaa gcaggcccga gccgcctgga taccgcagct 10500
    aggaataatg gaataggacc gcggttctat tttgttggtt ttcggaactg aggccatgat 10560
    taagagggac ggccgggggc attcgtattg cgccgctaga ggtgaaattc ttggaccggc 10620
    gcaagacgga ccagagcgaa agcatttgcc aagaatgttt tcattaatca agaacgaaag 10680
    tcggaggttc gaagacgatc agataccgtc gtagttccga ccataaacga tgccgactgg 10740
    cgatgcggcg gcgttattcc catgacccgc cgggcagctt ccgggaaacc aaagtctttg 10800
    ggttccgggg ggagtatggt tgcaaagctg aaacttaaag gaattgacgg aagggcacca 10860
    ccaggagtgg gcctgcggct taatttgact caacacggga aacctcaccc ggcccggaca 10920
    cggacaggat tgacagattg atagctcttt ctcgattccg tgggtggtgg tgcatggccg 10980
    ttcttagttg gtggagcgat ttgtctggtt aattccgata acgaacgaga ctctggcatg 11040
    ctaactagtt acgcgacccc cgagcggtcg gcgtccccca acttcttaga gggacaagtg 11100
    gcgttcagcc acccgagatt gagcaataac aggtctgtga tgcccttaga tgtccggggc 11160
    tgcacgcgcg ctacactgac tggctcagcg tgtgcctacc ctgcgccggc aggcgcgggt 11220
    aacccgttga accccattcg tgatggggat cggggattgc aattattccc catgaacgag 11280
    gaattcccag taagtgcggg tcataagctt gcgttgatta agtccctgcc ctttgtacac 11340
    accgcccgtc gctactaccg attggatggt ttagtgaggc cctcggatcg gccccgccgg 11400
    ggtcggccca cggccctggc ggagcgctga gaagacggtc gaacttgact atctagagga 11460
    agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt aaacgggaga 11520
    ctgtggagga gcggcggcgt ggcccgctct ccccgtcttg tgtgtgtcct cgccgggagg 11580
    cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgag gtgtctggag tgaggtgaga 11640
    gaaggggtgg gtggggtcgg tctgggtccg tctgggaccg cctccgattt cccctccccc 11700
    tcccctctcc ctcgtccggc tctgacctcg ccaccctacc gcggcggcgg ctgctcgcgg 11760
    gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacg aggggcggta cgtcgttacg 11820
    ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcg cgcgctttgc tctcccggca 11880
    cccatccccg ccgcggctct ggcttttcta cgttggctgg ggcggttgtc gcgtgtgggg 11940
    ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgcc acgcttttct ggcctcgcgt 12000
    gtcctccccg ctcctgtccc gggtacctag ctgtcgcgtt ccggcgcgga ggtttaagga 12060
    ccccgggggg gtcgccctgc cgcccccagg gtcggggggc ggtggggccc gtagggaagt 12120
    cggtcgttcg ggcggctctc cctcagactc catgaccctc ctccccccgc tgccgccgtt 12180
    cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagc cccctcgggc gccgtggggg 12240
    cccgacccgc gccgccggct tgcccgattt ccgcgggtcg gtcctgtcgg tgccggtcgt 12300
    gggttcccgt gtcgttcccg tgtttttccg ctcccgaccc tttttttttc ctccccccca 12360
    cacgtgtctc gtttcgttcc tgctggccgg cctgaggcta cccctcggtc catctgttct 12420
    cctctctctc cggggagagg agggcggtgg tcgttggggg actgtgccgt cgtcagcacc 12480
    cgtgagttcg ctcacacccg aaataccgat acgactctta gcggtggatc actcggctcg 12540
    tgcgtcgatg aagaacgcag ctagctgcga gaattaatgt gaattgcagg acacattgat 12600
    catcgacact tcgaacgcac ttgcggcccc gggttcctcc cggggctacg cctgtctgag 12660
    cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggt gctgcgcggc tgggagtttg 12720
    ctcgcagggc caacccccca acccgggtcg ggccctccgt ctcccgaagt tcagacgtgt 12780
    gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggag cctggtctcc cccgcgcatc 12840
    cgcgctcgcg gcttcttccc gctccgccgt tcccgccctc gcccgtgcac cccggtcctg 12900
    gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtc tttctcggtc ccgtgccccg 12960
    tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcg cgtccgcatc tgctctggtc 13020
    gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtgg tcggtcccct gcggccgcgg 13080
    ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcc tgcggtggtt gtctgtgtgt 13140
    gtttgggtct tgcgctgggg gaggcggggt cgaccgctcg cggggttggc gcggtcgccc 13200
    ggcgccgcgc accctccggc ttgtgtggag ggagagcgag ggcgagaacg gagagaggtg 13260
    gtatccccgg tggcgttgcg agggagggtt tggcgtcccg cgtccgtccg tccctccctc 13320
    cctcggtggg cgccttcgcg ccgcacgcgg ccgctagggg cggtcggggc ccgtggcccc 13380
    cgtggctctt cttcgtctcc gcttctcctt cacccgggcg gtacccgctc cggcgccggc 13440
    ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcac ccccgggtgt tgcgagttcg 13500
    gggagggaga gggcctcgct gacccgttgc gtcccggctt ccctgggggg gacccggcgt 13560
    ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagat cctccacccc cgccgccctc 13620
    ccctcccgcc ggcctctcgg ggaccccctg agacggttcg ccggctcgtc ctcccgtgcc 13680
    gccgggtgcc gtctctttcc cgcccgcctc ctcgctctct tcttcccgcg gctgggcgcg 13740
    tgtcccccct ttctgaccgc gacctcagat cagacgtggc gacccgctga atttaagcat 13800
    attagtcagc ggaggaaaag aaactaacca ggattccctc agtaacggcg agtgaacagg 13860
    gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgt gggaaatgtg gcgtacggaa 13920
    gacccactcc ccggcgccgc tcgtgggggg cccaagtcct tctgatcgag gcccagcccg 13980
    tggacggtgt gaggccggta gcggccccgg cgcgccgggc tcgggtcttc ccggagtcgg 14040
    gttgcttggg aatgcagccc aaagcgggtg gtaaactcca tctaaggcta aataccggca 14100
    cgagaccgat agtcaacaag taccgtaagg gaaagttgaa aagaactttg aagagagagt 14160
    tcaagagggc gtgaaaccgt taagaggtaa acgggtgggg tccgcgcagt ccgcccggag 14220
    gattcaaccc ggcggcgcgc gtccggccgt gcccggtggt cccggcggat ctttcccgct 14280
    ccccgttcct cccgacccct ccacccgcgc gtcgttcccc tcttcctccc cgcgtccggc 14340
    gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcg cgcgggcggg gccgggggtg 14400
    gggtcggcgg gggaccgccc ccggccggcg accggccgcc gccgggcgca cttccaccgt 14460
    ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggc ccggtgggga aggtggctcg 14520
    gggggggcgg cgcgtctcag ggcgcgccga accacctcac cccgagtgtt acagccctcc 14580
    ggccgcgctt tcgccgaatc ccggggccga ggaagccaga tacccgtcgc cgcgctctcc 14640
    ctctcccccc gtccgcctcc cgggcgggcg tgggggtggg ggccgggccg cccctcccac 14700
    ggcgcgaccg ctctcccacc cccctccgtc gcctctctcg gggcccggtg gggggcgggg 14760
    cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtc gggtcccggg gggaccgtcg 14820
    gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggc ggcgatgtcg gctacccacc 14880
    cgacccgtct tgaaacacgg accaaggagt ctaacgcgtg cgcgagtcag gggctcgtcc 14940
    gaaagccgcc gtggcgcaat gaaggtgaag ggccccgccc gggggcccga ggtgggatcc 15000
    cgaggcctct ccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgccgggg 15060
    aggtggagca cgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ttgggcaggg 15120
    cgaagccaga ggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 15180
    cgacctgggt ataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 15240
    tttccctcag gatagctggc gctctcgctc ccgacgtacg cagttttatc cggtaaagcg 15300
    aatgattaga ggtcttgggg ccgaaacgat ctcaacctat tctcaaactt taaatgggta 15360
    agaagcccgg ctcgctggcg tggagccggg cgtggaatgc gagtgcctag tgggccactt 15420
    ttggtaagca gaactggcgc tgcgggatga accgaacgcc gggttaaggc gcccgatgcc 15480
    gacgctcatc agaccccaga aaaggtgttg gttgatatag acagcaggac ggtggccatg 15540
    gaagtcggaa tccgctaagg agtgtgtaac aactcacctg ccgaatcaac tagccctgaa 15600
    aatggatggc gctggagcgt cgggcccata cccggccgtc gccgcagtcg gaacggaacg 15660
    ggacgggagc ggccgcgaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat 15720
    aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 15780
    tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 15840
    gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 15900
    atttccgtgt cgcccttatt cccttttttg cggcattttg cttcctgttt ttgctcaccc 15960
    agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 16020
    cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 16080
    aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 16140
    gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 16200
    agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 16260
    aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 16320
    gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 16380
    ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc 16440
    aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 16500
    aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 16560
    tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 16620
    agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 16680
    ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 16740
    ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 16800
    ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 16860
    acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 16920
    agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 16980
    ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 17040
    cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 17100
    gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 17160
    cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 17220
    gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 17280
    caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc cgaagggaga 17340
    aaggcggaca ggtatccggt aagcggcagg gtcggaacag gaga 17384
    <210> SEQ ID NO 119
    <211> LENGTH: 2814
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pLITMUS38 Plasmid
    <400> SEQUENCE: 119
    gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60
    tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120
    ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180
    ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240
    tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300
    gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360
    gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420
    acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480
    tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540
    caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600
    gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660
    cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720
    tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780
    agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840
    tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900
    ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960
    acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020
    ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080
    aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140
    aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200
    aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260
    ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320
    ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380
    aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440
    gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500
    cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560
    atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620
    ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680
    ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740
    ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800
    ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860
    ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920
    tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980
    tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040
    tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100
    tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160
    gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220
    tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280
    ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340
    accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400
    acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460
    ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gatatctgga 2520
    tccacgaatt cgctagcttc ggccgtgacg cgtctccgga tgtacaggca tgcgtcgacc 2580
    ctctagtcaa ggccttaagt gagtcgtatt acggactggc cgtcgtttta caacgtcgtg 2640
    actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 2700
    gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2760
    atggcgaatg gcgcttcgct tggtaataaa gcccgcttcg gcgggctttt tttt 2814
    <210> SEQ ID NO 120
    <211> LENGTH: 2847
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pLIT38attB Plasmid
    <400> SEQUENCE: 120
    gttaactacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 60
    tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 120
    ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 180
    ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 240
    tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 300
    gatccttgag agttttcgcc ccgaagaacg ttctccaatg atgagcactt ttaaagttct 360
    gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 420
    acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 480
    tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 540
    caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 600
    gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 660
    cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 720
    tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 780
    agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 840
    tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 900
    ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 960
    acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 1020
    ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg 1080
    aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta 1140
    aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1200
    aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1260
    ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1320
    ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta 1380
    aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcg aacgtggcga 1440
    gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 1500
    cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtaaaagg 1560
    atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 1620
    ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 1680
    ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 1740
    ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 1800
    ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 1860
    ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 1920
    tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 1980
    tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2040
    tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2100
    tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2160
    gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2220
    tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2280
    ttcctggcct tttgctggcc ttttgctcac atgtaatgtg agttagctca ctcattaggc 2340
    accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 2400
    acaatttcac acaggaaaca gctatgacca tgattacgcc aagctacgta atacgactca 2460
    ctagtggggc ccgtgcaatt gaagccggct ggcgccaagc ttctctgcag gattgaagcc 2520
    tgctttttta tactaacttg agcgaaatct ggatccacga attcgctagc ttcggccgtg 2580
    acgcgtctcc ggatgtacag gcatgcgtcg accctctagt caaggcctta agtgagtcgt 2640
    attacggact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 2700
    ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 2760
    ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcttc gcttggtaat 2820
    aaagcccgct tcggcgggct ttttttt 2847
    <210> SEQ ID NO 121
    <211> LENGTH: 4223
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pLIT38attBBSRpolyA2 Plasmid
    <400> SEQUENCE: 121
    accatgaaaa catttaacat ttctcaacaa gatctagaat tagtagaagt agcgacagag 60
    aagattacaa tgctttatga ggataataaa catcatgtgg gagcggcaat tcgtacgaaa 120
    acaggagaaa tcatttcggc agtacatatt gaagcgtata taggacgagt aactgtttgt 180
    gcagaagcca ttgcgattgg tagtgcagtt tcgaatggac aaaaggattt tgacacgatt 240
    gtagctgtta gacaccctta ttctgacgaa gtagatagaa gtattcgagt ggtaagtcct 300
    tgtggtatgt gtagggagtt gatttcagac tatgcaccag attgttttgt gttaatagaa 360
    atgaatggca agttagtcaa aactacgatt gaagaactca ttccactcaa atatacccga 420
    aattaaaagt tttaccatac caagcttggc tgctgcctga ggctggacga cctcgcggag 480
    ttctaccggc agtgcaaatc cgtcggcatc caggaaacca gcagcggcta tccgcgcatc 540
    catgcccccg aactgcagga gtggggaggc acgatggccg ctttggtccg gatctttgtg 600
    aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc 660
    tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt 720
    tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg gaatgccttt 780
    aatgaggaaa acctgttttg ctcagaagaa atgccatcta gtgatgatga ggctactgct 840
    gactctcaac attctactcc tccaaaaaag aagagaaagg tagaagaccc caaggacttt 900
    ccttcagaat tgctaagttt tttgagtcat gctgtgttta gtaatagaac tcttgcttgc 960
    tttgctattt acaccacaaa ggaaaaagct gcactgctat acaagaaaat tatggaaaaa 1020
    tattctgtaa cctttataag taggcataac agttataatc ataacatact gttttttctt 1080
    actccacaca ggcatagagt gtctgctatt aataactatg ctcaaaaatt gtgtaccttt 1140
    agctttttaa tttgtaaagg ggttaataag gaatatttga tgtatagtgc cttgactaga 1200
    gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca 1260
    cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 1320
    agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagatccaga 1380
    tttcgctcaa gttagtataa aaaagcaggc ttcaatcctg cagagaagct tggcgccagc 1440
    cggcttcaat tgcacgggcc ccactagtga gtcgtattac gtagcttggc gtaatcatgg 1500
    tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 1560
    ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attacatgtg 1620
    agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1680
    taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1740
    cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 1800
    tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 1860
    gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 1920
    gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 1980
    tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2040
    gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2100
    cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2160
    aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2220
    tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2280
    ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2340
    attatcaaaa aggatcttca cctagatcct tttacgcgcc ctgtagcggc gcattaagcg 2400
    cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2460
    ctcctttcgc tttcttccct tcctttctcg ccacgttcgc tttccccgtc aagctctaaa 2520
    tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2580
    tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2640
    gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2700
    ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 2760
    aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 2820
    aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 2880
    ggtaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 2940
    agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3000
    gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 3060
    ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3120
    gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3180
    cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 3240
    acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3300
    cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3360
    cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 3420
    ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 3480
    tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 3540
    acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggagaacgt 3600
    tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 3660
    actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 3720
    aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 3780
    ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 3840
    ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 3900
    cgaaaagtgc cacctgacgt agttaacaaa aaaaagcccg ccgaagcggg ctttattacc 3960
    aagcgaagcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 4020
    cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 4080
    taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtccg taatacgact 4140
    cacttaaggc cttgactaga gggtcgacgc atgcctgtac atccggagac gcgtcacggc 4200
    cgaagctagc gaattcgtgg atc 4223
    <210> SEQ ID NO 122
    <211> LENGTH: 2686
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pUC18 Plasmid
    <400> SEQUENCE: 122
    tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
    cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
    ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
    accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
    attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
    tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
    tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420
    actctagagg atccccgggt accgagctcg aattcgtaat catggtcata gctgtttcct 480
    gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540
    aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600
    gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660
    agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720
    gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780
    gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840
    cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900
    aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960
    tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020
    ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080
    ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140
    cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200
    ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260
    gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320
    atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380
    aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440
    aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500
    gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560
    cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620
    gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680
    tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740
    ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800
    ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860
    atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920
    cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980
    tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040
    aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100
    tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160
    ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220
    agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280
    gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340
    agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400
    accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460
    gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520
    cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580
    ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640
    atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686
    <210> SEQ ID NO 123
    <211> LENGTH: 8521
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCXeGFPattB(6xHS4)2 Plasmid
    <400> SEQUENCE: 123
    tacggggcgg gggatccact agttattaat agtaatcaat tacggggtca ttagttcata 60
    gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
    ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
    ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
    atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
    cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
    tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
    atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
    gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
    gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
    tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
    gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
    ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
    gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
    ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
    tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
    cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
    ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
    tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
    cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
    gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
    ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
    gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
    gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
    tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
    cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
    acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
    gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
    acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcgccacc atggtgagca 1740
    agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1800
    acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1860
    ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1920
    ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1980
    tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2040
    acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2100
    tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2160
    acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2220
    tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2280
    agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2340
    cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2400
    tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcactc 2460
    ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 2520
    aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 2580
    agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 2640
    ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg 2700
    agtatttggt ttagagtttg gcaacatatg ccatatgctg gctgccatga acaaaggtgg 2760
    ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga 2820
    aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta 2880
    acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta 2940
    ctcccagtca tagctgtccc tcttctctta tgaagatccc tcgacctgca gcccaagctt 3000
    ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 3060
    caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 3120
    cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg 3180
    gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3240
    ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 3300
    gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt 3360
    ggaggctagt ggatcccccg ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg 3420
    agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca 3480
    cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg 3540
    ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct 3600
    gtccccgtga gcggatccgc ggccccgtat cccccaggtg tctgcaggct caaagagcag 3660
    cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg 3720
    cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct 3780
    cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg 3840
    ctgtccccgt gagcggatcc gcggccccgt atcccccagg tgtctgcagg ctcaaagagc 3900
    agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc 3960
    cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg 4020
    ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg 4080
    ggctgtcccc gtgagcggat ccgcggcccc gtatccccca ggtgtctgca ggctcaaaga 4140
    gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc 4200
    cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc 4260
    ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg 4320
    ggggctgtcc ccgtgagcgg atccgcggcc ccgtatcccc caggtgtctg caggctcaaa 4380
    gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg 4440
    tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg 4500
    gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg 4560
    ggggggctgt ccccgtgagc ggatccgcgg ccccgtatcc cccaggtgtc tgcaggctca 4620
    aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc 4680
    tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc 4740
    gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg 4800
    ggggggggct gtccccgtga gcggatccgc ggggctgcag gaattcgatt gaagcctgct 4860
    tttttatact aacttgagcg aaatcaagct cctaggcttt tgcaaaaagc taacttgttt 4920
    attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 4980
    tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5040
    tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5100
    gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5160
    tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5220
    agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5280
    cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5340
    ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5400
    tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5460
    gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 5520
    gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5580
    gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5640
    ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5700
    ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5760
    ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5820
    gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5880
    ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5940
    tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6000
    ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 6060
    gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 6120
    tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 6180
    cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 6240
    ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 6300
    gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 6360
    caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6420
    gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 6480
    ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6540
    tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 6600
    caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6660
    tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6720
    cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6780
    ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6840
    aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6900
    tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 6960
    gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 7020
    gaaaagtgcc acctggtcga cggtatcgat aagcttgata tcgaattcct gcagccccgc 7080
    ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc 7140
    cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc 7200
    atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc 7260
    tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg atacggggcc 7320
    gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct 7380
    cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc 7440
    gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc 7500
    gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg ggatacgggg 7560
    ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc 7620
    ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 7680
    ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 7740
    tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg ggggatacgg 7800
    ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat gtaattacgt 7860
    ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc 7920
    ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg 7980
    gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tgggggatac 8040
    ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 8100
    gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 8160
    ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 8220
    gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 8280
    acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 8340
    acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 8400
    ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 8460
    acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 8520
    a 8521
    <210> SEQ ID NO 124
    <211> LENGTH: 8851
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18EPOcDNA Plasmid
    <400> SEQUENCE: 124
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420
    agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480
    gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540
    ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600
    gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660
    acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720
    acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780
    ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840
    acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900
    atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960
    ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020
    cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080
    gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140
    ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200
    aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260
    ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320
    tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380
    ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440
    gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500
    ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560
    ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620
    tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680
    atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740
    gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800
    aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860
    cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920
    tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980
    gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040
    cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100
    tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160
    aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220
    acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280
    actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340
    ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400
    cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460
    ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520
    gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580
    ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640
    ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700
    gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760
    gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820
    tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880
    gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940
    gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000
    ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060
    gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120
    ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180
    ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240
    gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300
    caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360
    gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420
    cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480
    tgaccggcgg ctctagaatg ggggtgcacg aatgtcctgc ctggctgtgg cttctcctgt 3540
    ccctgctgtc gctccctctg ggcctcccag tcctgggcgc cccaccacgc ctcatctgtg 3600
    acagccgagt cctggagagg tacctcttgg aggccaagga ggccgagaat atcacgacgg 3660
    gctgtgctga acactgcagc ttgaatgaga atatcactgt cccagacacc aaagttaatt 3720
    tctatgcctg gaagaggatg gaggtcgggc agcaggccgt agaagtctgg cagggcctgg 3780
    ccctgctgtc ggaagctgtc ctgcggggcc aggccctgtt ggtcaactct tcccagccgt 3840
    gggagcccct gcagctgcat gtggataaag ccgtcagtgg ccttcgcagc ctcaccactc 3900
    tgcttcgggc tctgggagcc cagaaggaag ccatctcccc tccagatgcg gcctcagctg 3960
    ctccactccg aacaatcact gctgacactt tccgcaaact cttccgagtc tactccaatt 4020
    tcctccgggg aaagctgaag ctgtacacag gggaggcctg caggacaggg gacagatgac 4080
    gtacaagtaa gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt 4140
    ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg 4200
    ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat 4260
    tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa 4320
    tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg ccatatgctg 4380
    gctgccatga acaaaggtgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 4440
    tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 4500
    ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 4560
    ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tgaagatccc 4620
    tcgacctgca gcccaagctt gcatgcctgc aggtcgactc tagtggatcc cccgccccgt 4680
    atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc 4740
    gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg 4800
    agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac 4860
    gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat ccgcggcccc 4920
    gtatccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc 4980
    ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg 5040
    ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg 5100
    acgtaattac atccctgggg gctttggggg ggggctgtcc ccgtgagcgg atccgcggcc 5160
    ccgtatcccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga 5220
    tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg 5280
    ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag 5340
    ggacgtaatt acatccctgg gggctttggg ggggggctgt ccccgtgagc ggatccgcgg 5400
    ccccgtatcc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc 5460
    gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc 5520
    ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg 5580
    agggacgtaa ttacatccct gggggctttg ggggggggct gtccccgtga gcggatccgc 5640
    ggccccgtat cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa 5700
    gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat 5760
    gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg 5820
    ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccccgt gagcggatcc 5880
    gcggccccgt atcccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga 5940
    aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg 6000
    atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg 6060
    ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtcccc gtgagcggat 6120
    ccgcggggct gcaggaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6180
    ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 6240
    taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6300
    aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6360
    attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6420
    cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6480
    gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6540
    ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6600
    agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6660
    tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6720
    ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6780
    gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6840
    ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6900
    gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6960
    aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 7020
    aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 7080
    ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7140
    gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7200
    gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7260
    tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 7320
    ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 7380
    ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 7440
    atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 7500
    ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7560
    tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7620
    attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7680
    tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7740
    ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7800
    gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7860
    gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7920
    gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7980
    aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 8040
    taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 8100
    tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 8160
    tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 8220
    atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 8280
    tttccccgaa aagtgccacc tgacgtagtt aacaaaaaaa agcccgccga agcgggcttt 8340
    attaccaagc gaagcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 8400
    tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 8460
    gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtccgtaat 8520
    acgactcact taaggccttg actagagggt cgacggtata cagacatgat aagatacatt 8580
    gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 8640
    tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc 8700
    gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac 8760
    gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg 8820
    tcagtcctgc tcctcggcca cgaagtgcac g 8851
    <210> SEQ ID NO 125
    <211> LENGTH: 10474
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18genEPO Plasmid
    <400> SEQUENCE: 125
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgatatc gaattcctgc 420
    agccccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac 480
    gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct 540
    ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac 600
    gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggat 660
    acggggccgc ggatccgctc acggggacag ccccccccca aagcccccag ggatgtaatt 720
    acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg 780
    ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc 840
    acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg 900
    atacggggcc gcggatccgc tcacggggac agcccccccc caaagccccc agggatgtaa 960
    ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg 1020
    cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg 1080
    gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg 1140
    ggatacgggg ccgcggatcc gctcacgggg acagcccccc cccaaagccc ccagggatgt 1200
    aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc 1260
    ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg 1320
    tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg 1380
    ggggatacgg ggccgcggat ccgctcacgg ggacagcccc cccccaaagc ccccagggat 1440
    gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt 1500
    ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa 1560
    ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc 1620
    tgggggatac ggggccgcgg atccgctcac ggggacagcc cccccccaaa gcccccaggg 1680
    atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 1740
    gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 1800
    aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 1860
    cctgggggat acggggcggg ggatccacta gttattaata gtaatcaatt acggggtcat 1920
    tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1980
    gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2040
    cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact 2100
    tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2160
    aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2220
    acatctacgt attagtcatc gctattacca tgggtcgagg tgagccccac gttctgcttc 2280
    actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 2340
    ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 2400
    cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 2460
    ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 2520
    gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 2580
    ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 2640
    ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 2700
    gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 2760
    gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg 2820
    tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg 2880
    gccgggggcg gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg 2940
    gtgtgtgcgt gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc 3000
    ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg 3060
    gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc 3120
    ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg 3180
    ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga 3240
    gggcgcaggg acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg 3300
    caccccctct agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg 3360
    gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc 3420
    cgcaggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 3480
    tgaccggcgg ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat 3540
    tcgccctttc tagaatgggg gtgcacggtg agtactcgcg ggctgggcgc tcccgcccgc 3600
    ccgggtccct gtttgagcgg ggatttagcg ccccggctat tggccaggag gtggctgggt 3660
    tcaaggaccg gcgacttgtc aaggaccccg gaagggggag gggggtgggg tgcctccacg 3720
    tgccagcggg gacttggggg agtccttggg gatggcaaaa acctgacctg tgaaggggac 3780
    acagtttggg ggttgagggg aagaaggttt gggggttctg ctgtgccagt ggagaggaag 3840
    ctgataagct gataacctgg gcgctggagc caccacttat ctgccagagg ggaagcctct 3900
    gtcacaccag gattgaagtt tggccggaga agtggatgct ggtagctggg ggtggggtgt 3960
    gcacacggca gcaggattga atgaaggcca gggaggcagc acctgagtgc ttgcatggtt 4020
    ggggacagga aggacgagct ggggcagaga cgtggggatg aaggaagctg tccttccaca 4080
    gccacccttc tccctccccg cctgactctc agcctggcta tctgttctag aatgtcctgc 4140
    ctggctgtgg cttctcctgt ccctgctgtc gctccctctg ggcctcccag tcctgggcgc 4200
    cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg aggccaagga 4260
    ggccgagaat atcacggtga gaccccttcc ccagcacatt ccacagaact cacgctcagg 4320
    gcttcaggga actcctccca gatccaggaa cctggcactt ggtttggggt ggagttggga 4380
    agctagacac tgccccccta cataagaata agtctggtgg ccccaaacca tacctggaaa 4440
    ctaggcaagg agcaaagcca gcagatccta cggcctgtgg gccagggcca gagccttcag 4500
    ggacccttga ctccccgggc tgtgtgcatt tcagacgggc tgtgctgaac actgcagctt 4560
    gaatgagaat atcactgtcc cagacaccaa agttaatttc tatgcctgga agaggatgga 4620
    ggtgagttcc tttttttttt tttttccttt cttttggaga atctcatttg cgagcctgat 4680
    tttggatgaa agggagaatg atcgagggaa aggtaaaatg gagcagcaga gatgaggctg 4740
    cctgggcgca gaggctcacg tctataatcc caggctgaga tggccgagat gggagaattg 4800
    cttgagccct ggagtttcag accaacctag gcagcatagt gagatccccc atctctacaa 4860
    acatttaaaa aaattagtca ggtgaagtgg tgcatggtgg tagtcccaga tatttggaag 4920
    gctgaggcgg gaggatcgct tgagcccagg aatttgaggc tgcagtgagc tgtgatcaca 4980
    ccactgcact ccagcctcag tgacagagtg aggccctgtc tcaaaaaaga aaagaaaaaa 5040
    gaaaaataat gagggctgta tggaatacat tcattattca ttcactcact cactcactca 5100
    ttcattcatt cattcattca acaagtctta ttgcatacct tctgtttgct cagcttggtg 5160
    cttggggctg ctgaggggca ggagggagag ggtgacatgg gtcagctgac tcccagagtc 5220
    cactccctgt aggtcgggca gcaggccgta gaagtctggc agggcctggc cctgctgtcg 5280
    gaagctgtcc tgcggggcca ggccctgttg gtcaactctt cccagccgtg ggagcccctg 5340
    cagctgcatg tggataaagc cgtcagtggc cttcgcagcc tcaccactct gcttcgggct 5400
    ctgggagccc aggtgagtag gagcggacac ttctgcttgc cctttctgta agaaggggag 5460
    aagggtcttg ctaaggagta caggaactgt ccgtattcct tccctttctg tggcactgca 5520
    gcgacctcct gttttctcct tggcagaagg aagccatctc ccctccagat gcggcctcag 5580
    ctgctccact ccgaacaatc actgctgaca ctttccgcaa actcttccga gtctactcca 5640
    atttcctccg gggaaagctg aagctgtaca caggggaggc ctgcaggaca ggggacagat 5700
    gacgtacaag taagaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 5760
    tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 5820
    tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt 5880
    cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc 5940
    aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgccatatg 6000
    ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa cagccccctg 6060
    ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt 6120
    gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca 6180
    gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatgaagat 6240
    ccctcgacct gcagcccaag cttgcatgcc tgcaggtcga ctctagtgga tcccccgccc 6300
    cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat 6360
    cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg 6420
    gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg 6480
    gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg gatccgcggc 6540
    cccgtatccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 6600
    atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 6660
    gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 6720
    gggacgtaat tacatccctg ggggctttgg gggggggctg tccccgtgag cggatccgcg 6780
    gccccgtatc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag 6840
    cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg 6900
    cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg 6960
    gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg agcggatccg 7020
    cggccccgta tcccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa 7080
    agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga 7140
    tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg 7200
    gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagcggatc 7260
    cgcggccccg tatcccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 7320
    aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 7380
    gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 7440
    gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc cgtgagcgga 7500
    tccgcggccc cgtatccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 7560
    ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 7620
    gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 7680
    gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagcg 7740
    gatccgcggg gctgcaggaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 7800
    tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 7860
    gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 7920
    ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 7980
    cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 8040
    cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 8100
    aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 8160
    gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 8220
    tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 8280
    agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 8340
    ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 8400
    taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 8460
    gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 8520
    gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 8580
    ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 8640
    ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 8700
    gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 8760
    caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 8820
    taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 8880
    aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 8940
    tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9000
    tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9060
    gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9120
    gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9180
    aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9240
    gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9300
    ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9360
    tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 9420
    atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 9480
    ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 9540
    ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 9600
    ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 9660
    atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 9720
    gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 9780
    tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 9840
    ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 9900
    acatttcccc gaaaagtgcc acctgacgta gttaacaaaa aaaagcccgc cgaagcgggc 9960
    tttattacca agcgaagcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat 10020
    cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 10080
    taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtccgt 10140
    aatacgactc acttaaggcc ttgactagag ggtcgacggt atacagacat gataagatac 10200
    attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 10260
    atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg 10320
    ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa 10380
    aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtagcac 10440
    gtgtcagtcc tgctcctcgg ccacgaagtg cacg 10474
    <210> SEQ ID NO 126
    <211> LENGTH: 6119
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: p18attBZeoeGFP Plasmid
    <400> SEQUENCE: 126
    cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg 60
    gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg 120
    tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg 180
    tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc 240
    tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg 300
    tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggatcc agatttcgct 360
    caagttagta taaaaaagca ggcttcaatc ctgcagagaa gcttgggctg caggtcgagg 420
    gatcttcata agagaagagg gacagctatg actgggagta gtcaggagag gaggaaaaat 480
    ctggctagta aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa 540
    caaaatataa aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca 600
    gcagggggct gtttcatata ctgatgacct ctttatagcc acctttgttc atggcagcca 660
    gcatatggca tatgttgcca aactctaaac caaatactca ttctgatgtt ttaaatgatt 720
    tgccctccca tatgtccttc cgagtgagag acacaaaaaa ttccaacaca ctattgcaat 780
    gaaaataaat ttcctttatt agccagaagt cagatgctca aggggcttca tgatgtcccc 840
    ataatttttg gcagagggaa aaagatctca gtggtatttg tgagccaggg cattggccac 900
    accagccacc accttctgat aggcagcctg cacctgagga gtgaattctt acttgtacag 960
    ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc 1020
    gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg 1080
    cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac 1140
    gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt tcttctgctt 1200
    gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt gccccaggat 1260
    gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc 1320
    ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg 1380
    ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc 1440
    ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg 1500
    cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc 1560
    gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac 1620
    caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg tggcgaattc 1680
    tttgccaaaa tgatgagaca gcacaacaac cagcacgttg cccaggagct gtaggaaaaa 1740
    gaagaaggca tgaacatggt tagcagaggc tctagagccg ccggtcacac gccagaagcc 1800
    gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc ccctgcggca gccccgaggc 1860
    tggagatgga gaaggggacg gcggcgcggc gacgcacgaa ggccctcccc gcccatttcc 1920
    ttcctgccgg cgccgcaccg cttcgcccgc gcccgctaga gggggtgcgg cggcgcctcc 1980
    cagatttcgg ctccgccaga tttgggacaa aggaagtccc tgcgccctct cgcacgatta 2040
    ccataaaagg caatggctgc ggctcgccgc gcctcgacag ccgccggcgc tccggggccg 2100
    ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc ccgccccgcc cggcaccccc 2160
    acctgccgcc accccccgcc cggcacggcg agccccgcgc cacgccccgc acggagcccc 2220
    gcacccgaag ccgggccgtg ctcagcaact cggggagggg ggtgcagggg ggggttacag 2280
    cccgaccgcc gcgcccacac cccctgctca cccccccacg cacacacccc gcacgcagcc 2340
    tttgttcccc tcgcagcccc cccgcaccgc ggggcaccgc ccccggccgc gctcccctcg 2400
    cgcacacgcg gagcgcacaa agccccgcgc cgcgcccgca gcgctcacag ccgccgggca 2460
    gcgcgggccg cacgcggcgc tccccacgca cacacacacg cacgcacccc ccgagccgct 2520
    cccccccgca caaagggccc tcccggagcc ctttaaggct ttcacgcagc cacagaaaag 2580
    aaacgagccg tcattaaacc aagcgctaat tacagcccgg aggagaaggg ccgtcccgcc 2640
    cgctcacctg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc gaggcggcgc 2700
    ggagcggggc acggggcgaa ggcaacgcag cgactcccgc ccgccgcgcg cttcgctttt 2760
    tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct 2820
    gattggctgc cgccgcacct ctccgcctcg ccccgccccg cccctcgccc cgccccgccc 2880
    cgcctggcgc gcgccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata 2940
    aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg 3000
    gggctcacct cgacccatgg taatagcgat gactaatacg tagatgtact gccaagtagg 3060
    aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac 3120
    gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta 3180
    ccgtaaatag tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac 3240
    atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt 3300
    taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat 3360
    tgattactat taataactag aggatccccg ggtaccgagc tcgaattcgt aatcatggtc 3420
    atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3480
    aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3540
    gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3600
    ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3660
    ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3720
    acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3780
    aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3840
    tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3900
    aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3960
    gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 4020
    acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4080
    accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4140
    ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4200
    gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4260
    gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4320
    ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4380
    gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4440
    cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4500
    cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4560
    gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4620
    tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4680
    gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4740
    agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4800
    tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4860
    agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4920
    gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4980
    catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5040
    ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5100
    atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5160
    tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5220
    cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5280
    cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5340
    atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5400
    aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5460
    ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5520
    aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtagttaa 5580
    caaaaaaaag cccgccgaag cgggctttat taccaagcga agcgccattc gccattcagg 5640
    ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 5700
    aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 5760
    cgttgtaaaa cgacggccag tccgtaatac gactcactta aggccttgac tagagggtcg 5820
    acggtataca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 5880
    gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5940
    aagctgcaat aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga 6000
    ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg 6060
    cggtggaatc gaaatctcgt agcacgtgtc agtcctgctc ctcggccacg aagtgcacg 6119
    <210> SEQ ID NO 127
    <211> LENGTH: 5855
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pCXLamInt Plasmid (Wildtype Integrase)
    <400> SEQUENCE: 127
    gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60
    gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120
    ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180
    ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240
    atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300
    cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360
    tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420
    atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480
    gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540
    gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600
    tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660
    gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720
    ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780
    gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840
    ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900
    tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960
    cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020
    ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080
    tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140
    cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200
    gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260
    ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320
    gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380
    gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440
    tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
    cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560
    acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620
    gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680
    acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggga agaaggcgaa 1740
    gtcatgagcg ccgggattta ccccctaacc tttatataag aaacaatgga tattactgct 1800
    acagggaccc aaggacgggt aaagagtttg gattaggcag agacaggcga atcgcaatca 1860
    ctgaagctat acaggccaac attgagttat tttcaggaca caaacacaag cctctgacag 1920
    cgagaatcaa cagtgataat tccgttacgt tacattcatg gcttgatcgc tacgaaaaaa 1980
    tcctggccag cagaggaatc aagcagaaga cactcataaa ttacatgagc aaaattaaag 2040
    caataaggag gggtctgcct gatgctccac ttgaagacat caccacaaaa gaaattgcgg 2100
    caatgctcaa tggatacata gacgagggca aggcggcgtc agccaagtta atcagatcaa 2160
    cactgagcga tgcattccga gaggcaatag ctgaaggcca tataacaaca aaccatgtcg 2220
    ctgccactcg cgcagcaaaa tcagaggtaa ggagatcaag acttacggct gacgaatacc 2280
    tgaaaattta tcaagcagca gaatcatcac catgttggct cagacttgca atggaactgg 2340
    ctgttgttac cgggcaacga gttggtgatt tatgcgaaat gaagtggtct gatatcgtag 2400
    atggatatct ttatgtcgag caaagcaaaa caggcgtaaa aattgccatc ccaacagcat 2460
    tgcatattga tgctctcgga atatcaatga aggaaacact tgataaatgc aaagagattc 2520
    ttggcggaga aaccataatt gcatctactc gtcgcgaacc gctttcatcc ggcacagtat 2580
    caaggtattt tatgcgcgca cgaaaagcat caggtctttc cttcgaaggg gatccgccta 2640
    cctttcacga gttgcgcagt ttgtctgcaa gactctatga gaagcagata agcgataagt 2700
    ttgctcaaca tcttctcggg cataagtcgg acaccatggc atcacagtat cgtgatgaca 2760
    gaggcaggga gtgggacaaa attgaaatca aataagaatt cactcctcag gtgcaggctg 2820
    cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc 2880
    tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg 2940
    gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 3000
    tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga 3060
    gtttggcaac atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat 3120
    cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga 3180
    ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt 3240
    tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct 3300
    gtccctcttc tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc 3360
    atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3420
    aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3480
    gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat 3540
    tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3600
    tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3660
    gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3720
    tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3780
    caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3840
    tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag 3900
    aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3960
    cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4020
    atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4080
    taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4140
    aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4200
    tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4260
    gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct 4320
    cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4380
    cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4440
    atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4500
    tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 4560
    ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4620
    acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4680
    aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4740
    aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4800
    tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4860
    cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4920
    catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4980
    ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5040
    aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5100
    ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5160
    caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5220
    attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5280
    agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5340
    actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5400
    ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 5460
    ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 5520
    gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5580
    atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5640
    cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5700
    gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5760
    gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5820
    ggttccgcgc acatttcccc gaaaagtgcc acctg 5855
    <210> SEQ ID NO 128
    <211> LENGTH: 303
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Human FER-1 Promoter
    <400> SEQUENCE: 128
    tccatgacaa agcacttttt gagcccaagc ccagcctagc tcgagctaaa cgggcacaga 60
    gacgccaccg ctgtcccaga ggcagtcggc taccggtccc cgctcccgag ctccgccaga 120
    gcgcgcgagg gcctccagcg gccgcccctc ccccacagca ggggcggggt cccgcgccca 180
    ccggaaggag cgggctcggg gcgggcggcg ctgattggcc ggggcgggcc tgacgccgac 240
    gcggctataa gagaccacaa gcgacccgca gggccagacg ttcttcgccg agagtcgggt 300
    acc 303
    <210> SEQ ID NO 129
    <211> LENGTH: 6521
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pIRES-BSR Plasmid
    <400> SEQUENCE: 129
    tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60
    ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120
    aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180
    gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240
    gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300
    agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360
    ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420
    cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480
    gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540
    caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600
    caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660
    cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720
    agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780
    agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840
    gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900
    ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960
    cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020
    aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080
    ataggctagc ctcgagaatt cacgcgtcga gcatgcatct agggcggcca attccgcccc 1140
    tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg 1200
    tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa 1260
    cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg 1320
    caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca 1380
    acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc 1440
    ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt 1500
    gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 1560
    ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca 1620
    tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac 1680
    gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccaccatg aaaacattta 1740
    acatttctca acaagatcta gaattagtag aagtagcgac agagaagatt acaatgcttt 1800
    atgaggataa taaacatcat gtgggagcgg caattcgtac gaaaacagga gaaatcattt 1860
    cggcagtaca tattgaagcg tatataggac gagtaactgt ttgtgcagaa gccattgcga 1920
    ttggtagtgc agtttcgaat ggacaaaagg attttgacac gattgtagct gttagacacc 1980
    cttattctga cgaagtagat agaagtattc gagtggtaag tccttgtggt atgtgtaggg 2040
    agttgatttc agactatgca ccagattgtt ttgtgttaat agaaatgaat ggcaagttag 2100
    tcaaaactac gattgaagaa ctcattccac tcaaatatac ccgaaattaa aagttttacc 2160
    ataccaagct tggcgggcgg ccgcttccct ttagtgaggg ttaatgcttc gagcagacat 2220
    gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 2280
    tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 2340
    agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 2400
    tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc cgataaggat cgatccgggc 2460
    tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 2520
    ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2580
    gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2640
    ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2700
    tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2760
    gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2820
    ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2880
    ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 2940
    aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3000
    tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat 3060
    ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc 3120
    agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa 3180
    gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 3240
    cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 3300
    taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 3360
    gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 3420
    agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagcttgat tcttctgaca 3480
    caacagtctc gaacttaagg ctagagccac catgattgaa caagatggat tgcacgcagg 3540
    ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3600
    ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3660
    gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3720
    ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3780
    ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3840
    cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3900
    ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3960
    cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 4020
    gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 4080
    tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 4140
    ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 4200
    agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 4260
    ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 4320
    ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc 4380
    tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatccg 4440
    cgtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4500
    cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4560
    acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4620
    acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4680
    aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4740
    tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4800
    gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4860
    tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4920
    aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4980
    cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5040
    agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5100
    ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5160
    tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5220
    tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5280
    caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5340
    accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 5400
    attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5460
    ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5520
    taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5580
    taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5640
    aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5700
    agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5760
    ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5820
    ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5880
    cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5940
    tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6000
    tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6060
    tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6120
    tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6180
    ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6240
    acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6300
    ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 6360
    gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6420
    ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6480
    ggccttttgc tggccttttg ctcacatggc tcgacagatc t 6521

Claims (123)

What is claimed is:
1. A eukaryotic chromosome comprising one or a plurality of att site(s), wherein:
an att site is heterologous to the chromosome; and
an att site permits site-directed integration in the presence of lambda integrase.
2. The eukaryotic chromosome of claim 1, wherein the att sites are selected from the group consisting of attP and attB or attL and attR, or variants thereof.
3. The eukaryotic chromosome of claim 1 that is an artificial chromosome.
4. The eukaryotic chromosome of claim 1 that is an artificial chromosome expression system (ACes).
5. The eukaryotic chromosome of claim 4 that is predominantly heterochromatin.
6. The chromosome of claim 1 that is an artificial chromosome that contains no more than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% euchromatin.
7. The chromosome of claim 1 that is a plant chromosome.
8. The chromosome of claim 1 that is an animal chromosome.
9. The chromosome of claim 7 that is a plant artificial chromosome.
10. The chromosome of claim 8 that is an animal artificial chromosome.
11. The chromosome of claim 8 that is a mammalian chromosome.
12. The chromosome of claim 11 that is a mammalian artificial chromosome.
13. The chromosome of claim 6 that is an artificial chromosome expression system (ACes).
14. A platform artificial chromosome expression system (ACes) comprising one or a plurality of sites that participate in recombinase catalyzed recombination.
15. The ACes of claim 14 that contains one site.
16. The ACes of claim 14 that is predominantly heterochromatin.
17. The ACes of claim 14 that contains no more than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% euchromatin.
18. The ACes of claim 14 that is a plant ACes.
19. The ACes of claim 14 that is an animal ACes.
20. The ACes of claim 14 that is selected from a fish, insect, reptile, amphibian, arachnid or a mammalian ACes.
21. The ACes of claim 14 that is a fish ACes.
22. The artificial chromosome expression system (ACes) of claim 14, wherein the recombinase and site(s) are from the Cre/lox system of bacteriophage P1, the int/att system of lambda phage, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Cin recombinase system, the Pin recombinase system of E. coli and the R/RS system of the pSR1 plasmid, or any combination thereof.
23. A method of introducing heterologous nucleic acid into a chromosome, comprising:
contacting a chromosome of claim 1 with a nucleic acid molecule comprising both the heterologous nucleic acid and a recombination site, in the presence of a recombinase that promotes recombination between the sites in the chromosome and in the nucleic acid molecule.
24. The method of claim 23, wherein the recombinase is selected from the group consisting of Cre, Gin, Cin, Pin, FLP, a phage integrase and R from the pSR1 plasmid.
25. The method of claim 23, wherein the nucleic acid molecule encodes a therapeutic protein, antisense nucleic acid, or comprises an artificial chromosome.
26. The method of claim 25, wherein the nucleic acid molecule comprises a yeast artificial chromosomes (YAC), a bacterial artificial chromosome (BAC) or an insect artificial chromosome (IAC).
27. A combination, comprising, the chromosome of claim 1 and a first vector comprising the cognate recombination site, wherein the cognate recombination site is a site that recombines with the site engineered into the chromosome.
28. The combination of claim 27, further comprising nucleic acid encoding a recombinase, wherein the nucleic acid is on a second vector or on the first vector, or on the ACes under an inducible promoter.
29. The combination of claim 28, wherein the recombinase and sites are from the Cre/lox system of bacteriophage P1, the int/att system of lambda phage, the FLP/FRT system of yeast, the Gin/gix recombinase system of phage Mu, the Pin recombinase system of E. coli and the R/RS system of the pSR1 plasmid, or any combination thereof.
30. The combination of claim 28, wherein a vector is the plasmid pCXLamIntR.
31. The combination of claim 27, wherein a vector is the plasmid pDsRedN1-attB.
32. A kit, comprising the combination of claim 27 and optionally instructions for introducing heterologous nucleic acid into the chromosome.
33. A method for introducing heterologous nucleic acid into a platform artificial chromosome, comprising:
(a) mixing an artificial chromosome comprising at least a first recombination site and a vector comprising at least a second recombination site and the heterologous nucleic acid;
(b) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the artificial chromosome.
34. The method of claim 33, wherein the artificial chromosome is an ACes.
35. The method of claim 33, wherein said mixing step (a) is conducted in cells ex vivo.
36. The method of claim 33, wherein said mixing step (a) is conducted extracellularly in an in vitro reaction mixture.
37. The method of claim 33, wherein the at least one recombination protein is encoded by a bacteriophage selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.
38. The method of claim 37, wherein the at least one recombination protein is encoded by bacteriophage lambda, or mutants thereof.
39. The method of claim 33, wherein at least one recombination protein is selected from the group consisting of Int, IHF, Xis and Cre, γδ, Tn3 resolvase, Hin, Gin, Cin and Flp.
40. The method of claim 32, wherein the recombination sites are selected from the group consisting of att and lox P sites.
41. The method of claim 33, wherein the first and/or second recombination site contains at least one mutation that removes one or more stop codons.
42. The method of claim 33, wherein the first and/or second recombination site contains at least one mutation that avoids hairpin formation.
43. The method of claim 33, wherein the first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:41-56:
a) RKYCWGCTTTYKTRTACNAASTSGB; (SEQ ID NO:41) (m-att) b) AGCCWGCTTTYKTRTACNAACTSGB; (SEQ ID NO:42) (m-attB) c) GTTCAGCTTTCKTRTACNAACTSGB; (SEQ ID NO:43) (m-attR) d) AGCCWGCTTTCKTRTACNAAGTSGB; (SEQ ID NO:44) (m-attL) e) GTTCAGCTTTYKTRTACNAAGTSGB; (SEQ ID NO:45) (m-attP1) f) AGCCTGCTTTTTTGTACAAACTTGT; (SEQ ID NO:46) (attB1) g) AGCCTGCTTTCTTGTACAAACTTGT; (SEQ ID NO:47) (attB2) h) ACCCAGGTTTCTTGTAGAAACTTGT; (SEQ ID NO:48) (attB3) i) GTTCAGCTTTTTTGTACAAACTTGT; (SEQ ID NO:49) (attR1) j) GTTCAGCTTTCTTGTACAAACTTGT; (SEQ ID NO:50) (attR2) k) GTTCAGGTTTCTTGTACAAAGTTGG; (SEQ ID NO:51) (attR3) l) AGCCTGCTTTTTTGTACAAAGTTGG; (SEQ ID NO:52) (attL1) m) AGCCTGCTTTCTTGTACAAAGTTGG; (SEQ ID NO:53) (attL2) n) ACCCAGCTTTCTTGTACAAAGTTGG; (SEQ ID NO:54) (attL3) o) GTTCAGCTTTTTTGTACAAAGTTGG; (SEQ ID NO:55) (attP1) p) GTTCAGCTTTCTTGTACAAAGTTGG; (SEQ ID NO:56) (attP2, P3)
and a corresponding or complementary DNA or RNA sequence, wherein R=A or G, K=G or T/U, Y=C or T/U, W=A or T/U, N=A or C or G or T/U, S=C or G, and B=C or G or T/U; and
the core region does not contain a stop codon in one or more reading frames.
44. The method of claim 33, wherein the first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.
45. The method of claim 33, wherein the vector comprising the second site further encodes at least one selectable marker.
46. The method of claim 45, wherein the marker is a promoterless marker, which, upon recombination is under the control of a promoter and is thereby expressed.
47. The method of claim 46, wherein the first recombination site is attP and is in the sense orientation prior to recombination.
48. The method of claim 46, wherein the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic, fluorescent, or capable of being bound by an antibody and FACs sorted.
49. The method of claim 48, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase (hisD).
50. A cell comprising, the chromosome of claim 1.
51. The cell of claim 50, wherein the cell is a nuclear donor cell.
52. The cell of claim 50, wherein the cell is a stem cell.
53. The stem cell of claim 52, wherein said stem cell is human and is selected from the group consisting of a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell and an embryonic stem cell.
54. The cell of claim 50, wherein the cell is mammalian.
55. The cell of claim 54, wherein the mammal is selected from the group consisting of humans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, hamsters, cats, dogs, and horses.
56. The cell of claim 50, wherein the cell is a plant cell.
57. A cell comprising the platform ACes of claim 14.
58. The cell of claim 57, wherein the cell is a nuclear donor cell.
59. The cell of claim 57, wherein the cell is a stem cell.
60. The stem cell of claim 59, wherein said stem cell is human and is selected from the group consisting of a mesenchymal stem cell, a hematopoietic stem cell, an adult stem cell and an embryonic stem cell.
61. A human mesenchymal cell comprising an artificial chromosome.
62. The human mesenchymal cell of claim 61, wherein said artificial chromosome is an ACes.
63. The human mesenchymal cell of claim 62, wherein the ACes is a platform-ACes.
64. A method for introducing heterologous nucleic acid into the mesenchymal cell of claim 63, comprising:
(a) introducing into the cell of claim 63, wherein the platform-ACes has a first recombination site, a vector comprising at least a second recombination site and the heterologous nucleic acid;
(b) incubating the resulting mixture in the presence of at least one recombination protein under conditions whereby recombination between the first and second recombination sites is effected, thereby introducing the heterologous nucleic acid into the platform-ACes within the mesenchymal cell.
65. A lambda-intR mutein comprising a glutamic acid to arginine change at position 174 of wild-type lambda-intR.
66. The lambda-intR mutein of claim 65, wherein the lambda-intR mutein comprises SEQ ID NO:37.
67. The method of claim 46 wherein the promoterless marker is transcriptionally downstream of the heterologous nucleic acid, wherein the heterologous nucleic acid encodes a heterologous protein, and wherein the expression level of the selectable marker is transcriptionally linked to the expression level of the heterologous protein.
68. The method of claim 67, wherein the selectable marker and the heterologous nucleic acid are transcriptionally linked by the presence of a IRES between them.
69. The method of claim 68, wherein the selectable marker is selected from the group consisting of an antibiotic resistance gene, and a detectable protein, wherein the detectable protein is chromogenic or fluorescent.
70. The method of claim 69, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and E. coli histidinol dehydrogenase.
71. The method of claim 67 further comprising expressing the heterologous protein and isolating the heterologous protein.
72. A method for producing a transgenic animal, comprising introducing a platform-ACes into an embryonic cell.
73. The method of claim 72, wherein the embryonic cell is a stem cell.
74. The method of claim 72, wherein the embryonic cell is in an embryo.
75. The method of claim 72, wherein the platform-ACes comprises heterologous nucleic acid that encodes a therapeutic product.
76. The method of claim 72, wherein the transgenic animal is a fish, insect, reptile, amphibians, arachnid or mammal.
77. The method of claim 72, wherein the ACes is introduced by cell fusion, lipid-mediated transfection by a carrier system, microinjection, microcell fusion, electroporation, microprojectile bombardment or direct DNA transfer.
78. A transgenic animal produced by the method of claim 72.
79. A cell line useful for making a library of ACes, comprising a multiplicity of heterologous recombination sites randomly integrated throughout the endogenous chromosomes.
80. A method of making a library of ACes comprising random portions of a genome, comprising introducing one or more ACes into the cell line of claim 79, under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites within the cell's chromosomal DNA; and isolating said multiplicity of ACes, thereby producing a library of ACes whereby multiple ACes have different portions of the genome within.
81. A library of cells useful for genomic screening, said library comprising a multiplicity of cells, wherein each cell comprises an ACes having a mutually exclusive portion of a chromosomal nucleic acid therein.
82. The library of cells of claim 81, wherein the cells of the library are from a different species than the chromosomal nucleic acid within the ACes.
83. A method of making one or more cell lines, comprising
a) integrating into endogenous chromosomal DNA of a selected cell species, a multiplicity of heterologous recombination sites,
b) introducing a multiplicity of ACes under conditions that promote the site-specific chromosomal arm exchange of the ACes into, and out of, a multiplicity of the heterologous recombination sites integrated within the cell's endogenous chromosomal DNA;
c) isolating said multiplicity of ACes, thereby producing a library of ACes whereby a multiplicity of ACes have mutually exclusive portions of the endogenous chromosomal DNA therein;
d) introducing the isolated multiplicity of ACes of step c) into a multiplicity of cells, thereby creating a library of cells;
e) selecting different cells having mutually exclusive ACes therein and clonally expanding or differentiating said different cells into clonal cell cultures, thereby creating one or more cell lines.
84. The method of claim 23, wherein the nucleic acid molecule with a recombination site is a PCR product.
85. Method of claim 23 wherein the recombinase is a protein and the recombination event occurs in vitro.
86. The method of claim 33, wherein the vector is a PCR product comprising a second recombination site.
87. The lambda-intR mutein of claim 65, wherein the mutein further comprises an amino acid signal for nuclear localization.
88. The lambda-intR mutein of claim 65, wherein the mutein further comprises an epitope tag for protein purification.
89. A modified iron-induced promoter comprising SEQ ID NO: 128.
90. A plasmid or expression cassette comprising the promoter of claim 89.
91. A vector, comprising:
a recognition site for recombination; and
a sequence of nucleotides that targets the vector to an amplifiable region of a chromosome.
92. The vector of claim 91, wherein the amplifiable region comprises heterochromatic nucleic acid.
93. The vector of claim 91, wherein the amplifiable region comprises rDNA.
94. The vector of claim 93, wherein the rDNA comprises an intergenic spacer.
95. The vector of claim 91, further comprising nucleic acid encoding a selectable marker that is not operably associated with any promoter.
96. The vector of claim 91, wherein the chromosome is a mammalian chromosome.
97. The vector of claim 91, wherein the chromosome is a plant chromosome.
98. A cell of claim 57 that is a plant cell, wherein the ACes platform is a MAC.
99. The plant cell of claim 98, wherein the MAC comprises transcriptional regulatory sequence of nucleotides derived from plants.
100. The plant cell of claim 99, wherein the regulatory sequence is selected from the group consisting of promoters, terminators, enhancers, silencers and transcription factor binding sites.
101. A cell of claim 57 that is an animal cell, wherein the ACes platform is a plant artificial chromosome (PAC).
102. The cell of claim 101 that is a mammalian cell.
103. The cell of claim 98, wherein the MAC comprises transcriptional regulatory sequence of nucleotides derived from plants.
104. The cell of claim 102, wherein the MAC comprises transcriptional regulatory sequence of nucleotides derived from plants.
105. The cell of claim 104, wherein the regulatory sequence is selected from the group consisting of promoters, terminators, enhancers, silencers and transcription factor binding sites.
106. A method, comprising:
introducing a vector of claim 91 into a cell;
growing the cells; and
selecting a cell comprising an artificial chromosome that comprises one or more repeat regions.
107. The method of claim 106, wherein sufficient portion of the vector integrates into a chromosome in the cell to result in amplification of chromosomal DNA.
108. The method of claim 106, wherein the artificial chromsome is an ACes.
109. A method for screening, comprising:
contacting a cell comprising a reporter ACes with test compounds or known compounds, wherein:
the reporter ACes comprises one or a plurality of reporter constructs;
a reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds; and
detecting any increase or decrease in signal output from the reporter, wherein a change in the signal is indicative of activity of the test or known compound on the regulatory region.
110. The method of claim 109, wherein the reporter is operatively linked to a promoter that controls expression of a gene in a signal transduction pathway, whereby activation or reduction in the signal indicates that the pathway is activated or down-regulated by the test compound.
111. The method of claim 109, wherein the reporter in the construct encodes drug resistance or encodes a fluorescent protein.
112. The method of claim 111, wherein the fluorescent protein is selected from the group consisting of red, green and blue fluorescent proteins.
113. The method of claim 109, wherein the ACes comprises a plurality of reporter-linked constructs, each with a different reporter, whereby the pathway(s) affected by the test compounds can be elucidated.
114. The method of claim 109, wherein a reporter is operatively linked to a promoter that is transcriptionally regulated in resopnse to DNA damage, and the test compounds are genotoxicants.
115. The method of claim 114, wherein the DNA damage is induced by apoptosis, necrosis or cell-cycle perturbations.
116. The method of claim 114, wherein unknown compounds are screened to assess whether they are genotoxicants.
117. The method of claim 114, wherein the promoter is a cytochrome P450-profiled promoter.
118. The method of claim 114, wherein the cell is in a transgenic animal and toxicity is assessed in the animal.
119. The method of claim 109, wherein:
the cell is a patient cell sample; the patient has a disease;
the regulatory region is one targeted by a drug or drug regimen; and
the method assesses the effectiveness of a treatment for the disease for the particular patient.
120. The method of claim 119, wherein the cell is a tumor cell.
121. The method of claim 109, wherein the cell is a stem cell or a progenitor cell, whereby expression of the reporter is operatively linked to a regulatory region exprssed in the cells to thereby identify stem cells or progenitor cell.
122. The method of claim 109, wherein the cell is in an animal; and the method comprises whole-body imaging to monitor expression of the reporter in the animal.
123. A reporter ACes comprises one or a plurality of reporter constructs, wherein the reporter construct comprises a reporter gene in operative linkage with a regulatory region responsive to test or known compounds.
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