Abstract:
Novel protein useful as a selectable marker resistant to the antibiotic nourseothricin and corresponding polynucleotides for insertion of genes and other genetic material into a variety of organisms, including plants are described.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to a novel protein useful as a selectable marker and corresponding polynucleotides for insertion of genes and other genetic material into a variety of organisms, including plants.  
         BACKGROUND OF THE INVENTION  
         [0002]    Selectable markers are genes that impart a characteristic to an organism to see the results of a biochemical or chemical assay or test. Such markers are known as labels. One of the basic principles of recombinant DNA technology is the use of biological markers to identify cells carrying recombinant DNA molecules. In bacteria, these are commonly drug resistance genes. In bacteria, drug resistance is used to select bacteria that have taken up cloned DNA from the much larger population of bacteria that have not. For example, a commonly used marker in mammalian cells is a bacterial drug-resistance gene that confers resistance to a neomycin-related drug, G418, which kills mammalian cells by blocking protein synthesis. The marker gene encodes an enzyme that destroys the drug. Although numerous markers exist for bacterial and mammalian cells, fewer gene markers are available for organisms such as plants. It would be desirable to provide a gene marker that could enable one to differentiate between plants that carry particular recombinant DNA molecules from plant that do not.  
         SUMMARY OF THE INVENTION  
         [0003]    In one embodiment, the present invention is directed towards a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1. The nucleic acid sequence may be selected from the group consisting of SEQ ID NOS. 2, 3 and 4.  
           [0004]    In another embodiment, the present invention is directed toward a DNA construct comprising a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1.  
           [0005]    In another embodiment, the present invention is directed towards a plasmid comprising a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1.  
           [0006]    In another embodiment, the present invention is directed towards a eukaryotic cell comprising a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1. The eukaryotic cell can be a plant cell, such as a dicot plant cell or a monocot plant cell.  
           [0007]    In another embodiment, the present invention is directed toward a plant or plant part having a eukaryotic cell comprising a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1.  
           [0008]    In another embodiment, the present invention is directed toward seed that can produce a plant comprising a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1. The present invention is also directed towards seed from the plant of this embodiment.  
           [0009]    In another embodiment, the present invention is directed toward a method of conferring resistance to the antibiotic nourseothricin, comprising providing to an organism t a nucleic acid sequence comprising a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID. NO. 1.  
           [0010]    In another embodiment, the present invention is directed toward a protein either comprising or consisting of the amino acid sequence of SEQ ID. NO. 1. The protein can be in an isolated or non-isolated form.  
           [0011]    In another embodiment, the present invention is directed towards a eukaryotic cell that can express a protein either comprising or consisting of the amino acid sequence of SEQ ID. NO. 1.  
           [0012]    In another embodiment, the present invention a plant or plant part having a eukaryotic cell that can express a protein either comprising or consisting of the amino acid sequence of SEQ ID. NO. 1.  
           [0013]    In another embodiment, the present invention is directed towards seed that can produce a plant comprising a protein either comprising or consisting of the amino acid sequence of SEQ ID. NO. 1.  
           [0014]    In any of the above embodiments, the eukaryotic cells, plant or plant part can be from an organism such as a microorganism or a plant, such as a dicot plant, e.g.  Arabidopsis thaliana  or a monocot plant, e.g.  Oryza sativa.    
         DETAILED DESCRIPTION OF THE INVENTION  
         [0015]    This invention describes the use of a novel nourseothricin N-acetyltransferase (NRG) with the aminoacid sequence SEQ. ID No. 1, encoded by a novel nucleotide sequence as exemplified, but not limited to SEQ. ID Nos. 2, 3 and 4, useful as a selectable marker in an organism such as microorganisms and plants. The conditions for its use as selectable marker with rice and  Arabidopsis thaliana  are described herein.  
           [0016]    Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.  
           [0017]    Ag7 term—the Ag7 terminator sequence is a sequence of 213 nucleotides from the 3′ end of the gene number 7 from  Agrobacterium tumefaciens.  The sequence is derived from plasmid vector pGPTV-HPT as described by Becker et al. in 1992 (Plant Mol Biol 20:1195-1197).  
           [0018]    aph4—selectable marker gene for hygromycin resistance. The sequence is derived from plasmid vector pGPTV-HPT as described by Becker et al. in 1992 (Plant Mol Biol 20:1195-1197).  
           [0019]    AvrII refers to a restriction enzyme site.  
           [0020]    “clonNAT” is the dihydrogen sulphate of the weakly basic antibiotic nourseothricin, consisting of streptothricin components F and D. The chemical name is 2-[4-O-Carbamoyl-2-deoxy-2-(3,6-diaminohexan-amido)-B-D-gulopyranoslamino)-3,3a,5,6,7,7a-hexahydro-5-hydroxy-4H-imidazo[4,5-c]pyridin-4-one dihydrogensulphate. The structural formula is set forth as follows:  
                         
 
           [0021]    GUS Intron refers to the GUS marker gene containing an intron, as disclosed in plamids pPG361 and pPG363.  
           [0022]    HindIII refers to a restriction enzyme site.  
           [0023]    nos prom—a promoter sequence from the nopaline synthase gene of  Agrobacterium tumefaciens,  as disclosed in plasmids pPG354, pPG361, pPG362, pPG363.  
           [0024]    nos term—a terminator sequence from the nopaline synthase gene of  Agrobacterium tumefaciens,  as disclosed in plasmids pPG354, pPG361, pPG362, pPG363.  
           [0025]    “Nourseothricin” refers to the streptothricin antibiotic components F and D, produced in cultures from a strain of  Streptomyces noursei.    
           [0026]    “nrg gene” refers to a generic group of nucleic acid sequence that can encode for the NRG protein. Selected species of the genus include, but are not limited to, nrg1, nrg2 and nrg3 described herein.  
           [0027]    “NRG protein” or “Nourseothricin Resistance Gene protein” refers to the polypeptide or amino acid sequence of SEQ ID. NO. 1. This protein has the ability to confer resistance to the antibiotic known as nourseothricin.  
           [0028]    35S prom—a promoter sequence from the genome of cauliflower mosaic virus, as disclosed in plasmids pPG361 and pPG363.  
           [0029]    ocs LB or “Left Border” refers to the DNA sequence that flanks the “left end” of the T-DNA and is disclosed in pPG361 and pPG363. The ocs LB is derived from octopine synthase (ocs) tumor inducing plasmids of  Agrobacterium tumefaciens.    
           [0030]    ocs RB or “Right Border” refers to the DNA sequence that flanks the “right end” of the T-DNA and is disclosed in pPG361 and pPG363. The ocs LB is derived from octopine synthase (ocs) tumor inducing plasmids of  Agrobacterium tumefaciens.  The osc RB and osc LB are recognized by Agrobacterium as sites for “cutting” or excision to enable the T-DNA to be inserted into a plant cell.  
           [0031]    PmII refers to a restriction enzyme site.  
           [0032]    The microorganism can be, for example, a fungus or bacteria. Where the organism is a fungus, the fungus can be from, but not limited to, any of the following genera: Magnaporthe, Mycosphaerella, Candida, Botrytis, Saccharomyces, Aspergillus, Peronaspora, Sclerotinia, Rhizoctonia, Phythium, Puccinia, Erysiphe, Ustilago, Fusarium, Phytophthora and Penicillium. Where the organism is a bacteria, the bacteria can be from Agrobacterium, Escherichia, Xanthomonas, Staphlococcus, Pseudomonas, Streptomyces and Bacillus.  
           [0033]    The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and is not intended to limit the scope of the invention. 
       
    
    
     EXAMPLE 1  
     Preparation of the nrg Gene and Protein  
       [0034]    Using the nat1 gene as the starting material from the plasmid pINS1, as delivered from the Hans Knoell Institute, the nrg gene is obtained in a PCR reaction, using the following primers:  
         [0035]    forward primer: 5′nat1 HindIII; ccgaagcttATGACCACTCTTGACGACACG [SEQ ID NO. 5] 
         [0036]    reverse primer: 3′nat1 AvrII; aaccctaggCTAGGGGCAGGGCATGCTCATG [SEQ ID NO. 6] 
         [0037]    These primers add a HindIII or a AvrII restriction site right upstream, or downstream of the coding region of the nat1 gene, respectively. The PCR products of two independent reactions are cloned into a pUC vector derived plasmid (pPG354, digested with HindIII and AvrII), adding a nos promoter and a nos terminator upstream, and downstream of the nat1 gene, respectively. The resulting plasmid is called pPG362. The resultant gene is sequenced in the resulting plasmid pPG362 [SEQ ID NO. 7], sequencing three plasmid clones, derived from two independent PCR reactions. Surprisingly and unexpectedly, the sequence of all three clones yields the following new resultant gene, hereafter called the nrg2 gene (SEQ ID NO. 3), with following three nucleotide changes: the nucleotide cytosine (“C’) is replaced with adenine (“A”) at position at position 209, the nucleotide guanine (“G”) is replaced with guanine (“G”) adenine (“A”) at position 569 and the nucleotide adenine (“A”) is replaced with guanine (“G”) at position 570. Also surprisingly and unexpectedly, in the resultant NRG protein, the amino acid, glycine (A), is replaced with arginine (D) at position 70 in the protein or polypeptide sequence. Also surprisingly and unexpectedly, the new NRG protein retains the ability to impart resistance to the nourseothricin antibiotic.  
       EXAMPLE 2  
     Cloning the nrg gene into a binary vector  
       [0038]    The nrg gene, controlled by the nos promoter and nos terminator elements from pPG362, is further cloned as a PmII fragment into the PmII sites of the binary vector pPG361, replacing the aph4 expression cassette, to give the new vector, pPG363, a binary vector containing the nrg expression cassette. In addition to the nrg expression cassette, the pPG363 plasmid [SEQ ID NO. 8] contains pRi Agrobacterium elements, ColEI elements for replication in  E. coli,  a kanamycin resistance gene, octopine-type left and right T-DNA border elements, and a GUS gene with an intron, controlled by the CaMV 35S promoter and the Ag7 terminator, as presented below. The description of the plasmid pPG363 is provided below and as SEQ ID NO. 8: 
         
 
         [0039]    wherein the ocs LB, PmI I, nos prom, nrg, nos term, 35S prom, GUS Intron, Ag7 term, ocs RB are known in the art or described herein. The numbers in parentheses indicate the nucleotide position within the plasmid at which the respective restriction enzymes cut the plasmid DNA.  
       EXAMPLE 3  
     Rice Transformation Using a Binary Vector with the nrg Marker Gene  
       [0040]    a) Dose response. For selection of transgenic calli based on resistance to clonNAT following Agrobacterium-mediated transformation of rice callus material, a dose response experiment is made to determine the concentration of clonNAT in callus growth medium in order to inhibit the growth of rice callus, or to kill the rice cells. Growth media (GM) used for rice callus comprise the following basic components: N6 salts (Duchefa) 3.95 g/l; B5 vitamins (Duchefa) 112 mg/l; proline (Duchefa) 500 mg/l; glutamine (Duchefa) 500 mg/l; casein hydrolysate (Duchefa) 500 mg/l. For the dose response experiment, medium plates are prepared that contain, apart from the basic components (GM), the following components: 2,4-D (2,4-dichlorophenoxyacetic acid; Duchefa) 2 mg/l; maltose (Sigma) 30 g/l; cefotaxime 200 mg/l; agarose (type I, Sigma) 5 g/l; pH 5.6. Medium plates are prepared with 25 ml of filter-sterilized medium per 10 cm petridishes. Variable amounts of a clonNAT stock solution of 200 mg/ml in water is added to these plates before filter-sterilization to obtain medium plates with clonNAT concentrations of 0, 5, 20, 100, 500, 1000 mg/l. Several pieces of rice callus, derived from immature rice embryos of the variety TP309 (National Small Grain Collection, USDA, ARS, Aberdeen, Id.) are put on these plates, the plates are incubated at 26° C. in the dark, and survival of the embryos and proliferation of the embryo cells are observed every day. Based on the survival rate of the embryos on the media plates containing different concentrations of clonNAT, a useful concentration of clonNAT for selection of transgenic rice is determined to be within a range of about 20 to about 1000 mg/l or more, preferably about 200 mg/l or less.  
         [0041]    b) Agrobacterium Transformation. For Agrobacterium-mediated rice transformation, the pPG363 plasmid is electroporated into electroporation-competent Agrobacterium cells of the strain LBA4404 (Life Technologies) and the electroporated cells are plated on LB medium (10 g/l Bactopeptone (Difco), 5 g/l Yeast Extract (Difco), 5 g/l NaCl, 15 g/l Bactoagar (Difco), 50 mg/l kanamycin; pH 7.0) and are incubated at 30° C. Two days after electroporation, a colony is picked from the LB plate and is used to inoculate 5 ml of liquid LB medium (LB medium without Bactoagar). The 5 ml culture is incubated at 30° C. on a shaker at 200 rpm. After 16 hours, 0.05 ml of this 5 ml culture is transferred to 100 ml of liquid LB medium and is then incubated at 30° C. on a shaker at 200 rpm. After 16 hours, the bacteria cells are spun down by centrifugation at 3000 rpm, resuspended in 100 ml of induction medium (GM basic medium with 2,4-D 2 mg/l; 10 g/l glucose; 120 g/l maltose; pH 5.2), and incubated at room temperature on a shaker at 100 rpm. After 1 hour, rice immature embryos that are cultured on GM plates for 6 days after isolation are immersed in the bacteria suspension. After 20 minutes, the embryos are transferred to cocultivation medium plates (GM basic medium with 2,4-D 2 mg/l; 10 g/l glucose; 120 g/l maltose; 50 g/l agarose; pH 5.2) and are incubated for 3 days at 24° C. in the dark. The cultivated embryos are transferred to growth medium (GM) plates (GM basic medium with 2,4-D 2 mg/l; maltose 30 g/l; cefotaxime 400 mg/l; agarose 5 g/l; pH 5.6) and incubated at 26° C. in the dark. After 5 days the cultivated embryos are transferred to a selection medium, known as clonNAT200 selection plates (GM basic medium with 2,4-D 2 mg/l; maltose 30 g/l; cefotaxime 200 mg/l; clonNAT 200 mg/l; agarose 5 g/l; pH 5.6) and are incubated at 26° C. in the dark. After 4-5 weeks, colonies of clonNAT-resistant callus are growing from the pieces of embryo-derived callus that died on the clonNAT-containing medium plates. These resistant callus colonies are picked from these plates and are transferred to fresh selection medium with increased maltose concentration (clonNAT200 6%M, GM basic medium with 2,4-D 2 mg/l; maltose 60 g/l; cefotaxime 200 mg/l; clonNAT 200 mg/l; agarose 5 g/l; pH 5.6). Small parts of the isolated callus colonies are used in a histological GUS assay to test for GUS activity. Positive GUS staining is a direct indication that these callus pieces are transgenic (and have been selected on clonNAT selection) with the nrg resistance gene. After 10 days, the resistant calluses are transferred to fresh medium of same composition to increase the callus mass. After 1 week, the callus is transferred to regeneration medium (GM basic medium with maltose 20 g/l; sorbitol 30 g/l; NAA (naphtalene acetic acid) 0.5 mg/l; BAP (6-benzylaminopurine) 3 mg/l; agarose 8 g/l; pH 5.6) and these plates are incubated at 25° C. under 16 hours light. After 4 weeks, small regenerated plantlets are then transferred to rooting medium (½-strength MS (Murashige &amp; Skoog) medium (micro and macro elements and vitamins), 2% sucrose, 0.15% phytagel (Sigma); pH 5.6) and grown to a height of 5-10 cm. Such plants are transferred to soil and are grown to maturity in the greenhouse. The transgenic state of these plants is tested by performing a histological GUS assay with leaf tissue, and a Southern analysis with plant genomic DNA probing with the nrg resistance gene.  
       EXAMPLE 4  
       Arabidopsis thaliana  Transformation with the nrg Marker Gene  
       [0042]    a) Dose response. The selection conditions for plants that are transgenic with the nrg gene are determined in a dose response experiment. From about 2000 to 3000 wildtype  Arabidopsis thaliana  seeds per pot are sown in 5″×5″ (13 cm×13 cm) soil pots. When the first true leaves of the seedlings have emerged after approximately 7 days, the seedlings are sprayed with a hand-held sprayer until all leaf material is completely wet on three consecutive days with a solution of 0.005% Silwet L-77 (50 ul/l) in water and variable concentrations of clonNAT (1, 5, 10, 20, 50, 100, 250, 500 mg/l). The results are assessed 36 hours after the last spray. A useful range of concentration of clonNAT for selection of transgenic Arabidopsis plants (as assessed as concentrations that kill non-transgenic Arabidopsis plants after applying the described sprayings) is determined within a range of about 20 to about 1000 mg/l or more, preferably about 200 mg/l or less.  
         [0043]    b)  Arabidopsis thaliana  transformation. The binary plasmid pPG363 is transformed into  Agrobacterium tumefaciens  strain, GV3101, and the transformed cells are plated on LB medium (10 g/l Bactopeptone (Difco), 5 g/l Yeast Extract (Difco), 5 g/l NaCl, 15 g/l Bactoagar (Difco), 50 mg/l kanamycin; pH 7.0) and are incubated at 30° C. Two days after transformation, a colony is picked from the LB plate and is used to inoculate 5 ml of liquid LB medium (LB medium without Bactoagar). The 5 ml culture is incubated at 30° C. on a shaker at 200 rpm. After 16 hours, 0.05 ml of this 5 ml culture is transferred to 100 ml of liquid LB medium and is then incubated at 30° C. on a shaker at 200 rpm. For Agrobacterium-mediated transformation of the T-DNA containing the nrg gene and the GUS gene from pPG363 to  Arabidopsis thaliana  via a flower dipping protocol (Clough S J, Bent A F, Floral dip: a simplified method for Agrobacterium-mediated transformation of  Arabidopsis thaliana.  Plant J 1998 Dec;16(6):735-43), the primary bolts of about five-weeks old  Arabidopsis thaliana  plants are removed. Five days later, subsequently emerged secondary bolts have grown. The leaves and bolts of these plants are dipped or submerged for five minutes in a suspension, consisting of 30 ml of an over-night culture of  Agrobacterium tumefaciens  (ecotype GV3101, containing the binary vector pPG363) in LB medium, diluted 3-fold with a 5% sucrose solution containing 0.005% Silwet L-77. The dipped plants are then kept for over-night in the dark at 22° C. and are then transferred back to a location in the growth room were they grow to maturity under normal growth conditions. The T1 seed is harvested and then used for selection. 2000-3000 seeds are sown on a 5″×5″ pot filled with soil and are stratified for 2 days at 4° C. The pots are then transferred to the growth room with 16 hours light and 22° C. The seedlings are sprayed with a solution containing 0.005% Silwet L-77 and 200 mg/l clonNAT on three consecutive days when the first true leaves have emerged. Surviving seedlings are transferred to individual pots one week after selection and are grown to maturity under normal growth conditions. The transgenic state of the plants and their progenies is tested by performing a histological GUS assay using leaf tissue and by performing Southern analysis using genomic DNA isolated from the Arabidopsis plants and probing the DNA blots with a labelled probe from the nrg gene.  
       Preparation of Starting Materials  
       [0044]    Origin, cloning, and sequence of the starting materials for preparing the nrg gene. The nat1 gene starting material is obtained from the Hans Knoell Institute, Jena, Germany, in plasmid pINS1. The sequence of the nat1 gene is described as the sequence X73149.1 (emb|X73149.|SNNAT1  S. noursei  gene for nourseothricin acetyltransferase) found in the database at the National Center for Biotechnology Information (NCBI).  
         [0045]    The binary vector pPG363 (SEQ ID. NO. 8) can be prepared from the vectors pPG361 (SEQ ID NO 9) and pPG362 (SEQ ID NO. 7).  
         [0046]    Methods for preparing DNA constructs or plasmids is known in the art. See, e.g., Sambrook et al.,  Molecular Cloning: A Laboratory Manual  (1989). The description of the plasmid pPG361 is provided below and as SEQ ID NO 9: 
         
 
         [0047]    wherein ocs LB, nos term, aph4, nos prom, 35S prom, GUS Intron, Ag7 term, ocs RB, and PmII are known in the art or as described herein. The numbers in parentheses indicate the nucleotide position within the plasmid at which the respective restriction enzymes cut the plasmid DNA.  
         [0048]    The description of the plasmid pPG362 is provided below and as SEQ ID NO 7: 
         
 
         [0049]    wherein the nos prom, the nrg gene, the Nos term, and PmII are known in the art or described herein. The numbers in parentheses indicate the nucleotide position within the plasmid at which the respective restriction enzymes cut the plasmid DNA.  
         [0050]    Plasmid pPG362 (SEQ ID NO 7) can be prepared from plasmid pPG354 and the nrg gene, as derived in a PCR reaction, by cloning the nrg gene PCR product as HindIII, AvrII-fragment into the HindIII and AvrII restriction sites of pPG354, replacing the aph4 gene.  
         [0051]    The description of the plasmid pPG354 is provided below and as SEQ ID NO 10: 
         
 
         [0052]    wherein nos term, aph4, nos prom, PmII, HindIII, and AvrII are as known in the art or as described herein. The numbers in parentheses indicate the nucleotide position within the plasmid at which the respective restriction enzymes cut the plasmid DNA.  
     
       
       
         1 
         
           
             10  
           
           
             1  
             189  
             PRT  
             Artificial Sequence  
             
               Novel protein conferring resistance to the 
      antibiotic known as nourseothricin  
             
           
            1 

Met Thr Thr Leu Asp Asp Thr Ala Tyr Arg Tyr Arg Thr Ser Val Pro 
 1               5                  10                  15 

Gly Asp Ala Glu Ala Ile Glu Ala Leu Asp Gly Ser Phe Thr Thr Asp 
            20                  25                  30 

Thr Val Phe Arg Val Thr Ala Thr Gly Asp Gly Phe Thr Leu Arg Glu 
        35                  40                  45 

Val Pro Val Asp Pro Pro Leu Thr Lys Val Phe Pro Asp Asp Glu Ser 
    50                  55                  60 

Asp Asp Glu Ser Asp Asp Gly Glu Asp Gly Asp Pro Asp Ser Arg Thr 
65                  70                  75                  80 

Phe Val Ala Tyr Gly Asp Asp Gly Asp Leu Ala Gly Phe Val Val Val 
                85                  90                  95 

Ser Tyr Ser Gly Trp Asn Arg Arg Leu Thr Val Glu Asp Ile Glu Val 
            100                 105                 110 

Ala Pro Glu His Arg Gly His Gly Val Gly Arg Ala Leu Met Gly Leu 
        115                 120                 125 

Ala Thr Glu Phe Ala Arg Glu Arg Gly Ala Gly His Leu Trp Leu Glu 
    130                 135                 140 

Val Thr Asn Val Asn Ala Pro Ala Ile His Ala Tyr Arg Arg Met Gly 
145                 150                 155                 160 

Phe Thr Leu Cys Gly Leu Asp Thr Ala Leu Tyr Asp Gly Thr Ala Ser 
                165                 170                 175 

Asp Gly Glu Gln Ala Leu Tyr Met Ser Met Pro Cys Pro 
            180                 185 

 
           
             2  
             570  
             DNA  
             Artificial Sequence  
             
               nrg1 - codons optimized for NRG (GC content 
      46.84%)  
             
           
            2 

atgactactc ttgatgatac tgcttaccgt taccgtactt ctgttcctgg agatgctgag     60 

gctatcgagg ctcttgatgg atctttcact actgatactg ttttccgtgt tactgctact    120 

ggagatggat tcactcttcg tgaggttcct gttgatcctc ctcttactaa ggttttccct    180 

gatgatgagt ctgatgatga gtctgatgat ggagaggatg gagatcctga ttctcgtact    240 

ttcgttgctt acggagatga tggagatctt gctggattcg ttgttgtttc ttactctgga    300 

tggaaccgtc gtcttactgt tgaggatatc gaggttgctc ctgagcatcg tggacatgga    360 

gttggacgtg ctcttatggg acttgctact gagttcgctc gtgagcgtgg agctggacat    420 

ctttggcttg aggttactaa cgttaacgct cctgctatcc atgcttaccg tcgtatggga    480 

ttcactcttt gtggacttga tactgctctt tacgatggaa ctgcttctga tggagagcag    540 

gctctttaca tgtctatgcc ttgtccttga                                     570 

 
           
             3  
             570  
             DNA  
             Artificial Sequence  
             
               nrg2 - codons not optimized for NRG (GC content 
      70.70%), after PCR reaction  
             
           
            3 

atgaccactc ttgacgacac ggcttaccgg taccgcacca gtgtcccggg ggacgccgag     60 

gccatcgagg cactggatgg gtccttcacc accgacaccg tcttccgcgt caccgccacc    120 

ggggacggct tcaccctgcg ggaggtgccg gtggacccgc ccctgaccaa ggtgttcccc    180 

gacgacgaat cggacgacga atcggacgac ggggaggacg gcgacccgga ctcccggacg    240 

ttcgtcgcgt acggggacga cggcgacctg gcgggcttcg tggtcgtctc gtactccggc    300 

tggaaccgcc ggctgaccgt cgaggacatc gaggtcgccc cggagcaccg ggggcacggg    360 

gtcgggcgcg cgttgatggg gctcgcgacg gagttcgccc gcgagcgggg cgccgggcac    420 

ctctggctgg aggtcaccaa cgtcaacgca ccggcgatcc acgcgtaccg gcggatgggg    480 

ttcaccctct gcggcctgga caccgccctg tacgacggca ccgcctcgga cggcgagcag    540 

gcgctctaca tgagcatgcc ctgcccctag                                     570 

 
           
             4  
             570  
             DNA  
             Artificial Sequence  
             
               nrg3 - codons optimized for NRG (GC content 
      46.84%)  
             
           
            4 

atgactactc ttgatgatac tgcttaccgt taccgtactt ctgttcctgg agatgctgag     60 

gctatcgagg ctcttgatgg atctttcact actgatactg ttttccgtgt tactgctact    120 

ggagatggat tcactcttcg tgaggttcct gttgatcctc ctcttactaa ggttttccct    180 

gatgatgagt ctgatgatga gtctgatgat ggagaggatg gagatcctga ttctcgtact    240 

ttcgttgctt acggagatga tggagatctt gctggattcg ttgttgtttc ttactctgga    300 

tggaaccgtc gtcttactgt tgaggatatc gaggttgctc ctgagcatcg tggacatgga    360 

gttggacgtg ctcttatggg acttgctact gagttcgctc gtgagcgtgg agctggacat    420 

ctttggcttg aggttactaa cgttaacgct cctgctatcc atgcttaccg tcgtatggga    480 

ttcactcttt gtggacttga tactgctctt tacgatggaa ctgcttctga tggagagcag    540 

gctctttaca tgtctatgcc ttgtccttga                                     570 

 
           
             5  
             30  
             DNA  
             Artificial Sequence  
             
               forward primer 5′nat1HindIII  
             
           
            5 

ccgaagctta tgaccactct tgacgacacg                                      30 

 
           
             6  
             31  
             DNA  
             Artificial Sequence  
             
               reverse primer 3′nat1AvrII  
             
           
            6 

aaccctaggc taggggcagg gcatgctcat g                                    31 

 
           
             7  
             4201  
             DNA  
             Artificial Sequence  
             
               plasmid pPG362 with nrg2 gene  
             
           
            7 

gtgggggata aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt     60 

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga    120 

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat    180 

gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag    240 

tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga    300 

cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc    360 

cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg    420 

cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc    480 

aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca    540 

ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa    600 

aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt    660 

ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca    720 

gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag    780 

ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc    840 

ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca    900 

gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt    960 

aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct   1020 

gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt   1080 

aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga   1140 

caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact   1200 

tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc   1260 

acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga   1320 

gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt   1380 

agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga   1440 

gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact   1500 

ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga   1560 

taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt   1620 

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca   1680 

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct   1740 

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta   1800 

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct   1860 

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc   1920 

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca   1980 

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga   2040 

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg   2100 

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt   2160 

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag   2220 

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt   2280 

tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt   2340 

tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga   2400 

ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta   2460 

atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa   2520 

tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat   2580 

gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta   2640 

cgccaagcta taccccacgt gcgtacgctc gagtcacgct gccgcaagca ctcagggcgc   2700 

aagggctgct aaaggaagcg gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc   2760 

cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag   2820 

caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca   2880 

agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta   2940 

aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atcatgagcg   3000 

gagaattaag ggagtcacgt tatgaccccc gccgatgacg cgggacaagc cgttttacgt   3060 

ttggaactga cagaaccgca acgttgaagg agccactcag ccgcgggttt ctggagttta   3120 

atgagctaag cacatacgtc agaaaccatt attgcgcgtt caaaagtcgc ctaaggtcac   3180 

tatcagctag caaatatttc ttgtcaaaaa tgctccactg acgttccata aattcccctc   3240 

ggtatccaat tagagtctca tattcactct caatccgtat accatggcta agcttatgac   3300 

cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat   3360 

cgaggcactg gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg ccaccgggga   3420 

cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga   3480 

cgaatcggac gacgaatcgg acgacgggga ggacggcgac ccggactccc ggacgttcgt   3540 

cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa   3600 

ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg   3660 

gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggcgccg ggcacctctg   3720 

gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga tggggttcac   3780 

cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct   3840 

ctacatgagc atgccctgcc cctagcctag gcaactctcc tggcgcacca tcgtcggcta   3900 

cagcctcggg aattgctacc gagctcgaat ttccccgatc gttcaaacat ttggcaataa   3960 

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg   4020 

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt   4080 

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc   4140 

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggaggcctca   4200 

c                                                                   4201 

 
           
             8  
             11978  
             DNA  
             Artificial Sequence  
             
               plasmid pPG363 - binary vector with the nrg2 
      gene  
             
           
            8 

tcccgcttcg ccggcgttaa ctcaagcgat tagatgcact aagcacataa ttgctcacag     60 

ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata agccctacac    120 

aaattgggag atatatcatg catgaccaaa atcccttaac gtgagttttc gttccactga    180 

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta    240 

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa    300 

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact    360 

gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca    420 

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt    480 

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg    540 

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag    600 

cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta    660 

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat    720 

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg    780 

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc    840 

ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac    900 

cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc    960 

gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg   1020 

tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag   1080 

ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc   1140 

cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac   1200 

aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac   1260 

gcgcgaggca gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt   1320 

ccgcgccctg gtagagcccg ggcgggtgtt ctgtcgtctc gttgtacaac gaaatccatt   1380 

cccattccgc gctcaagatg gcttcccctc ggcagttcat cagggctaaa tcaatctagc   1440 

cgacttgtcc ggtgaaatgg gctgcactcc aacagaaaca atcaaacaaa catacacagc   1500 

gacttattca cacgagctca aattacaacg gtatatatcc tgccagtcag catcatcaca   1560 

ccaaaagtta ggcccgaata gtttgaaatt agaaagctcg caattgaggt ctacaggcca   1620 

aattcgctct tagccgtaca atattactca ccggtgcgat gccccccatc gtaggtgaag   1680 

gtggaaatta atgatccatc ttgtctagag gcgcgccagg cctccatctt gaaagaaata   1740 

tagtttaaat atttattgat aaaataagtc aggtattata gtccaagcaa aaacataatt   1800 

tattgatgca aagtttaaat tcagaaatat ttcaataact gattatatca gctggtacat   1860 

tgccgtagat gaaagactga gtgcgatatt atgtgtaata cataaattga tgatatagct   1920 

agcttagctc atcgggccta ggtcattgtt tgcctccctg ctgcggtttt tcaccgaagt   1980 

tcatgccagt ccagcgtttt tgcagcagaa aagccgccga cttcggtttg cggtcgcgag   2040 

tgaagatccc tttcttgtta ccgccaacgc gcaatatgcc ttgcgaggtc gcaaaatcgg   2100 

cgaaattcca tacctgttca ccgacgacgg cgctgacgcg atcaaagacg cggtgataca   2160 

tatccagcca tgcacactga tactcttcac tccacatgtc ggtgtacatt gagtgcagcc   2220 

cggctaacgt atccacgccg tattcggtga tgataatcgg ctgatgcagt ttctcctgcc   2280 

aggccagaag ttctttttcc agtaccttct ctgccgtttc caaatcgccg ctttggacat   2340 

accatccgta ataacggttc aggcacagca catcaaagag atcgctgatg gtatcggtgt   2400 

gagcgtcgca gaacattaca ttgacgcagg tgatcggacg cgtcgggtcg agtttacgcg   2460 

ttgcttccgc cagtggcgcg aaatattccc gtgcaccttg cggacgggta tccggttcgt   2520 

tggcaatact ccacatcacc acgcttgggt ggtttttgtc acgcgctatc agctctttaa   2580 

tcgcctgtaa gtgcgcttgc tgagtttccc cgttgactgc ctcttcgctg tacagttctt   2640 

tcggcttgtt gcccgcttcg aaaccaatgc ctaaagagag gttaaagccg acagcagcag   2700 

tttcatcaat caccacgatg ccatgttcat ctgcccagtc gagcatctct tcagcgtaag   2760 

ggtaatgcga ggtacggtag gagttggccc caatccagtc cattaatgcg tggtcgtgca   2820 

ccatcagcac gttatcgaat cctttgccac gcaagtccgc atcttcatga cgaccaaagc   2880 

cagtaaagta gaacggtttg tggttaatca ggaactgttc gcccttcact gccactgacc   2940 

ggatgccgac gcgaagcggg tagatatcac actctgtctg gcttttggct gtgacgcaca   3000 

gttcatagag ataaccttca cccggttgcc agaggtgcgg attcaccact tgcaaagtcc   3060 

cgctagtgcc ttgtccagtt gcaaccacct gttgatccgc atcacgcagt tcaacgctga   3120 

catcaccatt ggccaccacc tgccagtcaa cagacgcgtg gttacagtct tgcgcgacat   3180 

gcgtcaccac ggtgatatcg tccacccagg tgttcggcgt ggtgtagagc attacgctgc   3240 

gatggattcc ggcatagtta aagaaatcat ggaagtaaga ctgctttttc ttgccgtttt   3300 

cgtcggtaat caccattccc ggcgggatag tctgccagtt cagttcgttg ttcacacaaa   3360 

cggtgatacg tacacttttc ccggcaataa catacggcgt gacatcggct tcaaatggcg   3420 

tatagccgcc ctgatgctcc atcacttcct gattattgac ccacactttg ccgtaatgag   3480 

tgaccgcatc gaaacgcagc acgatacgct ggcctgccca acctttcggt ataaagactt   3540 

cgcgctgata ccagacgttg cccgcataat tacgaatatc tgcatcggcg aactgatcgt   3600 

taaaactgcc tggcacagca attgcccggc tttcttgtaa cgcgctttcc caccaacgct   3660 

gatcaattcc acagttttcg cgatccagac tgaatgccca caggccgtcg agttttttga   3720 

tttcccgggt tggggtttct acctgaatta atttaccacg gttaatactc agatcaagat   3780 

ggtaaaaaaa tggcggtaag attaatctgc acactgtaat taataatgta ccggacgtaa   3840 

catatgaagc ttagccatgg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct   3900 

tatatagagg aagggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg   3960 

gagatatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg   4020 

atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg   4080 

atagcctttc ctttatcgca atgatggcat ttgtagtgcc accttccttt tctactgtcc   4140 

ttttgatgaa gtgacagata ggatcgggaa ttaattcgga tccgtacggc gcgccgcgcc   4200 

atttaaatca cgtgaggcct cccgatctag taacatagat gacaccgcgc gcgataattt   4260 

atcctagttt gcgcgctata ttttgttttc tatcgcgtat taaatgtata attgcgggac   4320 

tctaatcata aaaacccatc tcataaataa cgtcatgcat tacatgttaa ttattacatg   4380 

cttaacgtaa ttcaacagaa attatatgat aatcatcgca agaccggcaa caggattcaa   4440 

tcttaagaaa ctttattgcc aaatgtttga acgatcgggg aaattcgagc tcggtagcaa   4500 

ttcccgaggc tgtagccgac gatggtgcgc caggagagtt gcctaggcta ggggcagggc   4560 

atgctcatgt agagcgcctg ctcgccgtcc gaggcggtgc cgtcgtacag ggcggtgtcc   4620 

aggccgcaga gggtgaaccc catccgccgg tacgcgtgga tcgccggtgc gttgacgttg   4680 

gtgacctcca gccagaggtg cccggcgccc cgctcgcggg cgaactccgt cgcgagcccc   4740 

atcaacgcgc gcccgacccc gtgcccccgg tgctccgggg cgacctcgat gtcctcgacg   4800 

gtcagccggc ggttccagcc ggagtacgag acgaccacga agcccgccag gtcgccgtcg   4860 

tccccgtacg cgacgaacgt ccgggagtcc gggtcgccgt cctccccgtc gtccgattcg   4920 

tcgtccgatt cgtcgtcggg gaacaccttg gtcaggggcg ggtccaccgg cacctcccgc   4980 

agggtgaagc cgtccccggt ggcggtgacg cggaagacgg tgtcggtggt gaaggaccca   5040 

tccagtgcct cgatggcctc ggcgtccccc gggacactgg tgcggtaccg gtaagccgtg   5100 

tcgtcaagag tggtcataag cttagccatg gtatacggat tgagagtgaa tatgagactc   5160 

taattggata ccgaggggaa tttatggaac gtcagtggag catttttgac aagaaatatt   5220 

tgctagctga tagtgacctt aggcgacttt tgaacgcgca ataatggttt ctgacgtatg   5280 

tgcttagctc attaaactcc agaaacccgc ggctgagtgg ctccttcaac gttgcggttc   5340 

tgtcagttcc aaacgtaaaa cggcttgtcc cgcgtcatcg gcgggggtca taacgtgact   5400 

cccttaattc tccgctcatg atcttgatcc cctgcgccat cagatccttg gcggcaagaa   5460 

agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa   5520 

ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact   5580 

gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct   5640 

gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt tccgcttcct   5700 

ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgac tcgagcgtac gcacgtgggt   5760 

cctattttat aataacgctg cggacatcta catttttgaa ttgaaaaaaa attggtaatt   5820 

actctttctt tttctccata ttgaccatca tactcattgc tgatccatgt agatttcccg   5880 

gacatgaagc catttacaat tgaatatatc ctgccgccgc tgccgctttg cacccggtgg   5940 

agcttgcatg ttggtttcta cgcagaactg agccggttag gcagataatt tccattgaga   6000 

actgagccat gtgcaccttc cccccaacac ggtgagcgac ggggcaacgg agtgatccac   6060 

atgggacttt taaacatcat ccgtcggatg gcgttgcgag agaagcagtc gatccgtgag   6120 

atcagccgac gcagcccggg ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac   6180 

caggcctgaa tcgccccatc atccagccag aaagtgaggg agccacggtt gatgagagct   6240 

ttgttgtagg tggaccagtt ggtgattttg aacttttgct ttgccacgga acggtctgcg   6300 

ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa aagttcgatt tattcaacaa   6360 

agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt   6420 

ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat   6480 

caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt   6540 

tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac   6600 

aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga   6660 

cgactgaatc cggtgagaat ggcaacagct tatgcatttc tttccagact tgttcaacag   6720 

gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg   6780 

attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa   6840 

tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag   6900 

gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg   6960 

catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc   7020 

agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca   7080 

gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc   7140 

cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc   7200 

gcggcctcga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt   7260 

ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac   7320 

atcagagatt ttgagacaca acgaagcttt ctgagccgcc gattttcctc ctcgagttgg   7380 

atgaactcgc cgagttcatc gtcaactgaa acagacacgg ccggattctg tgagacaggt   7440 

tgaaccgcag ctctcttcca ttgataatag gtctgaacgg aaatacccac gatcttaacg   7500 

gcgtccttca aggttgcgcc gccagcgacc tgagcttcga tttgaccgat cttctccagt   7560 

ttttctcggt tgctgaggcc gcgggttttc ggcttcacgg atttgaacga tcccgtgcgg   7620 

gctgtttcgg ctggtgcttt ctttgctctt ctacctctag gagcagccgg ctcaacttcg   7680 

gcagcagcag taccgtccgg cggattctgg atctcttcgt cagccattaa tcgtcctctg   7740 

tgtgggttat tgctttgtct gccagctcga tccaagagtc aacgtttgtg cctagggcag   7800 

taaataggca gtgctccgcg actacatgcc tcggccggca aaataccgcc gcatgtagag   7860 

caggctctcc ttcacgatca acgatcggca tggggccttc gtgcttgttg agtaatgtta   7920 

tcgctcccat cagagcacgc ttggtactcc gggaatcgga tggtctgtcg atcatccaaa   7980 

aaacgctcat gttttcaacc tattaggtct gtggtcagct gaccacagac catcctgctc   8040 

catactcgct aattctagcc aaaccgcaac gtcccctgcc cgctagcctt caagagcgcc   8100 

attatcatcg ggccaagtga aaacttcccg agctcgctcc gccgtgtcag atctcggaga   8160 

tagcccccgg gcgaattgat gaagttcgct cgctccaaaa tgcacgccat cgctgctgcc   8220 

gcattctccg gtcccattgc ctcacacgcg tcttggtaag ccgacgggct gacccccagc   8280 

atagaccgaa ccaccaccgc agccgacatg aggtcacgcc agctagcaac cgcaccgctc   8340 

ggcccataat tgccaatggt cgggcatgct ttcaggatca tcccgagggg gaacgctttt   8400 

atcggctcgc tccttgcccg gtctatttca ctcggcttag cgccctgctc cttttcagag   8460 

cgaggttcaa gttcattaac ggattcgggt tttgaattct gtatgtgctg ctcgctctgg   8520 

gcagcattgg tgctattatt ttctgaattg tctctaattt ccaaccggtt gattatctct   8580 

tcctggagca tccacatctc ttcgagaatt gactctacat cagcaagcgt cggggcgcgt   8640 

ggaattctac ccacaagttc cacatagact tcctcgacag cttgccagtc gccctccgct   8700 

ccctcttcca tagctgccgt aattagcttc cgaacgtccc gtcggcaaat cgtcagactt   8760 

tctttggcca tcctgaatgc tgctcgatcg gccatcacct gctgtgccat catcgctagc   8820 

tcttcggacc gcgcgagaag cggagacaaa tcgaagccaa acgcgcgctc gatctgacca   8880 

gcgccatcct tacgagcgta acgctttccg ttggcgctat ccttccggac gatcaagcct   8940 

gactccacga gcatggcgat gtgcctacgc aaagtcgcgc cagccatccc atgcgcccga   9000 

agggcaagct gagcattcga cgggaagacg atcagctgtg cctcctgacg caactccgtt   9060 

tccgggtgaa agctcaatag cgcatcaagg acggcaagac tgttggactg gattccaagt   9120 

agttccatgg ccgcggacgc gtctctaaag accttccact tgtccgctgt cttgccttgt   9180 

ttgatatcgg ccagcgccgt ctggcgccgc acaagcgcaa gcgtcattgg ccgccgcccg   9240 

aatggcgtcg ttacacttcc tgtctgcatc atctttcacc tttcagcagg caaaggaaat   9300 

cagctcacca aaacggcgct aaaaactctt gacgaggatt cgaggaaatg cgattctgtt   9360 

cgcgctagag agacagaagg gcttccgcga cggcgacgtt gagggggctc ttttcttttg   9420 

cggtttactc tccccgtttc cgttggttct cagcgtggta cgcttgatac agcgctggca   9480 

catgatcgag cacgaaggtc gcaaaatcgg gcgtcgcctt cctgtcaatc gtgatttcca   9540 

gtttggcctt gctctgcgtc acctgtgcaa ttctggtgcc gtctggggtg gccatgacct   9600 

cgggaagtcc acgcgcaacc cgactgggct tcagactagc gatcaccgcc ttgaatcgtt   9660 

ctgccgatgg cagcgcttga acttcctccg acatagcata tttagccacg tcggccggtg   9720 

aagaaacttt ctcaatcagc tcggcaagtt gttgccaact cggccgtcca acaccaggag   9780 

cggcaccaat agcatcggtc agttcagagg ggagggcgtc gacgagcaga agcatcttgg   9840 

acaaattgct cttgtcgatc gacatcgcgg cgatgacaat ctctcgagaa aactgcctgt   9900 

tcaggcgatg tgcgaagcgc gccttttcga tgaaggtaag atcttcgcgc tcattgtttt   9960 

cctgaccctg tgctacgacc acttgctcgt ccgtcagttc gcgaacgacc gctctgaccg  10020 

gaagtccgag ttctgaaacg gcgcgtagcc ggcggtggcc gaaggcaacc tgatatcggc  10080 

ccggctggct cggatgcggt cgcacaagga ttgggacttg ctgtccttgt tcccggatcg  10140 

aagtaaggag cccgtcaatg tcccctcgca tacgatcctg cacgaaagac ggttctattg  10200 

acgaggcatc caactctatc actgcctgac cttcagcgag acgccgctcg atctcttcgg  10260 

cacggctaag acgatcgttt tgctctcgca gtgcgttacc aatgttcgct gtgagcttcg  10320 

ttgccggatc gcgctccttc cttgttacgc cgaggagcgg catggagcgg ttctttgccg  10380 

tcctattgtc ggcgggcgac gtctcagggg cgtcagttga gacgccaagg atgtgcttcc  10440 

ggctcatgtg ggcctacccc atgctttttt gatcagtgtt tcgatctcgt cgttgacggc  10500 

gttcatcgcc tccaaggctc gatcataggt cgagcgcgtg aacaggccac gctccacttc  10560 

gaatagagtc tggtttgtca ggccagcgtc cgaaaccgcg gtggttttaa gcatcggaaa  10620 

attgaggaca ttttcgccaa aaatcgaccg cagataacct accatttggt tctgtggtcc  10680 

gtcgctcggt tcgaaacggg ttatcagata gcgcatccaa ttaaacttga acttggcgcc  10740 

agcattctcg atttcacgca aaaggttcga tgtcattgcc agaaactggt tcatcgacat  10800 

cacatccagc atctgcggat ggaccgtgac aagaatggac gtcgccgcag tcaatgcgga  10860 

tagcgtgaga tacccaagct ggggagggca gtcgatgacc acgacgtcat agttatccgc  10920 

gatatcttca attacttggc tgatgcgacc ataaaagagc gtgtcgccct ctttgcggtt  10980 

catcagcgcg cgtggcgtat cgtgttcaaa ctccatcagc tcaaggttac caggaatcag  11040 

gtggaggtcg ggaatgtaag tccctcggac gactcgttcg attgccacct gctcatcatc  11100 

ataccttata gcgccgtaga gcgtttcgtt cgggccaacg tccgtctccg gttggctccc  11160 

aaagagtgca gaaaggctcg cttgaggatc gagatcaatg gccaagactc gatatccgcg  11220 

catagcgagg tactgcgcca gatgcgcggc ggtggtggtc ttacccgacc cacctttgaa  11280 

attcatcaca gagataacct gaagctgctc gccgcctcga cgatgtggca ggtagcgccg  11340 

gttcccgcgg ccgacctgat ccatatactt ccgaatcaca tggatatctt caattgagaa  11400 

cattcgcctg ccacccgggc tcatgctaac attcaactct ggcatctcag acgcggtctg  11460 

ccgtaaatat gactcgccaa cgccgagcag cttggacgcc tccgatggcc cgaatgttcg  11520 

aatgcccttc tcggaatgcg gcgggaaaac cttaagatga tgtgcttgaa gttggctcga  11580 

gagggcatcg gcatgacgct ccatcaaggc cgtcaaccct acaactacag gcgctgcttt  11640 

taggacagac ttcgccatct caaacccatt ccttgccagt ggcgatattt ttcgcgaaac  11700 

tggaaaagtt ccgccgctgg caattagcgc cgattctgct gtttgggcaa gagcttttag  11760 

gttaacagaa ggttaacgcc ctcaggtcga aaaactccac ccaactgtta tttgtattta  11820 

tttccaatgc cttagagaga ttgccatttg aatatgttca tgtattgttt tagtgataat  11880 

cctacaatcg taacccaaaa agaggtcgcc ctctgcgcgc cgtcgtccaa tataggcgaa  11940 

gtcacccttg cgactcaggc ggattctacc ttgtagga                          11978 

 
           
             9  
             12438  
             DNA  
             Artificial Sequence  
             
               Plasmid pPG361  
             
           
            9 

tcccgcttcg ccggcgttaa ctcaagcgat tagatgcact aagcacataa ttgctcacag     60 

ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata agccctacac    120 

aaattgggag atatatcatg catgaccaaa atcccttaac gtgagttttc gttccactga    180 

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta    240 

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa    300 

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact    360 

gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca    420 

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt    480 

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg    540 

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag    600 

cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta    660 

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat    720 

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg    780 

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc    840 

ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac    900 

cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc    960 

gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg   1020 

tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag   1080 

ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc   1140 

cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac   1200 

aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac   1260 

gcgcgaggca gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt   1320 

ccgcgccctg gtagagcccg ggcgggtgtt ctgtcgtctc gttgtacaac gaaatccatt   1380 

cccattccgc gctcaagatg gcttcccctc ggcagttcat cagggctaaa tcaatctagc   1440 

cgacttgtcc ggtgaaatgg gctgcactcc aacagaaaca atcaaacaaa catacacagc   1500 

gacttattca cacgagctca aattacaacg gtatatatcc tgccagtcag catcatcaca   1560 

ccaaaagtta ggcccgaata gtttgaaatt agaaagctcg caattgaggt ctacaggcca   1620 

aattcgctct tagccgtaca atattactca ccggtgcgat gccccccatc gtaggtgaag   1680 

gtggaaatta atgatccatc ttgtctagag gcgcgccagg cctccatctt gaaagaaata   1740 

tagtttaaat atttattgat aaaataagtc aggtattata gtccaagcaa aaacataatt   1800 

tattgatgca aagtttaaat tcagaaatat ttcaataact gattatatca gctggtacat   1860 

tgccgtagat gaaagactga gtgcgatatt atgtgtaata cataaattga tgatatagct   1920 

agcttagctc atcgggccta ggtcattgtt tgcctccctg ctgcggtttt tcaccgaagt   1980 

tcatgccagt ccagcgtttt tgcagcagaa aagccgccga cttcggtttg cggtcgcgag   2040 

tgaagatccc tttcttgtta ccgccaacgc gcaatatgcc ttgcgaggtc gcaaaatcgg   2100 

cgaaattcca tacctgttca ccgacgacgg cgctgacgcg atcaaagacg cggtgataca   2160 

tatccagcca tgcacactga tactcttcac tccacatgtc ggtgtacatt gagtgcagcc   2220 

cggctaacgt atccacgccg tattcggtga tgataatcgg ctgatgcagt ttctcctgcc   2280 

aggccagaag ttctttttcc agtaccttct ctgccgtttc caaatcgccg ctttggacat   2340 

accatccgta ataacggttc aggcacagca catcaaagag atcgctgatg gtatcggtgt   2400 

gagcgtcgca gaacattaca ttgacgcagg tgatcggacg cgtcgggtcg agtttacgcg   2460 

ttgcttccgc cagtggcgcg aaatattccc gtgcaccttg cggacgggta tccggttcgt   2520 

tggcaatact ccacatcacc acgcttgggt ggtttttgtc acgcgctatc agctctttaa   2580 

tcgcctgtaa gtgcgcttgc tgagtttccc cgttgactgc ctcttcgctg tacagttctt   2640 

tcggcttgtt gcccgcttcg aaaccaatgc ctaaagagag gttaaagccg acagcagcag   2700 

tttcatcaat caccacgatg ccatgttcat ctgcccagtc gagcatctct tcagcgtaag   2760 

ggtaatgcga ggtacggtag gagttggccc caatccagtc cattaatgcg tggtcgtgca   2820 

ccatcagcac gttatcgaat cctttgccac gcaagtccgc atcttcatga cgaccaaagc   2880 

cagtaaagta gaacggtttg tggttaatca ggaactgttc gcccttcact gccactgacc   2940 

ggatgccgac gcgaagcggg tagatatcac actctgtctg gcttttggct gtgacgcaca   3000 

gttcatagag ataaccttca cccggttgcc agaggtgcgg attcaccact tgcaaagtcc   3060 

cgctagtgcc ttgtccagtt gcaaccacct gttgatccgc atcacgcagt tcaacgctga   3120 

catcaccatt ggccaccacc tgccagtcaa cagacgcgtg gttacagtct tgcgcgacat   3180 

gcgtcaccac ggtgatatcg tccacccagg tgttcggcgt ggtgtagagc attacgctgc   3240 

gatggattcc ggcatagtta aagaaatcat ggaagtaaga ctgctttttc ttgccgtttt   3300 

cgtcggtaat caccattccc ggcgggatag tctgccagtt cagttcgttg ttcacacaaa   3360 

cggtgatacg tacacttttc ccggcaataa catacggcgt gacatcggct tcaaatggcg   3420 

tatagccgcc ctgatgctcc atcacttcct gattattgac ccacactttg ccgtaatgag   3480 

tgaccgcatc gaaacgcagc acgatacgct ggcctgccca acctttcggt ataaagactt   3540 

cgcgctgata ccagacgttg cccgcataat tacgaatatc tgcatcggcg aactgatcgt   3600 

taaaactgcc tggcacagca attgcccggc tttcttgtaa cgcgctttcc caccaacgct   3660 

gatcaattcc acagttttcg cgatccagac tgaatgccca caggccgtcg agttttttga   3720 

tttcccgggt tggggtttct acctgaatta atttaccacg gttaatactc agatcaagat   3780 

ggtaaaaaaa tggcggtaag attaatctgc acactgtaat taataatgta ccggacgtaa   3840 

catatgaagc ttagccatgg gtgatttcag cgtgtcctct ccaaatgaaa tgaacttcct   3900 

tatatagagg aagggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg   3960 

gagatatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg   4020 

atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg   4080 

atagcctttc ctttatcgca atgatggcat ttgtagtgcc accttccttt tctactgtcc   4140 

ttttgatgaa gtgacagata ggatcgggaa ttaattcgga tccgtacggc gcgccgcgcc   4200 

atttaaatca cgtgcgtacg ctcgagtcac gctgccgcaa gcactcaggg cgcaagggct   4260 

gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga   4320 

atgtcagcta ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag   4380 

cttgcagtgg gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac   4440 

cggaattgcc agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga   4500 

tggctttctt gccgccaagg atctgatggc gcaggggatc aagatcatga gcggagaatt   4560 

aagggagtca cgttatgacc cccgccgatg acgcgggaca agccgtttta cgtttggaac   4620 

tgacagaacc gcaacgttga aggagccact cagccgcggg tttctggagt ttaatgagct   4680 

aagcacatac gtcagaaacc attattgcgc gttcaaaagt cgcctaaggt cactatcagc   4740 

tagcaaatat ttcttgtcaa aaatgctcca ctgacgttcc ataaattccc ctcggtatcc   4800 

aattagagtc tcatattcac tctcaatccg tataccatgg ctaagcttat gaaaaagcct   4860 

gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac   4920 

ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt   4980 

ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat   5040 

cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggcattcagc   5100 

gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct   5160 

gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg   5220 

gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac   5280 

actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact   5340 

gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg   5400 

gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc   5460 

ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat   5520 

tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag   5580 

cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg   5640 

tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat   5700 

gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc   5760 

gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta   5820 

ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagaattcc   5880 

taggcaactc tcctggcgca ccatcgtcgg ctacagcctc gggaattgct accgagctcg   5940 

aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc   6000 

ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac   6060 

atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac   6120 

atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg   6180 

gtgtcatcta tgttactaga tcgggaggcc tcacgtgggt cctattttat aataacgctg   6240 

cggacatcta catttttgaa ttgaaaaaaa attggtaatt actctttctt tttctccata   6300 

ttgaccatca tactcattgc tgatccatgt agatttcccg gacatgaagc catttacaat   6360 

tgaatatatc ctgccgccgc tgccgctttg cacccggtgg agcttgcatg ttggtttcta   6420 

cgcagaactg agccggttag gcagataatt tccattgaga actgagccat gtgcaccttc   6480 

cccccaacac ggtgagcgac ggggcaacgg agtgatccac atgggacttt taaacatcat   6540 

ccgtcggatg gcgttgcgag agaagcagtc gatccgtgag atcagccgac gcagcccggg   6600 

ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc   6660 

atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt   6720 

ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat   6780 

ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc   6840 

agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg   6900 

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa   6960 

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc   7020 

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg   7080 

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat   7140 

ggcaacagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca   7200 

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga   7260 

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg   7320 

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg   7380 

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata   7440 

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca   7500 

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg   7560 

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat   7620 

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt   7680 

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt   7740 

attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca   7800 

acgaagcttt ctgagccgcc gattttcctc ctcgagttgg atgaactcgc cgagttcatc   7860 

gtcaactgaa acagacacgg ccggattctg tgagacaggt tgaaccgcag ctctcttcca   7920 

ttgataatag gtctgaacgg aaatacccac gatcttaacg gcgtccttca aggttgcgcc   7980 

gccagcgacc tgagcttcga tttgaccgat cttctccagt ttttctcggt tgctgaggcc   8040 

gcgggttttc ggcttcacgg atttgaacga tcccgtgcgg gctgtttcgg ctggtgcttt   8100 

ctttgctctt ctacctctag gagcagccgg ctcaacttcg gcagcagcag taccgtccgg   8160 

cggattctgg atctcttcgt cagccattaa tcgtcctctg tgtgggttat tgctttgtct   8220 

gccagctcga tccaagagtc aacgtttgtg cctagggcag taaataggca gtgctccgcg   8280 

actacatgcc tcggccggca aaataccgcc gcatgtagag caggctctcc ttcacgatca   8340 

acgatcggca tggggccttc gtgcttgttg agtaatgtta tcgctcccat cagagcacgc   8400 

ttggtactcc gggaatcgga tggtctgtcg atcatccaaa aaacgctcat gttttcaacc   8460 

tattaggtct gtggtcagct gaccacagac catcctgctc catactcgct aattctagcc   8520 

aaaccgcaac gtcccctgcc cgctagcctt caagagcgcc attatcatcg ggccaagtga   8580 

aaacttcccg agctcgctcc gccgtgtcag atctcggaga tagcccccgg gcgaattgat   8640 

gaagttcgct cgctccaaaa tgcacgccat cgctgctgcc gcattctccg gtcccattgc   8700 

ctcacacgcg tcttggtaag ccgacgggct gacccccagc atagaccgaa ccaccaccgc   8760 

agccgacatg aggtcacgcc agctagcaac cgcaccgctc ggcccataat tgccaatggt   8820 

cgggcatgct ttcaggatca tcccgagggg gaacgctttt atcggctcgc tccttgcccg   8880 

gtctatttca ctcggcttag cgccctgctc cttttcagag cgaggttcaa gttcattaac   8940 

ggattcgggt tttgaattct gtatgtgctg ctcgctctgg gcagcattgg tgctattatt   9000 

ttctgaattg tctctaattt ccaaccggtt gattatctct tcctggagca tccacatctc   9060 

ttcgagaatt gactctacat cagcaagcgt cggggcgcgt ggaattctac ccacaagttc   9120 

cacatagact tcctcgacag cttgccagtc gccctccgct ccctcttcca tagctgccgt   9180 

aattagcttc cgaacgtccc gtcggcaaat cgtcagactt tctttggcca tcctgaatgc   9240 

tgctcgatcg gccatcacct gctgtgccat catcgctagc tcttcggacc gcgcgagaag   9300 

cggagacaaa tcgaagccaa acgcgcgctc gatctgacca gcgccatcct tacgagcgta   9360 

acgctttccg ttggcgctat ccttccggac gatcaagcct gactccacga gcatggcgat   9420 

gtgcctacgc aaagtcgcgc cagccatccc atgcgcccga agggcaagct gagcattcga   9480 

cgggaagacg atcagctgtg cctcctgacg caactccgtt tccgggtgaa agctcaatag   9540 

cgcatcaagg acggcaagac tgttggactg gattccaagt agttccatgg ccgcggacgc   9600 

gtctctaaag accttccact tgtccgctgt cttgccttgt ttgatatcgg ccagcgccgt   9660 

ctggcgccgc acaagcgcaa gcgtcattgg ccgccgcccg aatggcgtcg ttacacttcc   9720 

tgtctgcatc atctttcacc tttcagcagg caaaggaaat cagctcacca aaacggcgct   9780 

aaaaactctt gacgaggatt cgaggaaatg cgattctgtt cgcgctagag agacagaagg   9840 

gcttccgcga cggcgacgtt gagggggctc ttttcttttg cggtttactc tccccgtttc   9900 

cgttggttct cagcgtggta cgcttgatac agcgctggca catgatcgag cacgaaggtc   9960 

gcaaaatcgg gcgtcgcctt cctgtcaatc gtgatttcca gtttggcctt gctctgcgtc  10020 

acctgtgcaa ttctggtgcc gtctggggtg gccatgacct cgggaagtcc acgcgcaacc  10080 

cgactgggct tcagactagc gatcaccgcc ttgaatcgtt ctgccgatgg cagcgcttga  10140 

acttcctccg acatagcata tttagccacg tcggccggtg aagaaacttt ctcaatcagc  10200 

tcggcaagtt gttgccaact cggccgtcca acaccaggag cggcaccaat agcatcggtc  10260 

agttcagagg ggagggcgtc gacgagcaga agcatcttgg acaaattgct cttgtcgatc  10320 

gacatcgcgg cgatgacaat ctctcgagaa aactgcctgt tcaggcgatg tgcgaagcgc  10380 

gccttttcga tgaaggtaag atcttcgcgc tcattgtttt cctgaccctg tgctacgacc  10440 

acttgctcgt ccgtcagttc gcgaacgacc gctctgaccg gaagtccgag ttctgaaacg  10500 

gcgcgtagcc ggcggtggcc gaaggcaacc tgatatcggc ccggctggct cggatgcggt  10560 

cgcacaagga ttgggacttg ctgtccttgt tcccggatcg aagtaaggag cccgtcaatg  10620 

tcccctcgca tacgatcctg cacgaaagac ggttctattg acgaggcatc caactctatc  10680 

actgcctgac cttcagcgag acgccgctcg atctcttcgg cacggctaag acgatcgttt  10740 

tgctctcgca gtgcgttacc aatgttcgct gtgagcttcg ttgccggatc gcgctccttc  10800 

cttgttacgc cgaggagcgg catggagcgg ttctttgccg tcctattgtc ggcgggcgac  10860 

gtctcagggg cgtcagttga gacgccaagg atgtgcttcc ggctcatgtg ggcctacccc  10920 

atgctttttt gatcagtgtt tcgatctcgt cgttgacggc gttcatcgcc tccaaggctc  10980 

gatcataggt cgagcgcgtg aacaggccac gctccacttc gaatagagtc tggtttgtca  11040 

ggccagcgtc cgaaaccgcg gtggttttaa gcatcggaaa attgaggaca ttttcgccaa  11100 

aaatcgaccg cagataacct accatttggt tctgtggtcc gtcgctcggt tcgaaacggg  11160 

ttatcagata gcgcatccaa ttaaacttga acttggcgcc agcattctcg atttcacgca  11220 

aaaggttcga tgtcattgcc agaaactggt tcatcgacat cacatccagc atctgcggat  11280 

ggaccgtgac aagaatggac gtcgccgcag tcaatgcgga tagcgtgaga tacccaagct  11340 

ggggagggca gtcgatgacc acgacgtcat agttatccgc gatatcttca attacttggc  11400 

tgatgcgacc ataaaagagc gtgtcgccct ctttgcggtt catcagcgcg cgtggcgtat  11460 

cgtgttcaaa ctccatcagc tcaaggttac caggaatcag gtggaggtcg ggaatgtaag  11520 

tccctcggac gactcgttcg attgccacct gctcatcatc ataccttata gcgccgtaga  11580 

gcgtttcgtt cgggccaacg tccgtctccg gttggctccc aaagagtgca gaaaggctcg  11640 

cttgaggatc gagatcaatg gccaagactc gatatccgcg catagcgagg tactgcgcca  11700 

gatgcgcggc ggtggtggtc ttacccgacc cacctttgaa attcatcaca gagataacct  11760 

gaagctgctc gccgcctcga cgatgtggca ggtagcgccg gttcccgcgg ccgacctgat  11820 

ccatatactt ccgaatcaca tggatatctt caattgagaa cattcgcctg ccacccgggc  11880 

tcatgctaac attcaactct ggcatctcag acgcggtctg ccgtaaatat gactcgccaa  11940 

cgccgagcag cttggacgcc tccgatggcc cgaatgttcg aatgcccttc tcggaatgcg  12000 

gcgggaaaac cttaagatga tgtgcttgaa gttggctcga gagggcatcg gcatgacgct  12060 

ccatcaaggc cgtcaaccct acaactacag gcgctgcttt taggacagac ttcgccatct  12120 

caaacccatt ccttgccagt ggcgatattt ttcgcgaaac tggaaaagtt ccgccgctgg  12180 

caattagcgc cgattctgct gtttgggcaa gagcttttag gttaacagaa ggttaacgcc  12240 

ctcaggtcga aaaactccac ccaactgtta tttgtattta tttccaatgc cttagagaga  12300 

ttgccatttg aatatgttca tgtattgttt tagtgataat cctacaatcg taacccaaaa  12360 

agaggtcgcc ctctgcgcgc cgtcgtccaa tataggcgaa gtcacccttg cgactcaggc  12420 

ggattctacc ttgtagga                                                12438 

 
           
             10  
             4297  
             DNA  
             Artificial Sequence  
             
               Plasmid pPG354  
             
           
            10 

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca     60 

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg    120 

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc    180 

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc    240 

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat    300 

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt    360 

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt tatcccccac gtgcgtacga    420 

tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc    480 

gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc    540 

tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc    600 

taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa    660 

attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcagat    720 

cctagacgat cgtttcgcca tggctaagct tatgaaaaag cctgaactca ccgcgacgtc    780 

tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga    840 

gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt    900 

aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc    960 

cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg   1020 

catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc   1080 

tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac   1140 

gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt   1200 

catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt   1260 

cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga   1320 

agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg   1380 

cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc   1440 

caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga   1500 

gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg   1560 

tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca   1620 

gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc   1680 

ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa   1740 

ccgacgcccc agcactcgtc cgagggcaaa ggaatagaat tcctaggatc gttcaaacat   1800 

ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata   1860 

atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat   1920 

gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa   1980 

aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg   2040 

aggcctcacg tggggtatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat   2100 

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg   2160 

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag   2220 

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt   2280 

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg   2340 

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg   2400 

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag   2460 

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga   2520 

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct   2580 

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc   2640 

tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg   2700 

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc   2760 

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca   2820 

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag   2880 

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct   2940 

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc   3000 

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga   3060 

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca   3120 

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat   3180 

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac   3240 

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt   3300 

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt   3360 

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag   3420 

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct   3480 

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt   3540 

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc   3600 

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt   3660 

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg   3720 

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg   3780 

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct   3840 

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc   3900 

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt   3960 

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt   4020 

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg   4080 

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat   4140 

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg   4200 

cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta   4260 

acctataaaa ataggcgtat cacgaggccc tttcgtc                            4297