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 
     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 
     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 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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.    
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 . is a represenation of the structure of the molecule “clonNAT”. 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-4one dihydrogensulphate. 
     FIG.  2 . is a diagram of the plasmid pPG363 (SEQ ID NO. 8) wherein the ocs LB, PmlI, 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. 
     FIG.  3 . ia a diagram of the plasmid pPG361 (SEQ ID NO 9) wherein ocs LB, nos term, aph4, nos prom, 35S prom, GUS Intron, Ag7 term, ocs RB, and PmlI 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. 
     FIG.  4 . is a diagram of the plasmid pPG362 (SEQ ID NO 7) wherein the nos prom, the nrg gene, the Nos term, and PmlI 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. 
     FIG.  5 . is a diagram of the plasmid pPG354 (SEQ ID NO 10) wherein nos term, aph4, nos prom, PmlI, 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. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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. 
     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. 
     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). 
     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). 
     AvrII refers to a restriction enzyme site. “clonNAT” is the dihydrogen sulphate of the weakly basic antibiotic nourseothricin, consisting of streptothricin components F and D. The chemical name is 2-[4O-Carbamoyl-2-deoxy-2-(3,6-diaminohexan-amido)-B-D-gulopyranoslamino)3,3a,5,6,7,7a-hexahydro-5-hydroxy4H-imidazo [4,5-c]pyridin-4-one dihydrogensulphate. GUS Intron refers to the GUS marker gene containing an intron, as disclosed in plamids pPG361 and pPG363. 
     HindIII refers to a restriction enzyme site. 
     nos prom—a promoter sequence from the nopaline synthase gene of  Agrobacterium tumefaciens , as disclosed in plasmids pPG354, pPG361, pPG362, pPG363. 
     nos term—a terminator sequence from the nopaline synthase gene of  Agrobacterium tumefaciens , as disclosed in plasmids pPG354, pPG361, pPG362, pPG363. 
     “Nourseothricin” refers to the streptothricin antibiotic components F and D, produced in cultures from a strain of  Streptomyces noursei.    
     “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. 
     “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. 
     35S prom—a promoter sequence from the genome of cauliflower mosaic virus, as disclosed in plasmids pPG361 and pPG363. 
     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.    
     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. 
     PmlI refers to a restriction enzyme site. 
     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. 
     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 
     Using the nat1 gene as the staring 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: 
     forward primer: 5′nat1HindIII; ccgaagcttATGACCACTCTTGACGACACG [SEQ ID NO. 5] 
     reverse primer: 3 ′nat1AvrII; aaccctaggCTAGGGGCAGGGCATGCTCATG [SEQ ID NO. 6] 
     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 209, the nucleotide guanine (“G”) is replaced with 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 (G), is replaced with aspartate (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 
     The nrg gene, controlled by the nos promoter and nos terminator elements from pPG362, is further cloned as a PmlI fragment into the PmlI 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 nag 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 in FIG.  2 . 
     EXAMPLE 3 
     Rice Transformation Using a Binary Vector With the nrg Marker Gene 
     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. 
     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); pH5.6) 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 
     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. 
     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 December, 1998 ; 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 
     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 pINS 1. The sequence of the nat1 gene is described as the sequence X73149.1 (emb|X73149.1|SNNAT1  S.noursei  gene for nourseothricin acetyltransferase) found in the database at the National Center for Biotechnology Information (NCBI). The binary vector pPG363 (SEQ ID. NO. 8) displayed in FIG. 2 can be prepared from the vectors pPG361 (SEQ ID NO 9) displayed in FIG.  3  and pPG362 (SEQ ID NO. 7) displayed in FIG.  4 . Methods for preparing DNA constructs or plasmids is known in the art. See, e.g., Sambrook et al.,  Molecular Cloning: A Laboratory Manual  (1989). Plasmid pPG362 (SEQ ID NO. 7) can be prepared from plasmid pPG354 (SEQ ID NO. 10) (FIG. 5) and the nrg gene, as derived from 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. 
     
       
         
           
             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