Patent Document

CROSS REFERENCE TO RELATED APPLICATION  
       [0001]    This application is a continuation in part of, and claims priority under 35 U.S.C. §120 to, U.S. application Ser. No. 09/923,773 filed Aug. 6, 2001, the disclosure of which are incorporated herein by reference in their entireties. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    Included in the disclosure are nucleic acid molecules representing the telomeric region of the linear chromosome of the bacterium  Agrobacterium tumefaciens  (hereinafter “ A. tumefaciens ”) and oligonucleotides based on the  A. tumefaciens  telomeric sequences and constructs comprising  A. tumefaciens  telomeric regions and methods of transforming plants using such constructs.  
         BACKGROUND OF THE INVENTION  
         [0003]    [0003] A. tumefaciens  is a gram negative aerobic rod grouped within Rhizobiaceae in the alpha subgroup of the proteobacteria. Agrobacterium species have a major impact as phytopathogens, and are the causative agent for a number of plant diseases. These diseases affect a wide range of dicotyledonous plants in more than 140 genera worldwide. Transmission of the disease occurs though soil contaminated with the bacterium that enters plants through fresh wounds or natural openings (such as lenticels). Infection results in the transfer and integration of the bacterial T-DNA into the plant genome (reviewed by Tinland, B., 1996,  Trends in Plant Science  1:178-184). Within the T-DNA is a set of oncogenes that upon expression result in a loss of cell division control. Treatment is preventative, such as the removal and burning of all infected plants and rotation of infected soil with non-susceptible plants, as once the bacterial DNA is integrated into the plant genome the disease can progress even without the bacterium.  
           [0004]    This loss of cell division control is manifested in different ways among Agrobacterium species.  A. tumefaciens,  for example, causes the formation of tumors at the crown, roots, or branches, known crown gall disease in numerous crop plants such as almond, peach, apricot, tomato, and grape. The proliferation of these tumors interrupts water and nutrient movement up the stem resulting in stunting, discoloration, and plant death. Plants that do survive have heightened sensitivity to winter injury and drought stress. While there is no cure for crown gall disease, preventative measures can be taken. In addition to the aforementioned measures, is the dipping of susceptible plants with the nonpathogenic  Agrobacterium radiobacter  (a species nearly identical to  A. tumefaciens  except it lacks the Ti plasmid), which works to prevent the disease by working antagonistically to  A. tumefaciens.    
           [0005]    Examples of diseases caused by Agrobacterium species are provided below.  
                                                       Agrobacterium Species   Host   Disease                             A. tumefaciens     dicotyledonous plants   crown gall             A. rhizogenes     dicotyledonous plants   hairy root             A. rubi     caneberry   cane gall             A. vitis     grape, chrysanthemum   crown gall                      
 
           [0006]    Plant diseases caused by Agrobacterium infection are induced by transfer of a defined segment of DNA, designated T-DNA, from an Agrobacterium plasmid into a plant (Chilton, M. D. et al.,1977, Cell. 11:263-271; Chilton, M. D. et al., 1982,  Nature.  295:432-434; reviewed by Ream, W. 1998, In: “Subcellular Biochemistry”, Biswas and Das (eds.) Plenum Press, New York, 365-384; Hansen, G. and M. D. Chilton, 1999, In:  Curr. Top. Microbiol. Immunol.  240:21-57). The pTi (tumor-inducing) plasmid is a large, self-transmissible plasmid harbored by infectious Agrobacterium, and Agrobacterium species are pathogenic only when a tumor-inducing plasmid is present. Such tumor inducing plasmids, referred to as pTi in  A. tumefaciens  and pRi in  A. rhizogenes,  contain two regions essential for the ability of Agrobacterium to cause disease. These are the virulence (vir regulon) and transferred DNA (T-DNA) regions. The virulence region is comprised of eight operons (virA-H), of which only virA, B, G, and D are necessary for tumorigenesis (reviewed by Hooykaas, P. J. J. and A. G. M. Beijersbergen, 1994,  Annual Review of Phytopathology  32:157-179). The remaining vir operons encode genes whose proteins affect the efficiency of tumorigenesis or host range. The T-DNA region is bordered by two direct repeats, e.g., of 23-25 bp, called the left border and right border. These borders delineate the segment of DNA which will be transferred into the host plant. The genes involved in stimulating tumor formation (specifically the plant growth hormones, cytokinin and auxin) as well as genes required for opine synthesis are located between the border sequences. Opines comprise a novel class of amino acid derivatives not normally present in plants, but whose synthesis in Agrobacterium infected plants provides a carbon and nitrogen source for the Agrobacterium. The particular type of opine produced is used as a distinguishing feature for classifying Agrobacterium strains (i.e., octopine, nopaline, succinamopine, agropine, cucumopine, agrocinopine and mannopine).  
           [0007]    The molecular processes by which  A. tumefaciens  infects plants are generally understood (reviewed by Hooykaas, P. J. J. and R. A. Schilperoort, 1992,  Plant Mol. Biol.  19:15-38; Winans, S. C. et al., 1994,  Res. Microbiol.  145:461-473; Hansen, G. and M. D. Chilton, 1999, In:  Curr. Top. Microbiol. Immunol.  240:21-57). The bacterium is attracted chemotactically to a wounded plant by responding to phenolic compounds, such as acetosyringone, released from the damaged plant cells. Acetosyringone triggers the induction of virulence proteins in  A. tumefaciens  through a two-component signal transduction pathway. This pathway is comprised of a receptor, VirA, and a transcriptional inducer, VirG. Detection of acetosyringone in the environment causes VirA to become autophosphorylated, leading to the phosphorylation of VirG at aspartic acid residue 52 (Jin, S. et al., 1990, J. Bacteriol. 172:4945-4950). Subsequently, phosphorylated VirG activates transcription of the vir regulon by interaction with a DNA consensus sequence, ryTncAaTTGnAaY (the “vir box”), found within the promoter of all vir regulon genes (Winans, S. C. et al., 1987,  Nucleic Acids Res.  15:825-837; Pazour, G. J. and A. Das, 1990,  Nucleic Acids Res.  18:6909-6913). In addition, the pH of the infection site (Mantis, N. J. et al., 1992,  J. Bacteriol.  174:1189-1196) and presence of monosaccharides (Huang, M. L. et al., 1990,  J. Bacteriol.  172:1814-1822) also effect the induction of virulence. Monosaccharides are sensed by ChvE protein, and ChvE also functions to activate vir gene transcription through VirA (reviewed by Winans, S. C. et al., 1994,  Res. Microbiol.  145:461-473).  
           [0008]    After induction of the vir regulon, a single-stranded version of the T-DNA, called the T-strand, is produced via nicking of the lower strand of T-DNA at the Right and Left Borders (Stachel et al., 1986,  Nature,  322:706-712; Reviewed by Zupan, J. R. and P. Zambryski, 1995,  Plant Physiology  107:1041-1047). This nicking is catalyzed by VirD1 and VirD2 proteins, and VirD2 becomes covalently attached to the 5′ end of the T-strand. The large gap is presumably filled by repair synthesis, allowing production of an additional T-strand (reviewed and discussed by Hansen and Chilton, 1999).  
           [0009]    Transfer of the T-strand, with VirD2 still attached, to the plant occurs through a type IV secretion system that is primarily encoded by the virB genes (reviewed by Christie, P. J., 1997,  J. Bacteriol.,  179:3085-3094). A single-stranded binding protein, VirE2, is also transferred to the plant, although it apparently is transported independently of the T-strand (Binns, A. N. et al., 1995,  J. Bacteriol.  177:4890-4899; Citovsky, V. et al., 1992,  Science,  256:1802-1804; Gelvin, S. B., 1998,  J. Bacteriol.  180:4300-4302). VirE2 coats the single-stranded DNA and, along with VirD2, targets the T-strand to the plant nucleus for integration (Citovsky, V. et al., 1992,  Science,  256:1802-1804; Tinland, B., 1992,  Proc. Natl. Acad. Sci. USA,  89:7442-7446). The processes required for T-DNA integration into the plant chromosome are not well understood, although both VirD2 and VirE2 probably play a role (Dombek, P. and W. Ream, 996,  J. Bacteriol.,  179:1165-1173; Rossi, L. et al., 1996,  Proc. Natl. Acad. Sci. USA,  93:126-130; Tinland, B. et al., 1995,  EMBO J.,  14:3585-3595). To date, the plant proteins necessary for these processes are essentially uncharacterized.  
           [0010]    The ability of  A. tumefaciens  to transfer T-DNA to plants has been adopted by biologists as a mechanism for production of transgenic plants. Because this process is unique in biology, and technically and economically important, much work has gone into elucidating the regulatory and functional processes involved in T-DNA transfer. Most of the critical transformation genes are located on the pTi, although a few (such as chvE) are located on a chromosome.  A. tumefaciens  also contains a second large plasmid, designated the “cryptic” plasmid, that is still largely uncharacterized. An inefficiency in plant transformation by  A. tumefaciens  is the propensity for constructs to “read through” beyond the T-DNA borders which results in transfer of more than intended DNA.  
           [0011]    [0011] A. tumefaciens  contains two chromosomes, one linear and one circular (Allardet-Servent, A. et al., 1993,  J. Bacteriol.  175:7869-7874), and a coordinated physical and genetic map of the  A. tumefaciens  C58 genome (Goodner, B. et al.,1999,  J. Bacteriol.  181:5160-5106) was recently published. The linear chromosome was initially predicted to contain at least one origin of replication, and at least one terminus site. Our sequence analysis has shown that the linear chromosome has a repABC-type replication system, which is characteristic of plasmids. This single origin is located slightly assymmetrically from the center of the chromosome. This arrangement predicts that DNA replication will initiate at a single site and proceed bidirectionally toward the chromosome termini (telomeres), and a specialized mechanism is required for replicating the chromosome termini. There are essentially two mechanisms by which prokaryotes have been previously shown to replicate the telomeres of linear plasmids or chromosomes (Volf, J-N., and J. Altenbuchner. 2000. FEMS Microbiol. Lett. 186:143-150; Casjens et al. 1997. Mol. Microbiol. 26:581; Hiratsu et al. 2000. Mol Gen Genet. 263:1015; Casjens et al. 2000. Mol Microbiol. 35:490; Wang, S-J. et al. 1999. Microbiology. 145:2209-20; Walther, T. C and Kennell J. C. 1999. Mol Cell. 4:229-38; Rybchin, V. N., and Svarchevsky, A. N. Mol Microbiol. 1999 33:895-903; Barreau, C. et al. 1998. Fungal Genet. Biol. 25:22-30).  
           [0012]    The first type of telomere is typified by Streptomyces, in which linear DNA molecules have open ends and carry proteins attached to the 5′ end of the DNA molecule. These terminal proteins serve to stabilize the ends and prime synthesis of the second strand, thereby allowing complete replication of the chromosomes (Qin Z. and Cohen, S. N. 1998. Mol. Microbiol. 28:893-903). In the second type of telomere, typified by Borellia and phage N15, the telomeres are covalently closed, and replication proceeds around the end, creating a large, double-stranded molecule with two repeats of the DNA. The two repeats must then be separated. This reaction is best characterized in the N15 system, in which the protelomerase enzyme has been shown to break the double stranded DNA at the telomeres, then re-join the ends of individual molecules to re-create the covalently closed ends (Deneke et al., 2000. Proc. Natl. Acad. Sci. USA, 97:7721-7726). A similar system may be used by other linear molecules with covalently closed ends.  
           [0013]    No orthologue of the N15 protelomerase has been identified in the  A. tumefaciens  C58 genome. However, the DNA near the telomeres are rich in IS elements, including several putative transposases. One or more of these transposases may play a role analogous to the role of N15 protelomerase, in which the telomeres of daughter molecules are separated by a transposase-type enzyme. Depending on the precise mechanism of replication of the telomeres, the transposase may also play a role in allowing replication of the lagging strand near telomeres by joining ends to allow priming of the lagging strand. Such a reaction would be similar to that catalyzed by IS3-type transposases when they form circles (Sekine, Y. et al. 1996. J. Biol. Chem. 271:197-202.) The telomerase would also be involved in separation of the telomeres upon completion of replication.  
           [0014]    The DNA has been assembled near the ends of the linear chromosome allowing identification and isolation of covalently-closed telomeres, apparently having hairpin turn ends. The nucleic acid sequences disclosed herein represent the telomeric regions of the linear chromosome which are useful in preparing linear “plasmid” constructs for Agrobacterium transformation.  
         SUMMARY OF THE INVENTION  
         [0015]    The present invention contemplates and provides nucleic acid molecules comprising a telomeric region of the linear chromosome of  A. tumefaciens.  More particularly this invention provides isolated telomeres from the linear chromosome of  A. tumefaciens  wherein the telomeres are isolated from restriction enzyme fragments at each end of the linear chromosome. In particular a fragment comprising the telomere is less than 4,000 nucleotide bases and comprises a segment of consecutive nucleotide bases having at least 90% identity, more preferably at least 95% identity, to SEQ ID NO: 1 or SEQ ID NO: 2. Moreover, each telomere is obtained by removing more or less of an identified segment of consecutive nucleotide bases from the restriction fragment leaving a covalently-closed double-stranded molecule. In a preferred aspect of the invention a telomere is obtained from a terminal fragment comprising consecutive nucleotide bases of SEQ ID NO: 1 which can be cut by any of the following restriction enzymes: ApaLI, AvrII, BamHI, KpnI, NdeI and NotI. In another preferred aspect of the invention a telomere is obtained from a terminal fragment comprising consecutive nucleotide bases of SEQ IS NO: 2 which can be cut by any of the following restriction enzymes: ApaLI, BamHI, EcoRI, MluI, PvuI and SpeI. Telomeres of this invention are preferably provided in a pair of isolated and distinct telomeres obtained from opposite ends of the linear chromosome.  
           [0016]    The telomeres of this invention are useful in DNA linear plasmid constructs for use in producing transgenic plants by  Agrobacterium tumefaciens  transformation. Such plasmids comprise an origin of replication and covalently-closed terminal regions obtained from telomeres of this invention. Such plasmids should inherently advantageously limit the maximum amount of “border read-through”. In preferred aspects of the invention the use of such linear plasmids advantageously improves the efficiency and quality of Agrobacterium transformation. Preferred aspects of this invention provide such DNA linear plasmid constructs with telomeric ends and further comprising DNA segments selected from the group consisting of promoters, selectable markers, screenable markers and polypeptide-encoding sequence. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    [0017]FIG. 1 illustrates restriction sites at the telomeres of the  Agrobacterium tumefaciens  linear chromosome and Southern blots of telomere fragments. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0018]    As used herein the term “plasmid” means an independently replicating, linear or circular piece of a DNA construct that can be transferred into an organism. The plasmids of this invention are preferably linear and capable of incorporating at least part of the DNA into the genome of the host organism.  
         [0019]    As used herein, a nucleic acid molecule, be it a naturally occurring molecule or a fragment of a naturally occurring molecule or a synthetic molecule, may be “isolated”, if the molecule is separated from substantially all other molecules normally associated with it in its native state. More preferably an isolated molecule is substantially purified and is the predominant species present in a preparation. A substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free from the other molecules (exclusive of solvent) present in the natural state. The term “isolated” is not intended to encompass molecules present in their natural or native state.  
         [0020]    The telomeres and other nucleic acid molecules of this invention will preferably be “biologically active” with respect to facilitating DNA replication.  
         [0021]    The agents of the present invention may also be recombinant. As used herein, the term recombinant describes (a) nucleic acid molecules that are constructed or modified outside of cells and that can replicate or function in a living cell, (b) molecules that result from the transcription, replication or translation of recombinant nucleic acid molecules, or (c) organisms that contain recombinant nucleic acid molecules or are modified using recombinant nucleic acid molecules.  
         [0022]    The term “oligonucleotide” as used herein refers to short nucleic acid molecules useful, e.g., as hybridizing probes, nucleotide array elements, sequencing primers, or primers for DNA extension reactions, such as polymerase chain reaction. The size of the oligonucleotide molecules of the present invention will depend upon several factors, particularly on the ultimate function or use intended for a particular oligonucleotide. Oligonucleotides, i.e. deoxyribonucleotides or ribonucleotides, can comprise ligated natural nucleic acid molecules or synthesized nucleic acid molecules and will generally comprise between 15 to 1000 nucleotides or between about 20 and about 100 nucleotides. The sequence of the oligonucleotides will ideally be identical or complementary to the sequence of a fragment of similar length in an Agrobacterium nucleic acid molecule provided herein.  
         [0023]    This invention provides oligonucleotides specific for nucleic acid molecules of the present invention. Such oligonucleotides find particular use as nucleic acid elements of solid arrays (e.g., synthesized or spotted), as hybridization probes, and as primers for amplification of telomeric regions of this invention. Oligonucleotides for use in polymerase chain reaction (PCR) as primers are preferably designed with the goal of amplifying nucleic acids from either the 3′ or the 5′ end of an Agrobacterium chromosome, e.g. about 500 to 800 bp of nucleic acids.  
         [0024]    The term “primer” as used herein refers to a nucleic acid molecule, preferably an oligonucleotide whether derived from a naturally occurring molecule, such as one isolated from a restriction digest, or one produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is oligomeric DNA. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains at least 15, more preferably 18 nucleotides, which are identical or complementary to the template and optionally a tail of variable length which need not match the template. The length of the tail should not be so long that it interferes with the recognition of the template. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.  
         [0025]    The primers herein are selected to be “substantially” complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. Computer generated search programs such as Primer3 (Steve Rozen, Helen J. Skaletsky (1996, 1997); code available at http://www.genome.wi.mit.edu/genome_software/other/primer3.html), STSPipeline (www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole et al.,  BioTechniques  25:112-123 (1998)), for example, can be used to identify potential PCR primers. Exemplary primers include primers that are 18 to 50 bases long, where at least between 18 to 25 bases are identical or complementary to a segment of corresponding length in the template sequence.  
         [0026]    Nucleic acid molecules or fragments thereof are capable of hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit “complete complementarity” i.e. each nucleotide in one sequence is complementary to its base pairing partner nucleotide in another sequence. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Conventional stringency conditions are described by Sambrook et al.,  Molecular Cloning,  A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and by Haymes et al.,  Nucleic Acid Hybridization, A Practical Approach,  IRL Press, Washington, D.C. (1985), the entirety of both of which are herein incorporated by reference. Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. Thus, in order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.  
         [0027]    Appropriate stringency conditions which promote DNA hybridization, for example, incubation in 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in  Current Protocols in Molecular Biology,  John Wiley &amp; Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.  
         [0028]    Preferred embodiments of the nucleic acid of this invention will hybridize to one or more of the nucleic acid molecules of this invention or complements thereof under low stringency conditions, for example at about 2.0×SSC and about 50° C. In a particularly preferred embodiment, a nucleic acid of the present invention will include those nucleic acid molecules that hybridize to one or more of the nucleic acid molecules of this invention or complements thereof under moderate stringency conditions. In an especially preferred embodiment, a nucleic acid of the present invention will include those nucleic acid molecules that hybridize to one or more of the nucleic acid molecules of this invention or complements thereof under high stringency conditions.  
         [0029]    As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100.  
         [0030]    Useful methods for determining sequence identity are disclosed in “Guide to Huge Computers”, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D.,  SIAM J Applied Math  (1988) 48:1073, each of which is incorporated herein by reference. More particularly, preferred computer programs for determining sequence identity include the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al.,  J. Mol. Biol.  215:403-410 (1990), incorporated herein by reference; version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for polypeptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.  
         [0031]    For purposes of this invention “percent identity” shall be determined using BLASTX version 2.0.08 for nucleotide translations of polypeptide sequences and BLASTN version 2.0.08 for polynucleotide sequences.  
         [0032]    DNA Constructs  
         [0033]    The present invention also encompasses the use of telomeres of the present invention in recombinant constructs. Using methods known to those of ordinary skill in the art, telomeres of this invention can be inserted into constructs also comprising an origin of replication and polypeptide-encoding sequence operably linked to a promoter. Such constructs can be introduced into a host cell of choice for expression of the encoded polypeptide. Potential host cells include both prokaryotic and eukaryotic cells. A host cell may be unicellular or found in a multicellular differentiated or undifferentiated organism depending upon the intended use. It is understood that useful exogenous genetic material may be introduced into any cell or organism such as a bacterial cell, fungal cell, fungus, plant cell, plant, mammalian cell, mammal, fish cell, fish, bird cell, bird or bacterial cell. Plant cells are a preferred target host organism.  
         [0034]    Depending upon the host, the regulatory regions for expression of transgenic DNA will vary and may include regions from viral, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters may be employed. Among transcriptional initiation regions which have been described are those obtained from bacterial and yeast hosts, such as  E. coli, B. subtilis,  and  Sacchromyces cerevisiae,  including genes such as beta-galactosidase, T7 polymerase and tryptophan E.  
         [0035]    Furthermore, for use in transformation of  A. tumefaciens,  constructs may include those in which a protein encoding sequence is positioned with respect to a promoter sequence such that production of antisense mRNA complementary to native mRNA molecules is provided. In this manner, expression of a native gene may be decreased. Such methods may find use for modification of particular functions of the targeted host, and/or for discovering the function of a naturally expressed protein.  
         [0036]    The present invention also encompasses the use of nucleic acid constructs of the present invention in constructs used for mutation of genes within  A. tumefaciens  using homologous recombination, e.g., as disclosed by Lloyd et al. in Chapter 119 of “ Escherichia coli  and Salmonella—cellular and molecular biology”, Second Edition, © 1996 by ASM Press, Washington D.C. Such constructs, for example, may contain two encoding segments of a protein encoding sequence harboring a heterologous portion of DNA (such as an antibiotic resistance marker) between the two encoding segments. Such constructs may also contain, for example, other deletions, insertions, or base changes, or combinations thereof, relative to the  A. tumefaciens -derived telomeric sequence. Introduction of these constructs into  A. tumefaciens  can be used to generate mutations in the DNA of  A. tumefaciens.  Such mutations are useful, for example, in functional analysis of the mutated genes.  
         [0037]    As used herein, a promoter region is a region of a nucleic acid molecule that is capable, when located cis to a nucleic acid sequence that encodes for a protein or polypeptide to function in a way that directs transcription of one or more mRNA molecules that encodes for the protein or polypeptide. Promoters may be located directly 5′ of the protein encoding sequence, for example where a promoter regulates transcription of a single gene. Alternatively, such as when a promoter regulates transcription of a group of genes in an operon, the promoter may be located some distance upstream from a particular encoding region. Promoters will generally be recognized by their presence 5′, or upstream, of the start site for a protein coding region and/or by the presence of the −10 and −35 consensus core promoter elements found in bacterial promoters. In addition, promoters may contain additional non-core sequences which can affect promoter strength. Such additional regulatory sequences may be located upstream of, downstream of, or between core promoter elements. Examples of additional regulatory elements include UP elements (−40 upstream region) and DSR elements (region immediately downstream of the transcription start site).  
         [0038]    The deduced structure of the linear chromosome suggests that it contains one origin of replication (a repABC-type replication system), and replication termini at each telomere. Due to its linear nature, the mode of replication of the linear plasmid will differ significantly from the mode of replication of the circular chromosome, with a specialized mechanism for replicating the chromosome termini. The telomeres at the chromosome termini provide the special structures needed for complete replication, and eventually separation, of the daughter chromosomes.  
         [0039]    The recombinant vector of this invention can be a single linear plasmid or a system of additional plasmids which together contain the total DNA to be introduced into the genome of the host. Methods which can be used to introduce recombinant vectors into Agrobacterium species include triparental mating (Ditta et al. (1985)  Plasmid  13:149-153; Ditta et al. (1980)  Proc. Natl. Acad. Sci. USA  77:7347-7351), electroporation (White et al. (1995)  Meth. in Mol. Biol.  47:135-141) and P1 transduction (Avery L. and Kaiser D. (1983)  Mol. Gen. Genet.  191:99-109).  
         [0040]    The constructs of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene whose product provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Various selectable markers may be used depending upon the host species to be transformed, and different conditions for selection may be used for different hosts.  
         [0041]    A construct of this invention may comprise polypeptide encoding sequence operably linked to a suitable promoter sequence and optionally to a suitable leader sequence. A leader sequence may be a nontranslated region of an mRNA which is important for translation by a host cell. A leader sequence is operably linked to the 5′ terminus of the nucleic acid sequence encoding the polypeptide. The leader sequence may be native to the nucleic acid sequence encoding the polypeptide or may be obtained from foreign sources. A polyadenylation sequence may also be operably linked to the 3′ terminus of the nucleic acid sequence of the present invention, particularly for use in eukaryotic host cells.  
         [0042]    To avoid the necessity of disrupting the cell to obtain the polypeptide, and to minimize the amount of possible degradation of the expressed polypeptide within the cell, it may be preferred that expression of the polypeptide gives rise to a product secreted outside the cell, especially in the case of expression in bacterial host cells of bacterium or bacteria. To this end, the polypeptide of the present invention may be linked to a signal peptide linked to the amino terminus of the polypeptide. A signal peptide is an amino acid sequence which permits the secretion of the polypeptide from the host into the culture medium.  
         [0043]    A nucleic acid molecule of the present invention which encodes a polypeptide may also be linked to a propeptide coding region. A propeptide is an amino acid sequence found at the amino terminus of apoprotein or proenzyme. Cleavage of the propeptide from the proprotein yields a mature biochemically active protein. The resulting polypeptide is known as a propolypeptide or proenzyme (or a zymogen in some cases). Propolypeptides are generally inactive and can be converted to mature active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide or proenzyme. The propeptide coding region may be native to the polypeptide or may be obtained from foreign sources.  
         [0044]    A nucleic acid molecule of the present invention which encodes a polypeptide may also be linked to a transit peptide coding region. A transit peptide is an amino acid sequence found at the amino terminus of an active protein which provides for transport of the protein into a plastid organelle, such as a plant chloroplast. The transit peptide coding region may be native to the type of cell to be transformed, or may be obtained from foreign sources.  
         [0045]    Plant Constructs and Plant Transformants  
         [0046]    Of particular interest is the use of DNA constructs of this invention for plant transformation or transfection. Exogenous genetic material may be transferred into a plant cell and the plant cell regenerated into a whole, fertile or sterile plant. Exogenous genetic material is any genetic material, whether naturally occurring or otherwise, from any source that is capable of being inserted into any organism. Such genetic material may be transferred into either monocotyledons and dicotyledons including but not limited to the plants, alfalfa,  Arabidopsis thaliana,  barley, broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape (canola), onion, flax, maize, ornamental plants, pea, peanut, pepper, potato, rice, rye, sorghum, soybean, strawberry, sugarcane, sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple, potato, lettuce, lentils, grape, banana, tea, turf grasses, sunflower, oil palm, etc.  
         [0047]    Many different methods for generating transgenic plants using  A. tumefaciens  have been described. In general, these methods rely on a “disarmed”  A. tumefaciens  strain that is incapable of inducing tumors, and a binary plasmid transfer system. The disarmed strain has the oncogenic genes of the T-DNA deleted. A binary plasmid transfer system consists of one plasmid with short, e.g. 23-25 base pair, T-DNA left and right border sequences, between which a gene for a selectable marker (e.g., an herbicide resistance gene) and other desired genetic elements are cloned and a second plasmid which encodes the  A. tumefaciens  genes necessary for effecting the transfer of the DNA between the border sequences in the first plasmid. When plant tissue is exposed to Agrobacterium carrying the two plasmids, the DNA between the left and right border repeats is transferred into the plant cells, transformed cells are identified using the selectable marker, and whole plants are regenerated from the transformed tissue. Plant tissue types that have been reported to be transformed using variations of this method include: cultured protoplasts (Komari, T., 1989,  Plant Science,  60:223-229), leaf disks (Lloyd, A. M. et al., 1986,  Science  234:464-466), shoot apices (Gould, J., et al., 1991,  Plant Physiology,  95:426-434), root segments (Valvekens, D. et al., 1988, PNAS, 85:5536-5540), tuber disks (Jin, S. et al., 1987,  Journal of Bacteriology,  169: 4417-4425), and embryos (Gordon-Kamm W., et al., 1990, Plant Cell, 2:603-618).  
         [0048]    In the case of  Arabidopsis thaliana  it is possible to perform in planta germline transformation (Katavic B., et al., 1994,  Molecular and General Genetics,  245:363-370; (Clough, S. and Bent, A., 1998,  Plant Journal,  16:735-743). In the simplest of these methods, flowering Arabidopsis plants are dipped into a culture of Agrobacterium such as that described in the previous paragraph. Among the seeds produced from these plants, 1% or more have integration of T-DNA into the genome.  
         [0049]    Monocot plants have generally been more difficult to transform with Agrobacterium than dicot plants. However, “supervirulent” strains of Agrobacterium with increased expression of the virB and virG genes have been reported to transform monocot plants with increased efficiency (Komari T. et al., 1986,  Journal of Bacteriology,  166:88-94; Jin S., et al., 1987,  Journal of Bacteriology,  169:417-425).  
         [0050]    Most T-DNA insertion events are due to illegitimate recombination events and are targeted to random sites in the genome. However, given sufficient homology between the transferred DNA and genomic sequence, it has been reported that integration of T-DNA by homologous recombination may be obtained at a very low frequency. Even with long stretches of DNA homology, the frequency of integration by homologous recombination relative to integration by illegitimate recombination is roughly 1:1000 (Miao, Z. and Lam, E., 1995,  Plant Journal,  7:359-365; Kempin S. A. et al., 1997, 389:802-803).  
         [0051]    Exogenous genetic material may be transferred into a plant cell by the use of a DNA vector or construct designed for such a purpose. Vectors have been engineered for transformation of large DNA inserts into plant genomes. Binary bacterial artificial chromosomes have been designed to replicate in both  E. coli  and  A. tumefaciens  and have all of the features required for transferring large inserts of DNA into plant chromosomes. BAC vectors, e.g., a pBACwich, have been developed to achieve site-directed integration of DNA into a genome.  
         [0052]    A construct or vector may also include a plant promoter to express the protein or protein fragment of choice. A number of promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter, a caulimovirus promoter such as the CaMV 19S promoter and the CaMV 35S promoter, the figwort mosaic virus 35S promoter, the light-inducible promoter from the small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll a/b binding protein gene promoter. For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Examples of such promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6-biphosphatase (FBPase) promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the phenylalanine ammonia-lyase (PAL) promoter and the chalcone synthase (CHS) promoter from  Arabidopsis thaliana.  Also reported to be active in photosynthetically active tissues are the ribulose-1,5-bisphosphate carboxylase (RbcS) promoter from eastern larch ( Larix laricina ), the promoter for the cab gene, cab6, from pine, the promoter for the Cab-1 gene from wheat, the promoter for the CAB-1 gene from spinach, the promoter for the cab1R gene from rice, the pyruvate, orthophosphate dikinase (PPDK) promoter from  Zea mays,  the promoter for the tobacco Lhcb gene, the  Arabidopsis thaliana  SUC2 sucrose-H+symporter promoter, and the promoter for the thylacoid membrane proteins from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other promoters for the chlorophyl a/b-binding proteins may also be utilized in the present invention, such as the promoters for LhcB gene and PsbP gene from white mustard (Sinapis alba). Additional promoters that may be utilized are described, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435.  
         [0053]    Constructs or vectors may also include, with the coding region of interest, a nucleic acid sequence that acts, in whole or in part, to terminate transcription of that region. For example, such sequences have been isolated including the Tr7 3′ sequence and the nos 3′ sequence or the like. It is understood that one or more sequences of the present invention that act to terminate transcription may be used.  
         [0054]    A vector or construct may also include other regulatory elements or selectable markers. Selectable markers may also be used to select for plants or plant cells that contain the exogenous genetic material. Examples of such include, but are not limited to, a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase gene which confers resistance to bromoxynil, a mutant acetolactate synthase gene (ALS) which confers imidazolinone or sulphonylurea resistance; and a methotrexate resistant DHFR gene.  
         [0055]    A vector or construct may also include a screenable marker to monitor expression. Exemplary screenable markers include a b-glucuronidase or uidA gene (GUS), an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a b-lactamase gene, a gene which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene, a xylE gene which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene, a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to melanin; an a-galactosidase, which will turn a chromogenic a-galactose substrate. Included within the terms “selectable or screenable marker genes” are also genes which encode a secretable marker whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected catalytically. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA, small active enzymes detectable in extracellular solution (e.g., a-amylase, b-lactamase, phosphinothricin transferase), or proteins which are inserted or trapped in the cell wall (such as proteins which include a leader sequence such as that found in the expression unit of extension or tobacco PR-S). Other possible selectable and/or screenable marker genes will be apparent to those of skill in the art.  
       EXAMPLE 1  
       [0056]    This example illustrates obtaining and characterizing isolated telomeres from the ends of the linear chromosome of  A. tumefaciens.  The genomic DNA sequence of the linear chromosome are derived from a double stranded library. The two basic methods for the DNA sequencing are the chain termination method of Sanger et al.,  Proc. Natl. Acad. Sci.  ( U.S.A. ) 74:5463-5467 (1977) and the chemical degradation method of Maxam and Gilbert,  Proc. Natl. Acad. Sci.  ( U.S.A. ) 74:560-564 (1977) using automated fluorescence-based sequencing as reported by Craxton,  Method,  2:20-26 (1991); Ju et al.,  Proc. Natl. Acad. Sci.  ( U.S.A. ) 92:4347-4351 (1995); and Tabor and Richardson,  Proc. Natl. Acad. Sci.  ( U.S.A. ) 92:6339-6343 (1995) and high speed capillary gel electrophoresis, e.g., as disclosed by Swerdlow and Gesteland,  Nucleic Acids Res.  18:1415-1419 (1990); Smith,  Nature  349:812-813 (1991); Luckey et al., Methods  Enzymol.  218:154-172 (1993); Lu et al.,  J. Chromatog. A.  680:497-501 (1994); Carson et al.,  Anal. Chem.  65:3219-3226 (1993); Huang et al.,  Anal. Chem.  64:2149-2154 (1992); Kheterpal et al.,  Electrophoresis  17:1852-1859 (1996); Quesada and Zhang,  Electrophoresis  17:1841-1851 (1996); Baba,  Yakugaku Zasshi  117:265-281 (1997). For instance, genomic nucleotide sequence traces are generated using a 377 DNA Sequencer (Perkin-Elmer Corp., Applied Biosystems Div., Foster City, Calif.) allowing for rapid electrophoresis and data collection. With these types of automated systems, fluorescent dye-labeled sequence reaction products are detected and chromatograms are subsequently viewed, stored in a computer and analyzed using corresponding apparatus-related software programs. These methods are known to those of skill in the art and have been described and reviewed (Birren et al.,  Genome Analysis: Analyzing  DNA, 1, Cold Spring Harbor, N.Y. (1998)).  
         [0057]    Quality genomic sequence traces are assembled generally as follows:  
         [0058]    (a) all traces are “vector-trimmed’ i.e., 5′ and 3′ vector and linker sequences are removed;  
         [0059]    (b) a PHRAP assembly is run using default assembly parameters;  
         [0060]    (c) contigs and singletons files and their corresponding quality files are united to create “islands” of contiguous sequence (contigs) from which genes are identified by sequence query against known gene databases.  
         [0061]    After genes are identified there is remaining DNA comprising the telomeric regions. The telomeric region at the left end of the linear chromosome comprises DNA in the sequence of SEQ ID NO: 1. The other telomeric region at the right end of the linear chromosome comprises DNA in the sequence of SEQ ID NO: 2.  
         [0062]    The telomeric region can be amplified by PCR using probes designed from SEQ ID NOs: 1 and 2. The telomeric regions can be sequenced. Alternatively, smaller telomeric regions can be isolated by cutting away DNA running toward the middle of the chromosome using a restriction enzyme which is selected to match a restriction site in SEQ ID NO: 1 or 2. Oligonucleotide probes having the sequence of SEQ ID NOs: 3 and 4 are used in a hybridization gel for Southern blots to identify progressively smaller restriction fragments of telomeric region comprising SEQ ID NOs: 1 and 2. In particular, restriction enzyme Kpn I can be used to cut a telomeric region of the left end of the linear chromosome which hybridizes to a probe of SEQ ID NO: 3; and restriction enzyme Eco RI can be used to cut a telomeric region of the right end of the linear chromosome which hybridizes to a probe of SEQ ID NO: 4.  
         [0063]    More particularly with reference to FIG. 1 there is shown restriction sites for the telomeres of the linear chromosome and Southern blots of the restriction fragments. Southern blot hybridization was performed on DNA fragments containing the Right (A) and Left (B) telomeres of the linear chromosome. Probes recognized DNA very near each telomere, and these were used to detect DNA fragments containing either intact telomeres (NdeI and MluI), or fragments lacking the telomere ends (DdeI and PvuII). A portion of the DNA was boiled for 10 minutes and allowed to cool slowly for various periods of time. DNA fragments were separated by agarose gel electrophoresis, then transferred to nylon membranes prior to hybridization. The mobility of fragments containing the telomeres was essentially identical regardless of whether the DNA samples had been denatered. These data indicate that the two strands of DNA containing the telomeres are covalently closed. Fragments lacking the telomere ends migrated faster following boiling, indicating denaturation creating two single-stranded DNA molecules. Slow cooling allowed renaturation of a portion of the denatured molecules. nb, not boiled; 0, 12, 24, 36 and 48 indicate the number of minutes that the DNA samples were allowed to cool before they were frozen in a dry ice-ethanol bath. After 48 minutes of cooling, the temparature of the DNA samples was approximately 50° C. Numbers on the right of each figure indicated the size, in kilobases, of a double-stranded DNA molecular weight standard. The Southern blots of denatured DNA fragments containing either telomere show a single molecule rather than two single-stranded molecules; the single fragments indicate that the two strands near the telomere are joined by a hairpin loop.  
         [0064]    The complete sequence of the telomeres can be deduced by using the DNA sequence present in SEQ ID NO:1 or 2 to prime PCR products that extend around the covalently-closed telomeres of the linear chromosome. The resulting DNA product may either be sequenced directly, or cloned and sequenced as described above. Alternatively, the sequence may be read directly from the chromosomal DNA of Agrobacterium, or from partially purified fragments thereof, as practiced by Fidelity Systems, Inc. (Gaithersburg, Md.). The proximal regions of both telomeres are similar in overall architecture, but are very different in sequence. The sequence near both telomeres contains several IS elements, with intervening DNA of additional repeated and unique sequence. The region is rich in potential secondary structure and contains numerous short sequence repeats.  
       EXAMPLE 2  
       [0065]    This example serves to illustrate the design of linear plasmid constructs of this invention. A linear plasmid is constructed comprising in order, a left telomere region of this invention, an origin of replication from  A. tumefaciens,  a promoter region, a polypeptide encoding region, a polyadenylation region, a selectable marker region, a screenable marker region and a right telomeric region of this invention. A construct may also be viable if it contains identical telomeres at both ends. Such a construct is used by transforming Agrobacterium and employing methods well known in the art to make transgenic plants such as of cotton, corn and soybean.  
         [0066]    All publications and patent applications are herein incorporated by reference in their entirely to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.  
         [0067]    Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.  
     
       
       
         1 
         
           
             4  
           
           
             1  
             20000  
             DNA  
             Agrobacterium tumefaciens  
           
            1 

cttcaccgtc tcaggcgtac cgatggtcac ggctgtctct tacaaatcct ttcaggaact     60 

gccgaaagcc aaagcaccag ctgtccttca aaagctcgcc caggccgtcg cggcagaagg    120 

tttttcaggt atccagatca acaaggcact gtcgtcaatc gatgcccatc aggaaaccag    180 

cggaagtggc aggattcaga cgctgcgggt tgtcgcccgc cagaaaggcg ccgctgtccg    240 

gatcgatgct gtcttcaata ttcaggcagg acagatcgcc gacaaagacg tcatccgcaa    300 

gggcatctgc gacatcataa aaggcgcgta aggcatggct taaagacact ccggtccagc    360 

agcacgagcc gtcgtgcaca ggaagccaat ctgtttcgcg caggatgtta cgaaatcgat    420 

gattgaagct tatcgctgac cgtatccgcg ggtagtctcc agtgttagat atgcgccgag    480 

cgaactcatc atgccgctac gccaaagacg gtcattgatc tcacgtgttt ggaccatatt    540 

ttctgtgcaa actaaacgat gacatagggc gatttttagt ggcggacaaa tacagacttc    600 

ccgaagagtt ttttaccact cggtttctcg ttagacgcat cgtacccaca gacgctgaag    660 

ctattttcga agggtggaac accgatcccg aggtgacgaa gtacctgacg tggaaacccc    720 

actccgagct tggccagaca cagcgggcga ttgaagaaaa ttatagtgcg tggaatgcag    780 

gtacatcgtt tccagctgtc atctgccatc gcgaacggcc acatgaacta atcggccgta    840 

ttgatgcacg tccgatgggc cacaaggtct cttacgggtg gcttgtccga agaacctggt    900 

ggggccgggg tgttgcaagc gaggtcgttc aactcgctgt agaacacgcg ttatcgcatc    960 

cgcgcatctt tcgcaccgaa gcatcctgcg acgttctgaa cacggcgtca gcaagagtga   1020 

tggaaaaagt agggatgaca aaggaggccg tgcttcgacg gtaccttttt caccccaatt   1080 

tttcgaatat gccgcgagac gccttcctgt attccaaggt acgttaactc agtgaaatca   1140 

cggggcgtcc aacttcctca taatcccgct attccaagac cggaaaagta cacgtcatcc   1200 

aaaaaagcaa tatgtgtcta tcgaacgcag ggaattaccg gattttccag aaagcgacgt   1260 

cggaaagttc ctgcaacaac cacacccgat ccaaatcctg aaatcaatct tgctagcaaa   1320 

aggtcaggta atgaatatga caccagccaa taatgggtcc caaatgccaa gagcgacgtc   1380 

gcgataaggt aaagttggcc gtccgtgcct gggcgacgaa aagccacgaa atgaaagccg   1440 

gcgacgctcc ccactaggca tgatacaaac tgaccgagaa ttatcgttgg ttttgcagtg   1500 

agcccatcca gcagacaaaa gacgaacgcc aagaatccag caccgagcaa ctgcaaaacg   1560 

gcggctccaa taaaacgtgt tccgcaggga tatttgggtt tcgaatagcg aaaatatgtg   1620 

accaagatac cctcctctga agtggtgcct ttgaaatcag ggcgaccatc tcaaaattcg   1680 

tacaacctca acttgagttg aggtcgaggg gggcggcgtc acaaggatta accgttgtgt   1740 

ggatagcggc acaaccgggt gtaactgtct ccgctttaca ttttctgact gcctaggcgg   1800 

gctggccaat aggaaaaatc gaagccatct ggcggcactc aaccgcggcg acacagtcgc   1860 

gctcaggcag gctagaggtg gttgtctaac gttcaacctg cgtcagacag ctacctggca   1920 

taactagatt gagccaacag ccgagattgc gtcagacgac ttctctcaat ggggttggtc   1980 

ataccgattg aaacgactgg aacccaaaaa tcagtgccat caaactaggc gcgttcgacg   2040 

ttgacggaac acatccaggt gattgtcgag gatggagcgc accgatccat aggttcgcgc   2100 

gccgatttcc aatgcccggc cacaggccgc attgaccctt tcccgctcaa aagctttgac   2160 

gagccgaata atgcctaggc aagctctgta gccctgttcg ggatgcgggc ggtcggcgag   2220 

aatgcggtcg cagagcaaag cgacgtccga tccgattgcc gacgcctcgc ggctgatacg   2280 

ctcgatcgtc cggtcggcaa accgccgatg ggaagatggc atgtgctccg gtgtcgtcgt   2340 

gtgcttgcca ttgccactcg atcgcctgtg agctgcgatc cgttcgccct tgtagaatat   2400 

ctcgatcgtg ttggcagtga tccgcgcctc gacctgctct cgggcaaacc ggtagggcac   2460 

ggagtaaaag tgcttgtcga tgtcgacatg ataatcgagg cctgcccggc gtatcttcca   2520 

ttcagcaaag acatatcgct cgaccggaag cggtcgcagc gcgggatagt cgagctcttc   2580 

aaacaactgt cgccttgtgc gcccaatgcg gcgcaagacg cgtttgtcgt tcagatcgtg   2640 

gaggagttcg ccaatcgctt tgttcacgtc agcaaggctg tagaagacgc ggtgacgcag   2700 

ccgacccagt agccagcgct cgacgatgcg aaccgccgcc tcgactttcg ccttgtcgcg   2760 

cggccgacgc ggtcgcgttg gaagaaccgc gctgccgtaa tgtgtcgcca tcccggtgta   2820 

ggtgcgattg acctgcggat caaaatggca tgccttgatg acggcaacct tggcattgtc   2880 

gggaacaagc agagccggtg cgccgccgaa tgtctcaagc gcaagcagat ggcattcgat   2940 

ccagtcggga agggtcccgc tccagcgggc atgggcaaat gaaaggctcg atgcgccgag   3000 

cactccaaca aacacatggg cctgccgcgt cttgcccgac aatctgtcga tgacaacaga   3060 

gaccgtgtcg cctgcataat cgacgaagag cttttcgcca gccgcatgat cctggcgcat   3120 

cgtcacaggc agcttcaaag cccaatggcg atacaaatca cagtaacggg aatagcggta   3180 

accctcagga tgaaggccga tatattcgtc ccacagaatc tgcagcgtca catgtttgcg   3240 

cttcagttcg cggtgaacct gcgcccaatc tggctcggcg atgcgccgat gacctgtctt   3300 

tgtcccggca gccttgtaaa gggcagcttc cagcaccgca tcgctgacat cctcagccaa   3360 

tggccacgac agcccggcac gctccagacg gcgcaatgtc tcacgcaccg tcgaaggagc   3420 

agcaccgacg cgcaccgcaa tcgacttgtg gccgagccct tcttcaaatc tgtgtcttaa   3480 

aatctcgcgg acacgccgca tcgctactct ctccgctggc attgcgttcc ttcctctcag   3540 

cacacctgaa agggacgaac ttctcaccag caggaaaccc cgacaaacac cccaatcagg   3600 

gggcgacatc atctcggaac aggggagcga ctaattatcg gaattggggg gcggcatcat   3660 

ttcggaatca gggggcgaca tgcctcggaa tttgcagtca ctcctgacga gcggcagggg   3720 

tggccctgtg aaacgcaggc cgcgccggac gacgaaccac tctcaagctc taaccattta   3780 

tgctgcacac gatctgtgcg gcattatttc aattaagcca aagatgccaa acagcaatat   3840 

ctccaggaag atccccgctt tggccaccat gcacatatgc gaaatagcgg ccatgatcgg   3900 

atctagcggc gtccggcaat gaaagcgcgc acttgtacgg caatgacacc actgcatatt   3960 

ctggcgggga aagccccatt gccgtcgcaa ggaacggact gagatggctt gccgtggagg   4020 

cggtgcccct aacataggca agcattacgg cctctctatt agtccaagca tagtcgccgg   4080 

tcgtaaaacg gctgagtcca ttccggcaat aaagatcgac tgtctttgcc tgtgctatgt   4140 

cgatgagctt gcactcaacg atcaagggga acaaatgctg tcgaccggtt agggaaattt   4200 

gaatatcggg ccgcatctca aaacgggttg cgtcgtaatt cttcgtttct gtgccacgcg   4260 

aaaccgcgcg aaccagcaaa ctcgccaaag ggtcagcccc caccagatgg ttcaagcgat   4320 

caaccatcaa actgttgagg tgcttctcgt caccgctaga aactgcagca ggataatccg   4380 

cttttattcc cgtgtaagcc tgttgaagca gctccagaat gaattcgaga tgcgaatggt   4440 

cgattgccgg tagaggcagt tcttgtccac gaatcaattc atacaggtga tcttgcaacc   4500 

cggagatcag tccactcata ctgcaggact ctccctaaaa aactccggtc gtgtccagat   4560 

tatacgttgc gcggcaaggc gggcctgtgt ctcgctccaa tatcgagatt gggcgaggag   4620 

catgatagcc aggctgtgct tgccctcatc tatgacaatt tcagtcgcac tggtgcgatc   4680 

agcaaccgca gtaagagcgc gaatatccgc aattgaatcc accgtctgat cttcggcttc   4740 

agtgttccag cacgcaacaa tacgaagcgc ctgccacggg gatgattgtc gatcaggtat   4800 

gtaatcgaca cggacggtgc atttgaaacg ttgagcaaac cttgtgagtt cggtttccag   4860 

cgttcggcaa taggttttga tctggtggat caacggctcg ctctgtgcca attcccgatt   4920 

tgaagcgtaa gggagattaa accgcagtgt atcgtcgata acagtcaggt cccgttcgcc   4980 

gagaccgtag agtttcgcta cccaagcatc gacatctggc caaacgtcgc cgccctcttt   5040 

ttccaaccgc gcaaaaagat tggttacctc ccgcacccga tcctgcgcga gtgactcgaa   5100 

gggcggaatc gccacctcct caaggataga tttctcaacc acctcacgct caaagccgaa   5160 

ttcgccgctg gttatcagcg atatccagag cgcaacgcga ctaccaagca caagtgcaag   5220 

atagcgcgtc aataacgacg cctcggcggc tcccttggaa ctaaagccat accaactctc   5280 

attgaatacg atgtcttgat cgcagactga catctgtatc cgctgtagct tggcaggcgg   5340 

ggatttatga acgagcaaga tcggcccgtt atagatagct ctatctcttg cgcgatgaac   5400 

gcgatcagtg tcgaaaactg gaagctgtga aggatcgata actaacgttg cggacgacac   5460 

gtcatcaaga tgaggaagtc catgcaggtg agtggcgtcg tcgccgatag tgccatcatc   5520 

atgcttcttg ctactatctc tgagcttctg atagccattt ccattcgcga gacctccatc   5580 

gcccaactcg cgccaatagc gtcccagcgt cgtgaaattg cctcgacgaa ccctgtcaat   5640 

tagcgaaaga tcagcctcac tgccgcgaaa taggattttt agggtctccg ggcgttcccg   5700 

caaatcctgc ggacggacca catacgcgtt cgtcgcatcg acccgcatta cgccggcatt   5760 

attgaaaccc ttttcatagc ggggcgtgag catccgaaat ccggacgctg tatgcgcagg   5820 

ccggttgacc gcaaaaagga ggcaaaacgg cgccgaaatg ctgggccaga cctttgtctg   5880 

gcgtagctca gagccgttga tgatcgaggt tacatccatc gcttcgagga ggctctgccg   5940 

cgccagcggc atgccatccc cttgctggaa tagaaaccgc gcatgcagcg caaatatgat   6000 

ttgagcgtca ggttttgccc atcgcatcgc ccgccagaca aatggcaaat cgagaactgc   6060 

gttgggcaaa ggaggagcgg ttataccgtc tccgagtctg ctccgagcga ttttatgaac   6120 

ctccgtaagc aggaggttcc agtcttccaa tccggtcgcg ctggcccacg gaggattgcc   6180 

aatcacgaga tcgtactggc cgtcgtgctc ctctccgata agcgagccga ggcttcccaa   6240 

ctgctttgct tcgggacgat caacatcggt ggtatcgact ggtcgatgca agacaacacc   6300 

gcgcaaatcg tcgaagtgca gcttgtccac gggcttggga tttggatcga gttcgatcga   6360 

tagcaagtag aggccgagcg cgcaaaagcg caaagcggcc tcattgatgt caaatccgcg   6420 

cacctgcttg taaaggattt ctcggagcgc tgctgtgtcc ggtctgtgcc cttctgatct   6480 

ccagcgcgcc gcaacgagtt cacgaaacgc cgcgagcaga aacacgcccg cgcccgcggc   6540 

tgggtctaat atgcgcgcgg ccgccgaaat accctgagca ttcagtccac gaaacgcggc   6600 

tgataccatc aggtttgcga tcggatgcgg cgtataaaag ccgccctcct gctgttgctg   6660 

ttttggggcg tgcttacgca ggtaatactc ataggcttgg ctcaacactc caactgttat   6720 

gtgcgagaag tcgagattat cccactgctc tttccaaccc agggacaatt gccccccctc   6780 

tgcgcgccgc agaatgttgc caatgaactg ccccccattt cgaccggaca gtcggcataa   6840 

gcagaaaggc tcaagcacag gcttgaggac aggcttatgt ctaacgacta tcgacacgtt   6900 

gaattgctga cgggtgatgt tcgccgcagg cggtggacaa ccgagcaaaa gctgacaatc   6960 

attgagcaga gttttgaacc cggcgagacg gtatcttcga ccgctcgccg tcatggcgtg   7020 

gcgcccaatt tgctttatcg gtggcgcagg ctcttgagcg agggaggtgc tgcagccgtg   7080 

gattctgacg agccggttgt cgggaattcg gaagtgaaga aactggagga tcgcgtccgg   7140 

gagttggagc gcatgctcgg tcgcaagacg atggaggtcg aaatcctccg cgaagccctt   7200 

tccaaagcgg actcaaaaaa acggatatcg cggccgatct tgttgccgaa ggacggttcg   7260 

cgatgaaggc cgtcgcagac acgctgggcg tctcccgttc caacctcatc gagcggctga   7320 

aaggcagatc aaagccgcgt gggccataca acaaggccga ggatgcagag cttctgcccg   7380 

ccatccgcag gctggtggat caaaggccaa cctatggcta tcggcggatc gccgcgctcc   7440 

tcaatcgcga aaggcgagcc gccgatcagc ctgtcgtcaa cgccaaacgg gtccatcgca   7500 

tcatgggtaa ccacgccatg ctactggagc acacagccgt tcgcaagggc cgcctccacg   7560 

atggcaaggt catggtcatg cgctccaacc tgcgctggtg ctcggacggc ctggagttcg   7620 

cctgctggaa tggcgaggtc attcgtctcg ccttcatcat cgacgccttc gaccgcgaga   7680 

tcatcgcctg gacggccgtt gccaatgcag gcatttccgg ctcagacgtg cgcgacatga   7740 

tgttggaggc ggtcgagaaa cgcttccatg caacccgagc cccgcatgct atcgagcatc   7800 

tctctgacaa tggctcggct tataccgcgc gggacacgag gctgtttgcg caagcactca   7860 

atctcacgcc ctgcttcacg ccggtcgcca gcccgcagtc gaacggcatg tcggaagcct   7920 

tcgtcaaaac gttgaagcgg gactatattc ggatatcagc tctaccggac gcccaaacag   7980 

cgctccggct catcgacgga tggatcgagg actacaacga aatccatccc cattccgcgc   8040 

tcaagatggc ttcccctcgg cagttcatca gggctaaatc aatctagccg acttgtccgg   8100 

tgaaatgggg tgcactccag ccaagaacat aacatgcttc caacgtgagc ttcggaaacg   8160 

cagcatccga caacggcagc aggtcaccat tgaacgtctt atcgagccat gcgcaggaaa   8220 

ggcgaactag atctggccgg tcaaacagtt catgatcttc gagaccagac tcatcgacag   8280 

aactctcact gccaacaatg ctactagttg atggcagcag gccacgatca gcaagaaagc   8340 

gcatgaagag cgccctgccc accaatgata tcgcgtcctc gccggagagg ccgctattgc   8400 

cttttaaaga cgatatggct tggtcaagca atttcagaat tacgccggtg atccaagtcc   8460 

tgtcattaat ctccgccgcc gggcgcagat ttgccaagcg tgcaaaggta agccagctct   8520 

cgttatcggc gacaccagcg tcaacgcgag ccttggaaag cgaaaggcga tcgagagcga   8580 

tccgatagac atccaatctt ccgggataga ccactccgag gtacggcgcg tcgcctcgca   8640 

ttgcgagaat ccgtcgaacc cgcttgagcc gatcaagatc tccgtttagc cggtcgccat   8700 

cgacaaggaa tacgagcgga gcttcctgcc actcgtagac accttcaacc agtcgcaaat   8760 

cctcatcatg gtcccgagcc acgaggatag tcgagtaggc aagcaactct ggtgccgaat   8820 

gatcgaaaag acgaacaccc ccgcgatcag cccccaattc atccagggca ttgtacatgc   8880 

cgatcataag cctgtcccca accaggggag ccagcccggc acagtagcac caaattccaa   8940 

aagggagctg aacgccgtca tcgatcgagg tccaatggca cttggcggtc gccgccaaac   9000 

tggccgggtt tcgcccgccc gcgaggcttc ttcacggtga gttctacctt cgatatcacg   9060 

ccccggaaat caaccccttg ggtgcccatt tcagcgacca acgcggcaat gatcgccaaa   9120 

tggtcgggaa cccctcctga acgggcataa tttgaaacag agttagggtt cattccaatc   9180 

aattccgcaa atgccctgat gctcaggcca gcgcgcgcga gatcgcggag aaacccctca   9240 

tagcccatcg ctccaaagcc ctaatcacat aattctatgt atactctcac atagaatcat   9300 

gtgttgcaat ccacaaatgc ggattgcgat gagaggtggc tttttggcgg atgcgcttgt   9360 

gtgaccgagc ttctgaaggg gcctttagcc cacgctaacg cctaacgccg catttccctt   9420 

ttcgccctca attatgcgct taacgccaga tgctggaaca ggctcttggc gggaggcttc   9480 

ttgccgatcc tgacatggcg agccgtgtcg gttgccgatg atctgacgcc acggcaggat   9540 

caggacaaga tttccgaaca cgcctatcat atcctgtgga agatacagca aggccacgtt   9600 

ccgaagtttc tgtggttgga ttgctgacga cgggcacaag cgttcaagca aggttgaacg   9660 

cggccggggc ggagccctac cttggccgta gcggtggatc gttttcgcgc gacggcccgt   9720 

cgcccgagcc ctcaatcgtt cccgtcgtcc ggccaaagcg gggttgggag cttttattgc   9780 

tgccttccag gttgatccgt ttccagccga cgacgccaga atttgcccga acgttcgtaa   9840 

accgcttcgg gcgtcaacgc attgaagatc ttcaaaacgc ctttgccgac acacagattt   9900 

gaagatgcgt cgaaatacgc caaggcttcg tcattgagtt ggaacaggtg gcttgcagcg   9960 

ctttcctgat ataaaaagcc cactgcttca tactctgcca acatccaacg ggctgcttct  10020 

tcttcggtca tgctttcttc ggcgcgacgt agggttgcaa cgtatcctct tcaaatacgg  10080 

aagtttcgtt ggaagcaccc ttgaaccatt tgcagcgata acttcccttg aaattgccgt  10140 

agtccttttg gacttccgaa atcgtcattg ctggcccacc ggacttaagt tgtacaacgt  10200 

cgccagtcga aaatttagct tccattatga ggcccctact ttgatgttac tttcgcctca  10260 

atctatggga aagttcggct ctgttccagc ttcaaacaag acggattcga tagaccacgt  10320 

caaggatcaa cgcgtttctt cgtcaaatcc gcttccgcac gcgaagccgc ctcgcagccg  10380 

ctcgacgccc gcataagctc atttcggcca cctgatttct ggtcgtaggt cgttagtata  10440 

ttttcgaaaa tcggaatttc acaataaatc aatattcgtg gctgaccggt tgggattggg  10500 

aaccatctca ttcccgttca ccatgaggat tgggcacagt aacatcctcc ggtatttctt  10560 

gacgaaggct cggccccttg gcgagacgtc gccctagcgc agtcacgaag gcgtcgtagc  10620 

gttcggcagc cttggcgcgc gcggcctgag cagctggttc cggcagggtt ggcgtggttg  10680 

caagccagaa gcgaacaaag acggcgatca tctcgatcga gatgccgacg tcgcgttcaa  10740 

gccgcatcat acgacgatcg acctgatcga gacgcttggt gatggcagct tcctgtcgtt  10800 

cggcggcgtc gggcgagagc gaggattcga tggcggcctc cgccaccagc gaacgggaat  10860 

ggtcgcggcg cgcggcatac tcggcgagct ggcgcatgac ggaaggatcg agatagaccg  10920 

agaggcggtg cttgcggggc ggttttgtca tgggtgcctc acagatccat gccgtcgccg  10980 

ggatcgagcg aaacctgccg cgcgacgctc tgcatgatcg tgcgcaggcg actggctgtc  11040 

caatcagacc ggggtcaccg aaaccctgat gggcctgacg gacaagatgg aactttatcc  11100 

gcgccttgct gaatgcatcg aacagataga gccgaccgcc tggaacgttg tgctcaaggc  11160 

aggcatcaag ttccatgtct tcgcgatagg cgttgccttg ttctggtatg cacaagcaca  11220 

ggtgcgctgg ttcatgcacg accttggcat aggcgcggca aaagcgcttt ccatcttcgc  11280 

cggagcattt ctgtttgcca cagtcgccgc attcgtcgtt gcgctattga tcactctcga  11340 

cttcaaaagc gtcgcgcccg cagcgagctg atccgccgcg ccgcctgcgg cgtaaccccg  11400 

agcgttttcg tgaccgcccc ggcatgaaca agcggcgttg ccatgacaag atcaacgagc  11460 

tccggcaact tcgatgaaag atatgtattc cgcgtggcgc ttccggcctg tgagggatgc  11520 

ggcatttaac tgccgccaca tcatatatcc ctcaggcacg ccgcatcgtc aacgcaaacg  11580 

tgcaacccgc tttgcacctg ttgattttag aattagtgtt ttatgtcggg gatttagcct  11640 

gtttgattgt agcgttttgg cgtactggat cgaagttttg gctattgttg gatagtgtag  11700 

cgtaggattt ggttgcgggg gcctgagtcc acggagactt gtaattttca gacggatagg  11760 

atgccgctaa aatgacttct gctttgcgcg aataacaagg gcagttggcc tctattcctg  11820 

ctgtcgaact cgtttcgatg cgatacctat ccattcataa aggatgccct ggtccagttt  11880 

cgaaaagcgc gctcgatgtt ttaaccactc cagcgagtca ccaggattac aacgggcata  11940 

gtcctcccga ataagtgcct cacaatcttc gcgaagcctg gggtcagttc tcagtggcaa  12000 

tcaacagtct ggtggctgca cgaggcaaag cagtcaatcg ccgtgtcgtc ccttctgttt  12060 

caagcacgga ccttagacag ttctgccatg atcagcagcg aattgctatc cacggcagtg  12120 

ctcacgccaa tggcagcaga tcttggcgtc acgaaaggcg cggctggtca gtcggtgaac  12180 

cggctgtcga gcgctgggct tttcgtctaa cgtcctcatc catcctacct cgccggccgt  12240 

caaatcattt cactgttggg gacggttggt acgtacttga cggtgacaaa cacgccgcag  12300 

cgtcggtttc gagcaatatc ttcaatgcgt ggctgccacc gcgccgactt tactgaccca  12360 

ggttcaaaga tagcggagga aacacatcaa ctcactggtg ccaagggttc aatccactgc  12420 

ttgggcgatt ggcgcaaagc attcgcatgc ttggcgggcg acatgccgaa aaccctgcca  12480 

tattctctcg aaaactggga cggactcgca tagccgacct ggaaacctgc ggttgctgcg  12540 

tccagacctt cactaagcat cagacgacga gcctcctgaa gcctaagctg tgtgcggaat  12600 

tctagtggac tcatattggt gaccgctttg aaatgcgcgt gaaacgtaga gcggctcatc  12660 

ccggccactt cggcgccagc ttccaccgaa caggtctcgc gaaagtgctt cctgatccac  12720 

acaatagacc gtgcgatctg attgacatgc gcgccagctt gcacgatctg tcgcaacgcc  12780 

gctcctccaa caccggtaat gagacgatat aatatctcac gcttcgccag cggtgaaagg  12840 

gcgtcgatat ctcgaggcgt gtccagcagc gcaaggagac gcgaagcggc atcaatgata  12900 

ccctcatcca ccttgttaag taacaggccg atagggccgt tgattggcgc gtgctgttct  12960 

gaaggatagc gaattgccag atccgccaac tccacggtgt caaggtcgag ttgcatcgaa  13020 

agataaggtt tgtctttcga tgcttcgatt accgatccca tgaccggaag atcaacggag  13080 

gcgaccagat agctggaggg atcatagaca aagcttgtct gcccgagaac cgcctgcttt  13140 

cgcccctgtg cgacgaggca aagtgtcggc tcgtagacga caggcatcgg caatgtcgtc  13200 

tcggaacagc gaatgagctt caaccccggc agctgcgttt caaacgtacc gtcgtgaggt  13260 

gcatggcgag caatggtggc ttcgatggtg tgttcacttt tcataatgct tttatgccat  13320 

gccttcttac caacggaaag tctgagcaag cgatatttgg acgatcaagc aagcatatcg  13380 

gacattaaag gagtcagtgg tctcccaccc agaaatcttc catttcccga tagaagcacc  13440 

gatctcgtca ccgttttccc cctggatcga ccagaacacg aaagttgcag cttcagacaa  13500 

ttcggcaagg ataacggaat atctgtctac cgctaaatcg cattcccggc acatctttct  13560 

ttctgccagc tcgaacgcaa agaccgtttg tccacggaca gggggcacgg ccgattaacg  13620 

atgagtacct gcaagatcgt caaatagaaa ggaaacgcca atgcagccga cagatttgca  13680 

agtgagccag caggtttccc ggctcatgcc actgtcaagt ggttcccaaa ccgcaaaccg  13740 

aaccgaactt ccggaaacaa ttcgggaatt ctttctgcta ctaaacaacc gtcaaattga  13800 

cgcgcttcca cagcttctcg cccccggcgc tctgatcgtt gacggtaaag gtcgaggaga  13860 

caccaacgcg tctccccgcg agaggctgaa cgacacgctt tcctttttgc gttcgccggt  13920 

tgtcgtgctt agatctgagc cattgttcga aggttgccgc gttacggtcg tatcaccgtc  13980 

cggcagcgac cttcgatatt acaagctgaa cttccagatg atctccggaa agattgtgct  14040 

tttgaaaatc ctgccagcaa aaccagacgc ttatgacttt ccgacatctg cccaagtggc  14100 

catcgtcaat gtttcgtcgc agtgaaccgc taatgggaaa aagtgtccaa tcgcgagtga  14160 

cctgacttcc ggctgtgtcc cggtacacgc tcgcttgctc cgccgtgggc aaccgcgatc  14220 

acatgaaacc tgacgcgaga ggacgacttg gcgatggagt tcactgtgac ctctttgcgt  14280 

acttccagtc ccggcgcgga agagggccgc ttttctcttc cgcgtggacc acattaattg  14340 

cctgctaaga cggctgaaat gctctccaga gcgcgtatga atcctcaata aagttgtagg  14400 

cgctcacttt gccatctctc accgagcaga ttgccgcgaa gtcgctaaca aaatctttgc  14460 

ctgttctacg aatgcgatgc ttgaaattgc cgatgatcac gaccttgtcg ccgctttcaa  14520 

aggtacgctc cacttgaaaa tcgagcggct cgaaattgac aggaaatagc gccatccaat  14580 

cgcgcacctg atcccgtccg tgcctgaggc cgatggtggg cacatcactg gccccttgaa  14640 

ccttccatac aacatcctca gtcaggcatt gaagggtctt ttcgacatcc ttcttgaaga  14700 

acgtctcgag ataatgctgg acgacctgtg tgggtgtcat ctcggtataa gttgtcatct  14760 

gatatctcct tgcaggatta gttgcgcatt aaggggtgat tagcgatggc caccggcgag  14820 

gatgaaaaat tcgccagtcg tccagctcga ttcgtccgat gcgaggaaaa tcgctgcagg  14880 

tataatgtcc tcaggaacgc cgagacgctt gagcggcgtc tgcgccatga tcatggggcc  14940 

gaagtcaaag tggagaaagc ctccggcctg cacgccgtca gtgtcgacca cgccgggctt  15000 

gattgcgttg acacgaatct tgcggggggc gagctcgttt gacaacgcgc gcgtcaaacc  15060 

gtcgattgca cctttgcttg ccgtatagac cgatgcgttt tctgggccga aaatcgttac  15120 

cgtcgaggac atgttcacaa tgctgccgcc ctccgccatg tatttcacag cttctttgat  15180 

cgcgagcagg tagccaaaga cattaagctc catatgcttg cggaacagat cgatagacag  15240 

gttttccagc ggctggaagt catagacacc ggcgttgttg accaggatgt caacgcggcc  15300 

gaaagctgcg tttgttgccg caaacagctc ttcaacttcc gcgggctcac tcatgtcggc  15360 

cttcacggcg atggctgtgc caccagcgtc gatgatctcc tcaacaactg cttcggccgc  15420 

agatttgctt gacgagtagt ttactacgac tgatgcaccc tctgcggcga aacttcgcgc  15480 

aattgccgcg ccaagtcctt tggaagcgcc ggtgatgaca gcgattttac cttgcaatcg  15540 

attgctcatg tttctttcct tgaggctcgt tggcccattg gtttcatgct tggcgtcatg  15600 

cgtaattttc gccagcgctt atgcaaaaga ggcgtaaccg cgtatgtctg tcttggtgca  15660 

agagacgcct attcggcgtc tctcgtttgg atcgacagaa tcaagaggtc tgagaggtct  15720 

tgcctgacca cagatccaca ggccccaacc ggtcagcgcc ctgcgtctcg agcatccaac  15780 

ctggatactc gggcggcagc tcgcttacag catccagccg cgcaacttcg tcgtcactca  15840 

gcacggtgcc gaccgcggcg atattatctt cgagctggct aagccgtttt gctccgacaa  15900 

tgaccgatgt cacgaccggc ttggcgagga gccaggccag cgctattcgt gccgggctgc  15960 

aaccgtgcgc ctttgctatg ggcttcagga cgtcgatgac gttccaggcc cgctccttgt  16020 

cgacaatcgg gaaatcaaaa gaggagcggc gcgagccttc cggagactga tttgcgcggc  16080 

tgaatctccc agatagcagc ccgccagcaa gcgggctcca gacaagcaag cccatctttt  16140 

ctgcatcgag caatggcttg agttcccgtt caaggtctcg acccgcaatc gagtaatatg  16200 

cctggagtgt attgaatcgt tccagcccga ggcgggcaga aacgccaaga gccgtcgcaa  16260 

tgcgccacgc ctgccagttg gatacgccga cgtaacgtac cttgccctgt cgaaccagat  16320 

catcaagcgc acgcagagtt tcctcgacag gagtgagggt gtcggttgcg tggatttggt  16380 

acaaatcaat gtgatcggtc tgcaggcgct tcaaactggc ttcaacagcg tccatgatat  16440 

gtccgcgcga tgcgccgacg tcgtttcggc ctgaacccat ccgactatag accttcgttg  16500 

ccaacacgat ctcgtttcgg gcaataccga ggttcttgaa cgattgtccg agcgttcgct  16560 

cgctcgcacc ggctgaatag acgtcggcgg tgtcgaagaa gttgatcccg gcgtcgatgc  16620 

tggctttgac caattcgtcc gccccggtct ggccgacatc gccgatatgc tcgtagatgc  16680 

ccgtgccgtc gctgaacgtc atggttccga ggcaaagttg tgagacaaga agtccggtgt  16740 

ttccaagtgt cttgtatttc atgtcagtcg ctcctgaaga cgcaagcttg cgtacgagcc  16800 

gaagatctcg gttcgctgtc gcttgctgat ggtagattga tggtccgggg cctcgctaag  16860 

tggtgagacg aatgctcatg gagagcccca gcaaacgcct caggtgccag gctgaatcta  16920 

ttggcccggc gccccgaaga tttcgttgat gtgcagcgag tcggtaaatt cccgaccgct  16980 

cgcgatcttg ccgtcgcgga agtgaagaag aaaatggtag ttgtttcgat agtgccggcc  17040 

gtccttcatc ggcagatcac ccttcgcgac gaccgcgagc ctgtcttctt ccccggtgac  17100 

ctcgtggtac tccatcttca gagggcccgc ggcgtcatcc agaagtcgag gtaagttctc  17160 

gaggtaaggg cgcttcgcat gcgtgccgga aaacttgttg ccgggcatga tccaccacgt  17220 

catatcgtcc gtgctgaggt tcgacatggt ttcagtgtcg cagcgcccca gtgcgtccag  17280 

aaatgctttg gcgattctct ctttctgctc tctactcatg gcgtgtctcc ttcttagcca  17340 

tttgttgttt gatgttggta aaatgaggtc gtttcctcga gaagaaaacg caaaagaatg  17400 

gcggaccgaa ttccaatttt ggaaaacctt attatgtgcg ggttcagggc cgatgtgatg  17460 

ccatcgggca ggtttagccg gtcctcgtag atctgcgaga caaacgcgca agcgtttcca  17520 

gccggtaccc aatatcgatc agcaacacgt tggagagggc ttgctcactg gcacaccagt  17580 

tcaatcaacg gctcggtaag ctacggaagg tggccccgca tcttcgaaac cattgctcca  17640 

agcgcttagc gcacgtctct accgagcatg cacctgatgc aaattcggcg agtataccgg  17700 

gaagcttgcg attttcgtcg ggacttacat cactgacctg gtcgcgacga accgccagcc  17760 

cacgacggaa gcttcgcgat gaggttcagc ttgcaacgcg tattccggtg tgtcgattat  17820 

tgagcacagg tccgcgaagc tgtcgtagcg gacgatgacg atgtcgtccc atacctcgct  17880 

ctcttttgga agcaatgtca tcaattggga gccagcgtag ataagctccg ttgaaatatt  17940 

gagcgccgtc gcgatctgct ggaaggccga ggcataaccc tcatagtatg cggatcgaga  18000 

cgttgtcttc acatgttcga acccgtcgga atatagcgga cggtctctga agcggaggaa  18060 

gttgatcatg acaaccggcc caccgcctaa ccgcttagca gcgtcggcaa gtgtttcagg  18120 

aatgagagct tcaagttcca ttttttgact ttctgacact ttatgttgat atgtcagaca  18180 

ttgacacttt tcgtcttttt gtcaagttag gggaatggag aagttttcaa ataaagaaga  18240 

acgggtcctg accgctgcgg aagatgtgtt cgcccggtac ggattcgcgc gcacgacgat  18300 

gggcgacatc gcaaaagcgg ctgccatttc ccgtccggcg ctctatttaa tatttccaga  18360 

caaagaggca atcttcaccc gtgtgatcga gatgatggac gccagatccc tggacctgat  18420 

ccagaatgag gtcgatcaga tcgcggctat tgatcagaag ctgctacacg cctgtactat  18480 

ttggggtctg catggagtgg aactcgctac cgcatatccc gacgctgccg atctattcga  18540 

tttccaattt ccggccgttc gtcaagttta cgaacggttt cagcagtttc tcgttcgaat  18600 

attggaaaaa ggaacggctt cttgggaatt gccggtgtca cgtgcagact ttgcgacgac  18660 

gctcacctat ggtctacgag gccttcgata cgcagcgact gacgttaatc acatgaggca  18720 

cctcattgaa gttcacgtaa ccgtgtacgg actgaccctt acgcgaacgg acgcttcaat  18780 

cggctgagaa aacgatgacg tcaaatccac gtatgctgct caaccatacg tgcgtgcctt  18840 

cgtttaaatg gacttcaaga ttgcacaact gcggtaccgg ttttcattgc gttcaggcaa  18900 

tgacaatagg attctaagtc actccctcct ttttgaaatg ctcgatgagt gcgcgaaacg  18960 

ctggcgaagt tctgcgtcgg ctcggataat aaaggctgaa acctgggagg tccgttagcc  19020 

aatctgtgag aacctcccgg agattgccgg cttcaagatc ggaggccact tgcgagcgaa  19080 

gcagaaagcc aataccgagc ccgtccatta cggctgatcg gatcgccgct ccgtcgtcga  19140 

ggatcaacgc tgggtctccc ttgtattcaa acttctgact accctttgcc agcggccacc  19200 

ggaatagccg tgacgagctc agatagcgat ataatatgca tcggtgacca gccagatgtt  19260 

ccggagccaa gggaattggg tagttctcaa agtaagatgc agagccagca atcacgatag  19320 

gggtagacgc cgtcaaggga agcgccacca tatctttatc gatgagcgta ccgaaacgga  19380 

tcccaccgtc aaacccttct gtgacgatgt catccagtct ctcactcgta gagatttcga  19440 

acacgacatc ggggtagcgc tgacaaaagc ttggcagcat tggccgaacg atgctttcaa  19500 

aggcgaccgg gatcatcgtc agccgaacga cgcctgcgat cgtctccctt gcttcctgta  19560 

aacgcaggag ctcttcccgg agatcgcgca gtgccggacc gagtatctcc aataggcgta  19620 

accctgagcc ggttggagcc acgcttctcg tggttcttgc aagcagagca accccgattt  19680 

tccgttcgag ttggctaatc gtgtagctca aagtggactg cttcacacca agttcggatg  19740 

ccgcatcggt gaagctctgg tgtcgcgcaa ccacttcgaa gactgctaaa tcgcggagga  19800 

tatgccggtc catttatcga tcctatctat agagcttctt gaggcgcagt atcttatcaa  19860 

tatgcggatg gacgtctatg cttcttcgac atcaagtttt gtcgacagcc tgcaagggat  19920 

ggatcgtgcc tctaaacagg tgagttgtga acgagggcgc gatttgccgc cgcacaggcc  19980 

cagcgataat taaaaaggaa                                              20000 

 
           
             2  
             12588  
             DNA  
             Agrobacterium tumefaciens  
           
            2 

gcgcgcccag gaagtagttt gacttccaca atgagcttgc ggagcagcgc atggatccga     60 

ttggtcatgt tgccgccctc cggcaattcg tcgggagtaa ccaactcgga caggggcagg    120 

gatggaaact cgaggggatt agacatatca aatccataac tgagatatca gaagagtttc    180 

tgacatatcg gaatgcgaaa tttctgtcaa gcggtcagtt tgccgcgata tcggcccaaa    240 

ggacgaagtg cagtgcttag ctggttcgag gagcgaaatg gctggaacca agttaaattc    300 

gtgagagtgc gcgcaagggc acgatctaaa ccaaccctgg ctttaccgga gacagtttga    360 

tttaagttat gtgctggtgc gtccagcatg gcgtgatatc gttcttcggc cacggccgga    420 

gggatgtttc cgatggctcc agaaggggct tcggacgtgg cggtttgact ccatggacga    480 

tgacgaatcg gagaagagaa tgagaaacgc agagcgagca ggtccgcttc ggtatcttga    540 

ggtatcggcc atcggcagtt tttgctctgc atttttttat ttcggggtgg ccatggcaat    600 

gccggcggac atgacggcgg agcgccttct caatatctgt gaagcgccca ccatgcaggc    660 

cgcgatgatc aagggtgacg aacttggctg gccgcggctg accgccgcgg aaacggagga    720 

atggcgtcgt agtttcgtcg catataatga gggttcggtg gtggtcgtgg gctggcgggg    780 

cgagaacgcc ggcagagccg agtcattgtc tttttgggtt gcgactggtc caaacggaca    840 

caaggcatgc gcctattcca cggcaaggcc tgccggtttt ctggatgcct tgtcggagcg    900 

gcttggtgca ccggataatc tcgacaaaaa tgacgcgata gaaagtacga cagcctggtg    960 

gaaacgaggt gcggtcgagt attcttttgt ccagatcggc tcatccgctg tcgtcaatat   1020 

ccgttcaagt cagtgaggta gtggcgtaaa cttcaggtaa attgtcgcat aatccgacag   1080 

gccgcttccc gtggaactta agattgtcag ccaagctggc gctcaattgc gaccttgtcg   1140 

aggaccgacc agaccctacg gatttttgcc tgttcgaagt cgtagaaaac atgctcgcag   1200 

aattgaacgc gccggccatt gaccggaagg tccatgaaga tggatttggg cgtacagtca   1260 

aaaaacagcc gggcggcaag ccgcgtcgcg tcgctgacaa gaatctcggc ctcaaatcgc   1320 

aggtcgggaa tgtcggcaaa gtccttgacc agcatatcgc gatagcccga cagcccgaac   1380 

ggccggccat tgtgttcgac attgtcgtcg acgaaggtgc cgagttcatc gaaggcttgg   1440 

tgattaaggg agtcgaggta agcgagatag atgtcgttga gtgtttgcaa gttgtcttcc   1500 

tctaccagta tggtttgtgc tgcccgcaac tctggagtta ccgaatcata ctggtgcagg   1560 

tcaaactgtg atgagctcgc acctgccatc atctagccgg taaaacccat cctgcggaac   1620 

gggtagctcg agcatttcct gctttaaaag cccgagaaaa acaccccgaa ggatccgccc   1680 

gaacagacca tgggaaatgg cgatggtcgg atgccggaca tccaggatcc atgacgtcgc   1740 

gcgcttgcag gaatcatcaa aactttcgcc atctggtgcc ctgaaatacc agtcgaacgc   1800 

attggacccg tcaagatggc cgggaaactc gttgtcgatt tcaaaacgcg tcaacccgtc   1860 

ccaggaccct gtcgtgacct caaccagtcg gtcatcttcg atatgcggga gcggcagctt   1920 

cgcctgtact gtacccgttc tgccgtttgt cgcacgcgcc cgagcggact gatctgcatc   1980 

tggaacaact ggttatcatt gttgagtgcg tcgcggagca ggcgagcaac ctggtccgcc   2040 

tgttcaacgc cccgtggcgt caagggtgaa tccaattgcc cctgaaaccg gccaagagag   2100 

ttccagagag tttcgccgtg gcgaaggagg taaatggtcg gtagagccac gcttgtttca   2160 

tcctgcacgt tcgttatcga ttgctgtcac taggaagcat caatacggaa acgtcccgat   2220 

tgcagcgcat tgatggccga ctgcaaagcc tgatccgtgt ccttgcctga cactggcagg   2280 

acatggtgct caaaggcgcc gagatcggcg aactgggagt gaagatcggc gaccaccagt   2340 

ggatcgctga ggctgtcgcc accccggtcc agacagcgtt cgatcgcctc agccgctgtc   2400 

gtgcgcagca caatgtaatg cagcggccgg gcaagtgccg taaaggccgg cagccagtcc   2460 

ggtcggacga cgccgtcaag gatgacgaag tagccctcct tggcgtaacg accggcaaca   2520 

tcggcggcga tctgcatgat catgcggttc tgctgatggg attgcggcag ccatggatcg   2580 

atgcggccgt gcttgatata tccccacaga tcatcgctgt gaaaatgcac ctttggaacg   2640 

ccgggaaggt tcgctagcgc ttcggcgatt gtggatttgc cagagccggg gtgcccggag   2700 

agaagcagga tattaccgcc aagatcgtcc gtcatgttca ttcactgacg ttgggcatgc   2760 

gtaaagcggc taatgatgta atcattcgtc tatcgacgtt accaccctgg tgtttcctat   2820 

cttggctcgc aggcaaatga ggccccctat ctggcaaagc ttatcacaag ataagcagac   2880 

gccagaagca gggtgacgat tgtaaagaat atcagacctg ccgccagttc atcgaactgg   2940 

ccgtcgcgtc cattgcggtc ccagtcgtaa gccatagcca acctccatcg ttgctgtaag   3000 

ggaagggtgg tcccgtatgg ttaacgaaag cctaatggcg gcaggctgaa tgtttccggt   3060 

atgatctgat atctgggtcg tagcgaccgt ttcgtaacga gacgagttgc aaaggaggca   3120 

cagaagtaat accgttggtt acgacggtat tacggaaatt tgacatgaca acaacggtaa   3180 

cggccaaagg gcaggtcacc attccaaaag ctgtacgcga gcttttaggg atctcaccgg   3240 

gaagctcggt cgattttgtc cgggctcccg atggacggat cgttctcgtc agggcggaca   3300 

agaaacagcc actgacgcgt tttgctaagc tgcgcggaca tgccggtgaa ggtcttggta   3360 

ccgacgccat catggctttg acccgtggtg acgagtgacg cttgtcgata caaatgtgct   3420 

gcttgatctt gtgacggacg acccggtttg ggccgattgg tcaatcgagc agctcgaact   3480 

ggcaagcgtt tcaggttcgc tgtacatcaa tgacgttgtc tacgcggaac tatctgttcg   3540 

atatgagcgg atagaagagc ttgacgcttt tgttgatcag gcggggttga agttcacccc   3600 

ttttcctcgc gcagcgttat ttctggcagg taaggccttt accaagtatc accggggcgg   3660 

cgggacccgt accggtgttt tgcctgactt ttttatcggc gcccacgcag caatacaaaa   3720 

ccttcccttg ttgacgcgag atgtggctcg ttaccgatcg tattttccaa ctgtcacttt   3780 

gatctcacct gaagtttagg aatgcgcggg ccttagcact tgtgagccga cttcggtttt   3840 

cgaagtagtt tcgcgctctt cgcctgaggt caggcgagcc gcaatgcttt caactggctg   3900 

gcttgacgag gctgatttcg gttttgctgt cgaggacgat atgcaggtcg cggatcatgc   3960 

cggctgcctg cagcagggcg tttcgcacgt cgtcgtcgct gcgccatgac gtcgcgaatt   4020 

atctgtcaca agatgaatat tttgacgagg cagagaccgc gagcttctat atcaggcaca   4080 

tgccaaagca ggcgttgcgt tcgaccgatc cgattcacga ctacttcact gtccttcttc   4140 

cgagcgtcgg ataccgcggt ggtaacgggc ggctgatgta tcaggcgcgg ctatcggaat   4200 

tatcacgcaa tccaaccggc atggttcgcc tctttgttga tgcttgccgc gatcttcggg   4260 

ttttttatgc agtgtccggc atgtcgcttt tcttctgttc ctatgcggca ggtgcgactg   4320 

aaaaagcata tcctttgttt cgtcggtttc caggccttct ttacgaagat ggcagcaatt   4380 

tcacgttgga aatattgcgt cgcaccgatg tgatccgaga cgtgaattgg ttgacagcca   4440 

tcaacgatga actgctggcg cgggtcggtg gcttagagaa agcaagagac gtgctgggag   4500 

acgagattgt cctccatccc tacgaaggag gcgtcgtttt ccaggcgggt gcccgcggcg   4560 

taccgcgcag tcaacgattt tctcaagccc ctgcgttagg aggcctggga ttatccctat   4620 

atatgagcgc cttacggtgt cgatgagatg gagtgtacgc aatggtggac tcatcgattt   4680 

gacgatggtg aacgttaagc tcgcccagcc tcaacgattt catcaccccg atggccatcc   4740 

ttaaggagcg gaggtgaaac gtctgggcga cactaaggca tctcataccc ggagatcatt   4800 

cctgccgata aaacttagtt gatttgctaa gttttccctt gctccgccac gatgaagcat   4860 

gatagttagc aatatgacta agcatcatcg cgaactttca ctgatcttcc aggctctggc   4920 

cgatccgacg cggcgggcga ttctggcgcg cctcggcggc gggccggcac cggtcatgga   4980 

gctgtccgct cccacggggc tgcgtctgcc cacggtcatg cggcaccttt ccgtgctgga   5040 

ggaggcgggg ttgatcatca cgtccaagga tggtcgggtg cgcacctgcg ccatcgtgcc   5100 

ggaggcgctg gagccggtat gcacgtggct cgatgagcag cgggcgatgt gggagagccg   5160 

gcttgaccgg ctggaggcat ttgcaatgca ggccatgaag gaggattccg aatgacaacg   5220 

aaaccgggtc aacaaagcgc acacgctgaa ccgcatcagc cacaagacgg ttttgcgacg   5280 

ctcagctttg aacgggaaat tgccgttccg ctatcggctc tctggcaggt ctggctgtca   5340 

cccgccgccc gggcggtgtg ggcttctccc tcaccctcgg tcaccgtgga gttcctggag   5400 

gcggacagca ggctgggcgg tcgcgaagtg tcgctctgca aggtcgccgg ccagccggat   5460 

attcgctgtg aatgcggctg gctggagctg cagccaaccc gccgcagcgt gaattacgag   5520 

gtggtctcat ccggcggcgt aacccagtcg gcagcgctgg tcactgccga ttttcagtct   5580 

gcggaagagc ggagccgctt gaccgtaacg gtgcagcttt cctctctggc cagggatatg   5640 

cgcgatggct atcacgaagg tttcggcgcg ggtctcaaca atctggccag cgtggccggg   5700 

cggaccatgg tgctggaacg agtgataaag gtgccgcgaa acatcgtctg gaaagcctgg   5760 

atgaacgaga agacgctgcc gcaatggtgg ggccctgaag gcttttcctg ccgcacgaag   5820 

aggattgatc tgcgcaccgg cggcgaatgg gtctttgaca tgatcggccc tgacggcaca   5880 

gtcttcccga accatcatcg ttatgtcgag atccggcctg aagagcggct tgcctatacg   5940 

ctgctgtggg gcgaaaacgg tccgaaacat gccgatgcct gggcctcctt cgaagatcag   6000 

gatggcgcga cgaaagttgt gctgggcatg gtgttcagca cggacgccga gttccagcaa   6060 

gcgaagggtt tcggcgccgt ggagctgggt cagcaaacgc tgggcaaact ggagcgcttc   6120 

gccaagtttt tttaaatgga ccgatcctgc atcgacattt tctatatccg aaatccgagc   6180 

tcctttatac tctgtttctt gatcgttatt cggcgataac cttgatttat aggatactca   6240 

atgaatttat aggaggccaa cccaaatatg gtggtgagcg ctaaagcaca tagagccatt   6300 

atccctagtg cgactaagcc gggcgtaaaa aatagaccta ccgctttttc agaaacttga   6360 

atcattaaat tgtgaacgag atagattgaa taacttgcgt ttccaaggtg gataacgagt   6420 

ctatttcgcg caaccaagcc gttttcctca aggaatacgc acccggagat aagcattaaa   6480 

ccagctaatc cccaagtcat tggtcgaaat atattgttac tataacttag acccaaaccg   6540 

ttgcccgtga cagcaataat aagtgctcca ctggcaatta aaatccaagc aattgttgga   6600 

ttggctgaag gagaattttc gcgtctcgaa acataaaaag caccaaatgc tgctcctatg   6660 

gcaaaatcaa ggattactgg gctggtataa tattctagat atgggttttt cgcgagaatt   6720 

attcctgaac tagccaggca aatcagaatt gttaccaaaa gcgctattct cattccgaaa   6780 

ttaggaacaa aaagtagcaa agcaaacagc gaatagaaga aaatttcaaa attcagcgtc   6840 

caaccaacag acaaaattgg ctcccacagg ccgttgcgcg tgaatgggat aaagaaaaga   6900 

gatttccaaa tgtagctgag ttgcagatcg atcacgccaa tggggcgaaa cccaatgagg   6960 

aacagggcta cgatagccaa cgtcattatc cagtatatag gaacgatacg aataattcgg   7020 

tttaagaaga attttgccgg gtctctgtct ttcccgaacg tggtcaacac cataatgaat   7080 

ccactgagaa cgaaaaatat atcaaccccg gcggctccga attcatagac tttggccccc   7140 

ggaaacaaac gatctacgta gggtaagaaa tgatgaaaga atactaggaa agccgccact   7200 

gctctcaaag cctgcagatt gataatcatt ttatggcctc aaaataccgt cgtagaaaat   7260 

tatcgaatag taacctatcg gtctaaaata ttgctctaat ttgatattat agtgggacga   7320 

agttgttcta ttactctcta aagccctatt ctcagaaatt ttttatctaa tcaattgtcg   7380 

tcacgcctac ttcaagaact gccttgaagg agatagtctg actggtgact tttatgaaac   7440 

tgaaatggtg gcgcgcaaaa agtaaatggt tggacggctg ccaagaagca aaatcctttt   7500 

cctgaagagg tacacaaatt tctaatgaat ttgcaagctg agaatgatgg acccgcggca   7560 

acagccctag cgacgcgagg gttcttgatt agagtggatt cctagagtaa aagttttttt   7620 

tgatattttt gatatttttt ttaaaagatt ggaatgacaa tgagtgcact cgttgtgccg   7680 

tcatttttct atcgcaagtt tctgacgata gctgcattat ttaaaaacaa caagcggtgg   7740 

cgccaacccg acgaatggat acgtgtgcac gttttttttc ctgtaacttc ggtggatggg   7800 

gaaaggctga ctcgtgtggt catgcggcga ggcgaggcca acaactatga ataccgggcc   7860 

ttaaacgagg aagaaaaaga agagatgttt tgcgatctag cttggtgaga cgtcatagca   7920 

ttattgcatg aggcttttca aattgcatcg acgaaggctt aacaaattgc ggcgttacaa   7980 

tgatctacct agcttcattg aagctttacc attatatccg acgcggataa ttgtatcccg   8040 

aggtattgtc cgtttatata tatttttgtt tggtttcttg tcccagtgta atgtaattcc   8100 

gacagccttt ggattgccat ttcttagaat tacagacatg ttagtcatat cgacgctgcc   8160 

aatattgtat ttggtatata taaggctatt taattcggcg ctgattttga ctcatattgc   8220 

tccgattatg tttgcattct taacctgtct attttttttc accgataggc ggcaaggcga   8280 

actatattcg tctgtcttta catttttata tttactttat tttcttccct ttacgtttgc   8340 

tttgctccgt gaggggtgct tggatttgtt ttgttgggga atcctggcag gattcgcgac   8400 

gacgacaatt tttttgttga ttgatttgta tgttccttac actcttacga agataggtct   8460 

cgcccttgtg tttgactatg aagcggtcgc tgacgcagcg gcaaagggcg actacaatgc   8520 

gcctctgctt cgcttcgaaa aagccggcgg actgtggacg cacggaaatg aagctggtcc   8580 

agtgtttgcc ctagccgctg ccgctgcggc gtctttaacg gaaaggcgtc gcaatttttt   8640 

tatatttgca tcatttgtag cgatatacct tgtttcgttt agcgccacac taaatagatc   8700 

cgggatgggt acggtcgtat tgataggact gatatcgtac atgtcttcgt tctcgaaccg   8760 

aaaaataact ttgacattgg tttatggctg cattggaaca ttaattggct caatggtgat   8820 

tttctttggc tcgttcgatc tactcgatgg cgcgatctca aagagatttc ttgaggacga   8880 

aaatgctagc tcgaatattt tcgaacggct tgaatctctc gggtatggca tacaagtggc   8940 

cctccataac ccttttggta taggacttgc cgctaggatg gctgcaatga atgcgttcag   9000 

taacttgggg acgcctcaca atggttttgt ttcgactgct tttacttcgg gtattttagt   9060 

tggttttttt gttgtgtcct cagtcgcata tatgttgttt cgttatagaa aggtgcgctt   9120 

ttttctttac gtggcagtta cactgacttg cggttatttt tttgaggaac tggacttcaa   9180 

cgccgccttt atgtgttggg ccggtttgtt gataggttac acctgtttgg atctcgatta   9240 

tcgcttggtt cgggggcggg caattttgcg gtacttgttg aaactgttta aaagtcagca   9300 

gtctgtatgt cctgaagcca ctagtttatc ttccgtgaac tcatgaatag aataatagag   9360 

tgaaaaccat gtgtcgtttc catcaattca aatattgacg ttaggaatct cgtagaggtt   9420 

taagatgtca cgcgggagca ttgtcatttt cgtcccaagg gtgttggttg atgccttgcg   9480 

atagaaggtg tctctcgcgt cactactgac ttgacgactt ttatcgctca aaaaagggga   9540 

cattgcgtgt ttgatcactt ggatttttag ttgttctccg ggcctgtagt ggtttctcat   9600 

tcgttttgtt tttcagctac ttgacgccaa tttgttcctt aatttacgcc tgctccgctt   9660 

tatatggcga actttttatt tcctcattct ttcccatccc tcgctggttg acctatattg   9720 

agccgtgaac ccataaacgg gcggcgaggc gattaagggc ggtcagcctg taagtaccaa   9780 

ataaaacaca tgcggtcgcg gacgttgcaa ccagcagcgt cgtaagctgc aaaagagtcg   9840 

gcaaacgctt ttcgcccttc ttcgataaag tcgtaacgaa ggctgatatc aaccatctgc   9900 

tgtcgtgcgg ttgttgcgga gagtgcgacg cgctcggccc aagttaaacc gtctggctcg   9960 

ctcatcaacg tgtttacctc tacccaataa cattcgcagg tagcttccgg cagcaggaat  10020 

tgcagtggcc cgtcaacgtt aagaaggcct aatttctcgg cttcattgat aaccagagat  10080 

attgtttcag gttgccaacc ccattcaccg ccgctgcgac aagcggcaga tcgcagtgta  10140 

tctggaagga gcatctcagg gttgtctttc atgcggcaag gctatacatc ttccaagagc  10200 

gatcaacgtc tgattcagtg ccgtcacggg ccgatacgat ctgaccgacc acgagtggcg  10260 

cgtgattcag ccgctgttgc ccaacaagcc gcgaggtgtg cctcgtgttg atgaccgtcg  10320 

cgcgctgaac ggcattttct gggttctgcg atcgggtgct ccttggcgtg aggtgccaca  10380 

gcgctatggc cccctacatc acctgctaca atcgcttccg acgctggatg aaagccggaa  10440 

tctgggacag catgatggac ggcctcacca gcacatcgca tgatcggatc acgatgatcg  10500 

acggcacttc aattcgcgta catcattcag cggcaacatt gaggacggat cacccagatc  10560 

gctgccttgg aaaaagtcgc ggcggtctca caaccaaaat ccatgcttta accgatggaa  10620 

aagggctgcc aatcaagatt gccattacac ccggtcatgc ccatgacctt acggcagcgg  10680 

gcgaactact cgataatctt tccgtcaggt gcgatgcttc ttgcggacaa tgcatatgat  10740 

gccaactggc tacgctcaaa gatgagcgcg caacgctcgt gggctaatat tccgcgaaag  10800 

tctaatcaaa aggaggcaat cgtcttcagc ccttggctgt acaaaaaagc gcaacctcat  10860 

gagcgcttct taacaagctc aaatacttca gacgggttgc aacccgatac gacaaacttg  10920 

gaatgacgtt tctcgcaatg acgaagctag cttgcattcg catcgtactc cgtcataacg  10980 

agtccacggc ctagttatgg ctgctcacaa agaggaattt aagagtactt gcaataatat  11040 

ttggagaatt gattttggca atgttttgat aatggataat tcatgaaata tattttctta  11100 

ttatcggctt ttttgatatt tttattttcg gcttttattt acttttttcc gatctttggc  11160 

gcggttgttt gtccgccttg ttttggtttt gtcgaggttg aaggggggat ttatattgac  11220 

gcgagcctaa atgcggatct tgagaaagat aagattttga gtaatctcga tgccgcacat  11280 

ttgttattga gagaggtcta cggtgaggtt gaggctccgc tacccgcaat atttttgtgc  11340 

gtgtccaaga attgcgccac gtacctaggt agacgtgggg agaaggcttc gtcgtttggg  11400 

cattgggcta ttgttgtcta tcgtgatgga aataactctg gcattttggc tcatgagctt  11460 

tctcatattg agattggctt taggctaggc ttttataata tgtcgtctgt ccccatttgg  11520 

tttgatgaag gggtagcagt ggtcgcatcg caagatcgta gatatttgaa cgttgaccct  11580 

tccggtagac tttcgtgtaa agagggtgtc accgggccgg tgatagccga tctcgatgag  11640 

tggtgtcggc gcgctagcat cggggacgtc ggcatataca gcgctgctgc ttgcgaagtt  11700 

atgaaatgga tggaccgtag gggcaatgaa ggttcattgg ttagactctt ggatttatta  11760 

cgatcgggcg agtcgtttga tgtggctttt gaatagtggt gtaggcagtt agttttttac  11820 

gggtttattt cgtttatttc atttcctcgc acatccccac cctttttctc tggtttatac  11880 

ttcttccttg ttcggcgatg aataggaaga cattcctgtt ttgaaactga accagcgccg  11940 

gctgtgagag aaagacgtcg ctctcatatt tcgcggtctg acaggtaaag tcctctggcc  12000 

gtgaccggag gagagatgga atctggtttg tggtgggcct ttctatttct ccgaaacgca  12060 

ttggctgaag gacacgctct gacaggcata aaccgctgat gcggggcact gataagtgtc  12120 

tttattttcc ctaggcggca attttcagtg ccccgttgca atgcccatcg agctgaaagg  12180 

ctgatggaaa aagagatgga gttcacaaca cggtcgcagc tgcgtcgcgt gataggaaag  12240 

gtgtgttcaa atgggtctct atgaaatgta cgtccttgct actcgtgcag ataccgccct  12300 

gatgaggaga aggctggcac gatctctttt tgccagggca aagcttcgaa atcggaagct  12360 

caagaagtag tatttacgca accagaagga gagttttgat tcgatggtca ataggaagag  12420 

gggagggcag gtgagttgcg acctgagcgg cttctgcaaa catatattta tataataacc  12480 

agcgcaaaac atataaaata tatgcgctgt tagattgtat atttttgaat gagggcggat  12540 

gagaatctgc ggacaaacct gattttactg acctgcaatt tgcggatt               12588 

 
           
             3  
             659  
             DNA  
             Agrobacterium tumefaciens  
           
            3 

ttttcaggta tccagatcaa caaggcactg tcgtcaatcg atgcccatca ggaaaccagc     60 

ggaagtggca ggattcagac gctgcgggtt gtcgcccgcc agaaaggcgc cgctgtccgg    120 

atcgatgctg tcttcaatat tcaggcagga cagatcgccg acaaagacgt catccgcaag    180 

ggcatctgcg acatcataaa aggcgcgtaa ggcatggctt aaagacactc cggtccagca    240 

gcacgagccg tcgtgcacag gaagccaatc tgtttcgcgc aggatgttac gaaatcgatg    300 

attgaagctt atcgctgacc gtatccgcgg gtagtctcca gtgttagata tgcgccgagc    360 

gaactcatca tgccgctacg ccaaagacgg tcattgatct cacgtgtttg gaccatattt    420 

tctgtgcaaa ctaaacgatg acatagggcg atttttagtg gcggacaaat acagacttcc    480 

cgaagagttt tttaccactc ggtttctcgt tagacgcatc gtacccacag acgctgaagc    540 

tattttcgaa gggtggaaca ccgatcccga ggtgacgaag tacctgacgt ggaaacccca    600 

ctccgagctt ggccagacac agcgggcgat tgaagaaaat tatagtgcgt ggaatgcag     659 

 
           
             4  
             787  
             DNA  
             Agrobacterium tumefaciens  
           
            4 

tactacttct tgagcttccg atttcgaagc tttgccctgg caaaaagaga tcgtgccagc     60 

cttctcctca tcagggcggt atctgcacga gtagcaagga cgtacatttc atagagaccc    120 

atttgaacac acctttccta tcacgcgacg cagctgcgac cgtgttgtga actccatctc    180 

tttttccatc agcctttcag ctcgatgggc attgcaacgg ggcactgaaa attgccgcct    240 

agggaaaata aagacactta tcagtgcccc gcatcagcgg tttatgcctg tcagagcgtg    300 

tccttcagcc aatgcgtttc ggagaaatag aaaggcccac cacaaaccag attccatctc    360 

tcctccggtc acggccagag gactttacct gtcagaccgc gaaatatgag agcgacgtct    420 

ttctctcaca gccggcgctg gttcagtttc aaaacaggaa tgtcttccta ttcatcgccg    480 

aacaaggaag aagtataaac cagagaaaaa gggtggggat gtgcgaggaa atgaaataaa    540 

cgaaataaac ccgtaaaaaa ctaactgcct acaccactat tcaaaagcca catcaaacga    600 

ctcgcccgat cgtaataaat ccaagagtct aaccaatgaa ccttcattgc ccctacggtc    660 

catccatttc ataacttcgc aagcagcagc gctgtatatg ccgacgtccc cgatgctagc    720 

gcgccgacac cactcatcga gatcggctat caccggcccg gtgacaccct ctttacacga    780 

aagtcta                                                              787

Technology Category: 8