Abstract:
A male sterile plant free from any undesirable characteristic is constructed by anther-specifically expressing barnase gene alone. A barnase gene, which has a mutation and thus sustains its activity at a lowered level, is transferred into a plant and anther-specifically expressed, thereby efficiently providing a male sterile transgenic plant.

Description:
[0001]    The present application claims priority from Japanese Patent Application No.: Hei 10-220060, the disclosure of which is incorporated herein by reference.  
         FIELD OF THE INVENTION  
         [0002]    This invention relates to a mutant barnase gene which makes it possible to efficiently yield a male sterile transgenic plant when expressed in a specific site of a plant, in particular, anther-specifically. The present invention further relates to a recombinant vector capable of expressing the mutant barnase gene of the invention in host cells, a plant transformed by this vector and a method for constructing a transgenic plant.  
         PRIOR ART  
         [0003]    Barnase is an RNase originating in  Bacillus amyloliquefaciens  (S. Nishimura and M. Nomura, Biochem. Biophys. Acta 30, 430-431:1958; R. W. Hartley, J. Mol. Biol., 202, 913-915:1988). This enzyme has 110 amino acid residues and hydrolyzes RNA. When expressed in cells, this enzyme inhibits the functions of the cells as a result of its potent RNase activity and thus causes cell death in many cases. By using this characteristic, it is therefore expected that the function of the specific site can be selectively controlled by expressing the barnase gene in a specific site of a plant.  
           [0004]    PCT International Publication WO89/10396 discloses a technique whereby a male sterile plant is obtained by constructing a male sterility gene by ligating the above-described barnase gene to the downstream of an anther tapetal cell-specific expression promoter and introducing the thus obtained gene into a plant. Male sterilization techniques are of great value in efficiently developing an F1 hybrid variety.  
           [0005]    When the barnase gene is employed as a male sterility gene, however, it is frequently observed that resulting male sterile transgenic plants exhibit unfavorable characteristics. PCT International Publication WO96/26283 refers to this problem in rice. It is also reported that similar phenomena are observed not only in rice but in lettuce (Scientia Horticulturae 55, 125-139:1993; Arlette Reymaerts, Hilde Van de Wiele, Greta De Sutter, Jan Janssens: Engineered genes for fertility control and their application in hybrid seed production). According to this report, a plant with depressed activity was constructed by introducing a male sterility gene comprising a tobacco anther-specific promoter (TA29) and a barnase gene into lettuce.  
           [0006]    Although reasons for these phenomena have not been accurately clarified so far, it is assumed that the so-called “position effect” of the site in which the gene is transferred may account for the mechanism (PCT International Publication WO96/26283). More specifically, a desired male sterile plant should be constructed if the male sterility gene is expressed exclusively in the target site (i.e., anthers). However, there is a possibility that the barnase might be expressed also in tissues other than anthers although in a very small dose under the action of expression regulators (for example, an endogenous enhancer) existing in the vicinity of the gene transfer site. In such a case, the unfavorable characteristics as described above can appear because barnase has a strong enzymatic activity.  
           [0007]    To overcome this problem, the method disclosed in PCT International Publication WO96/26283 exploits the character of cauliflower mosaic virus 35S promoter (hereinafter referred to as CaMV35S promoter) of being expressed potently in tissues other than anthers. Namely, barstar, i.e., an inhibitory protein against barnase is employed therein. The barstar gene ligated to a CaMV35S promoter is transferred into a plant simultaneously with a barnase gene and then the barstar gene is constitutively expressed in tissues other than the anthers, thereby eliminating the effects of the barnase in tissues other than the anthers. However, it is necessary in this method to transfer not only a barnase gene but also a barstar gene, and hence, the problem is that the application of this technique to the breeding of F1 varieties of rice or corn seeds may give rise to a gene silencing. “Gene silencing” is a phenomenon wherein the expression of a gene is inhibited when plural copies of the foreign gene are introduced. It is known that this problem frequently occurs when the expression of a foreign gene is effected by a 35S promoter, though the detailed mechanism thereof has not been clarified yet (R. B. Flavel, Proc. Natl. Acad. Sci. USA 91, 3490-3496:1994; J. Finnegan, Bio. Technology 12, 883-888:1994; M. A. Matzke and A. J. M. Matzke, Plant Physiol., 107, 679-685:1995). Since, in rice and corn, a barstar gene is used as a “fertility-restoring gene” in the pollen parent (father) so as to allow the pollen in the F1 generation to restore the fertility (C. Mariani, et al., Nature 357, 384-387:1992), if an MS plant (mother) is constructed by the method with the use of a barstar gene as reported in WO96/26283, the F1 plant has plural copies of the barstar gene. In such a case, there is a possibility that the expression of the gene may be inhibited due to the gene silencing.  
         SUMMARY OF THE INVENTION  
         [0008]    The present invention provides a method for constructing a male sterile plant by using a barnase gene without resort to a barstar gene.  
           [0009]    The present invention further provides a mutant barnase gene to be used in the above method and a process for producing the same.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0010]    In the present invention, the DNA sequence of barnase gene (R. W. Hartley, J. Mol. Biol. 202, 913-915:1988) is mutated at least in part and then the thus obtained mutant barnase gene is anther-specifically expressed in a plant so as to make the plant substantially male sterile without any substantially disadvantageous effect on the tissues other than anthers.  
           [0011]    The mutation can be performed by a known method such as site-specific mutagenesis, deletion of a fragment by using restriction enzymes or the low fidelity PCR method. Among all, it is preferred to use the low fidelity PCR method. This method is described in detail in D. Leung, E. Chen and D. Goedda, Technique 1, 11-15:1989; Y. Z. Xiaoping and R. H. Ebright, Nucleic Acid Res. 19, 6052:1991; G. C. Rice et al., Proc. Natl. Acad. Sci. USA 89, 5467-5471: 1992 and the contents of these documents are incorporated herein by reference. By using this technique, PCR is carried out under such conditions as to induce some errors during the amplification reaction. Thus, random mutations can be introduced into the target DNA fragment.  
           [0012]    Primers to be used in the low fidelity PCR method in the present invention are selected as in the conventional PCR method. It is preferred that each of these primers has a similar number of nucleotides as in the conventional PCR method.  
           [0013]    The present inventors performed PCR by using a DNA containing the sequence represented by SEQ ID NO: 1 as a template with the use of a combination of primer 1 (5′-CGTTCGGCTC GATGGTACCG GTTATCAACA CGTTTGA-3′: SEQ ID NO: 6) and primer 2 (5′-CCTCTAGATT ATCTGATTTT TGTAAAGGTC TGATAATG-3′: SEQ ID NO: 7) under such conditions as to induce errors. Thus, a mutant barnase gene having the DNA sequence represented by SEQ ID NO: 3 was isolated. The sequence represented by SEQ ID NO: 1 employed herein is the sequence of the coding region of the barnase gene contained in the known plasmid pVE108 (WO92/13956). This sequence corresponds to the wild type barnase gene from which the unnecessary moiety corresponding to the secretion signal in the N-terminal side has been deleted.  
           [0014]    It is also possible to acquire different mutant barnase genes by a similar method.  
           [0015]    The PCR amplification product is cloned in a host (for example,  Escherichia coli ) and clones containing a mutant barnase gene is isolated. In this cloning, clones having a mutant barnase gene may be screened by assaying the RNase activity expressed by the gene. However, it is advantageous to screen the clone by the method consisting of the following two steps by taking advantage of the fact that the RNase activity of barnase affects the growth of  E. coli.    
           [0016]    In the first step, a plasmid is prepared by using the mutant barnase gene obtained above and then  E. coli  is transformed thereby. The growth of the  E. coli  transformant thus obtained will be inhibited by the activity of the mutant barnase. Based on this fact, a colony growing slowly (i.e., a small colony), compared with  E. coli  having a control vector free from any barnase gene, is selected. It is expected that the thus selected  E. coli  strain will contain the mutant barnase gene. To confirm that the inhibition of the growth of  E. coli  is due to the expression of the mutant barnase gene, the second step is then carried out.  
           [0017]    In the second step, a barstar gene is employed. As described above, barstar is an inhibitory protein against barnase. In  E. coli  which expresses barstar, the enzymatic activity of barnase is inhibited and thus the degradation of mRNA, which otherwise takes place in the presence of barnase, can be inhibited. Therefor if,  E. coil  which expresses a barstar gene is transformed with a barnase gene, the growth will not be inhibited. Accordingly, it is expected that the thus obtained  E. coli  transformant will produce little difference in growth rate from the transformant constructed by the control plasmid which is free from any barnase gene and, therefore, these transformants will produce little difference in colony size too. Based on this principle, a plasmid is prepared from the  E. coli  strain selected in the first step. The plasmid is then used to transform another  E. coli  strain, in which the barstar gene is constitutively expressed. Thus, mutant barnase gene-containing colonies having almost the same size as the one of the  E. coli  transformed by the control plasmid are selected. The  E. coli  with the constitutive expression of the barstar gene to be used herein can be prepared by, for example, the method as will be described in Example 1 hereinafter.  
           [0018]    The DNA sequence of the thus obtained mutant barnase gene may be analyzed by a conventional method, if necessary, to thereby examine the mutation in detail.  
           [0019]    A preferred example of the mutant barnase gene of the present invention is one which has the DNA sequence represented by SEQ ID NO: 3. Compared with the nucleotide sequence of SEQ ID NO: 1 coding for the wild type activity, the nucleotide sequence encoding this gene has an insertion of “T” at the 15-position from A in the initiation codon ATG and a deletion of “A” at the 333-position. According to the normal translation manner, translation will be initiated from ATG at the 1-position of this DNA sequence represented by SEQ ID NO: 3, and terminated at the ninth codon which has been converted to a termination codon as a result of the insertion of the “T” at the 15-position. It is unlikely that the thus formed oligopeptide consisting of 8 amino acids has the barnase activity. Further, there is no other ATG or GTG codon from which the translation can be initiated in the correct frame in the vicinity thereof. However, the fact that the growth rate of  E. coli  was restored by the barstar protein strongly suggests that the translation of the barnase protein from the gene of SEQ ID NO: 3 proceeded in the correct frame. This is seemingly due to a phenomenon called “frame shift re-coding” whereby the ribosome controls the translation while automatically shifting the reading frame during the course of translation into the protein. This phenomenon, which has been reported in several genes of viruses,  E. coli  and animals, generally depends exclusively on an individual nucleotide sequence and neither any special protein nor translation device is required. The frame shifting efficiency during the translation varies from gene to gene, i.e., ranging from several % to 50%. In the case of the mutant barnase gene, it is considered that the efficiency of the shifting of the reading frame during the translation is not high and thus the translation product in the correct frame is formed in a smaller amount, thereby lowering the activity of the gene.  
           [0020]    Moreover, there is a high possibility that other mutant barnase genes of which the translation efficiency is lowered due to the shifting of the reading frame, as in the case of the DNA sequence represented by SEQ ID NO: 3, or some other reasons can be obtained by mutating the barnase gene by, for example, the low fidelity PCR method. Furthermore, there is a high possibility that a gene having a DNA sequence derived from the DNA sequence represented by SEQ ID NO: 3 by substitution, deletion, insertion or addition of one or more nucleotides will be a mutant barnase gene which provides a lowered translation efficiency due to the shifting of the reading frame as a result of the insertion of T at the 15-position from A in the initiation codon ATG and/or the deletion of A at the 333-position, as in the DNA sequence represented by SEQ ID NO: 3. It is considered that each of these genes, likewise the gene of SEQ ID NO: 3, is capable of making a plant substantially male sterile when it is anther-specifically expressed in the plant. Accordingly, these genes are also included in the scope of the present invention similar to the gene of SEQ ID NO: 3.  
           [0021]    The plant which is to be male sterilized by the mutant barnase gene is not particularly limited, so long as the gene can be transferred into the plant to provide a male sterilized plant. Examples of the plants include rice, corn, tobacco, lettuce and Brassica. Among all, rice and corn are preferred.  
           [0022]    In order to male sterilize a plant, the mutant barnase gene is expressed specifically in the anthers of the plant to inhibit the anther functions by the RNase activity of barnase. Such an anther-specific expression can be achieved by using the method described in WO92/13957. In brief, the mutant barnase gene is ligated to the downstream of an anther-specific promoter and then incorporated into plant cells with the use of an expression vector.  
           [0023]    The incorporation may be performed by using the Agrobacterium method, the electroporation method, the particle gun method, etc.  
           [0024]    From the transformed plant cells, a complete plant can be formed by the regeneration from a callus of the transformed plant cells in accordance with, for example, the method reported in Y. Hiel et al., Plant J. 6, 271-282:1994.  
           [0025]    The male sterility of the thus constructed plant can be confirmed by the inability of the plant to fertilize unless it is pollinated from another fertilizable plant. 
       
    
    
     EXAMPLE 1  
       [0026]    Preparation of Mutant Barnase Gene Low Fidelity PCR  
         [0027]    Primer 1 (CGTTCGGCTC GATGGTACCG GTTATCAACA CGTTTGA: SEQ ID NO: 6) and Primer 2 (CCTCTAGATT ATCTGATTTT TGTAAAGGTC TGATAATG: SEQ ID NO: 7) were synthesized by a DNA synthesizer (manufactured by Applied Biosystems) in accordance with the method described in S. L. Beaucage et al., Tetrahedron Lett., 22. 1859-1862:1982. Known plasmid pVE108 (WO92/13956) was used as a template and PCR was carried out with the use of the combination of Primers 1 and 2. SEQ ID NO: 1 shows the sequence of the coding region of the barnase gene which is contained in pVE108. The reaction was performed for 50 cycles with each cycle consisting of 1 minute at 94° C., 1 minute at 57° C. and 1 minute at 72° C. in 10 mM Tris-HCl (pH 9.5), 50 mM KCl, 2 mM MgCl 2 , and 1 mM each of dNTP, 10 ng of the template DNA and 0.5 U of Taq DNA polymerase.  
         [0028]    After the completion of the reaction, the amplification product was separated by electrophoreses on agarose gel (2% SeaKem GTG agarose, 1×TAE) and purified by the DEAE-cellulose method (M. Muramatsu, Labo-manual Idenshikogaku (Gene Engineering Labo-Manual), Maruzen, pp.111:1988). By using the purified product thus obtained as a template, PCR was performed again under the conditions as defined above.  
         [0029]    Ligation of Mutated Barnase Gene Fragment into Plasmid Vector  
         [0030]    The reaction product from the above step was digested with SacI and XbaI in a conventional manner and then purified by agarose gel electrophoresis to give an “insertion fragment”. A plasmid to be used for transferring this insertion fragment into  E. coli  can be appropriately selected. For example, use may be made of plasmid pHM1 therefor. The plasmid pHM1 was constructed as follows. Plasmid pBR322 was cleaved at the EcoRI site and blunt-ended by using T4 DNA polymerase (manufactured by Takara Shuzo). Separately, lacZ expression cassette (322 bp) was excised from pUC18 with PvuII and the ends thereof were blunted. The cassette was integrated into the restriction site of the blunt-ended plasmid pBR 322 to give plasmid pHM1.  
         [0031]    Plasmid pHM1 was digested with SacI and XbaI followed by dephosphorylation with calf intestine alkaline phosphatase (manufactured by Takara Shuzo) to give a restriction enzyme-treated plasmid fragment. Into this restriction enzyme-treated plasmid pHM1, the above “insertion fragment” containing a barnase gene was ligated by using Takara Ligation Kit ver.1 (manufactured by Takara Shuzo).  
         [0032]    Introduction into  E. coli  and Selection of Barnase-active Clone  
         [0033]    The  E. coli  having the barnase gene introduced thereinto may be selected by, for example, the following method. Since mRNA is degraded in cells by the barnase activity, the synthesis of protein is suppressed and, in its turn, the growth of  E. coli  is inhibited. Thus, the  E. coli  transformed by the barnase gene can be selected by taking advantage of the fact that the desired transformant colony has a smaller size than the  E. coli  colony transformed by the control plasmid free from the mutant barnase gene. When the wild type barnase or a mutant barnase still maintaining the activity comparable thereto is integrated into pHM1,  E. coli  cannot form any colony. Thus, clones having a sufficiently weakened barnase activity can be exclusively selected.  
         [0034]    Thus, the plasmid having the mutant barnase gene ligated thereto was precipitated from ethanol and then introduced into  E. coli  LE392 strain by the electroporation method with the use of GenePulser (BioRad) in accordance with the manufacture&#39;s instruction. Also, the control plasmid free from mutant barnase was introduced into the  E. coli  LE392 strain in the same manner.  
         [0035]    Next, these  E. coli  transformants were plated onto an LB agar medium containing tetracycline (25 μg/ml) and incubated at room temperature (25° C.) for 72 hours. Then colonies of a smaller size than the colony of  E. coli  having the control plasmid pHM1 introduced thereinto were screened as colonies of the  E. coli  transformants having the mutant barnase transferred thereinto (Table 1, A).  
         [0036]    Selection of Mutant Barnase Clone by Barstar Gene  
         [0037]    As described above, barstar is an enzyme acting as an antagonist to barnase. In  E. coli  wherein barstar is expressed, the enzymatic activity of barnase is inhibited and thus the degradation of mRNA, which otherwise takes place in the presence of barnase, can be inhibited. Therefore, if  E. coli  expressing barstar is transformed by a plasmid containing a barnase gene, the growth thereof will not be inhibited. Accordingly, it is expected that the thus obtained  E. coli  transformant will show little difference in growth rate from the transformant constructed by using the control plasmid free from a barnase gene and, therefore, these transformants will show little difference in colony size too.  
         [0038]    An  E. coli  transformant which expressed barstar was constructed in the following manner. A barstar gene described in R. W. Hartley, J. Mol. Biol. 202, 913-915:1998 was excised with HindIII and XbaI and ligated in frame to a tac promoter (de Boer et al., Proc. Natl. Acad. Sci. USA 80, 21-25:1983). The gene, together with a chloramphenicol tolerance gene (N. K. Alton and D. Vapnek, Nature 282, 864-869:1979), was ligated into the defective transposon which lacked the transferase in a vector described in Herrero et al., J. Bacteriol. 172, 6557-6567:1990. Subsequently, the thus obtained plasmid was introduced into  E. coli  MC1061 strain thereby to transfer the transposon, which contained the barstar gene cassette, into the chromosome of the  E. coli . It was confirmed based on the chloramphenicol tolerance that this  E. coli  was able to maintain the barstar gene cassette in a stable state. Since the  E. coli  MC1061 strain lacked the lacI gene, the tac promoter was continuously active and thus the barstar gene was constitutively expressed.  
         [0039]    The plasmid containing the mutant barnase gene as the insertion fragment and the control plasmid free from the same were each introduced into the above  E. coli  transformant by the electroporation method. Then the  E. coli  transformants were plated onto an LB agar medium containing IPTG (1 mM) and tetracycline (25 μg/ml) and incubated at 25° C. for 72 hours. Among the colonies of the  E. coli  transformed by the mutant barnase gene, a colony being comparable in size to the colony of the  E. coli  having the control plasmid pHM1, which had been simultaneously plate-cultured, was screened and named “#4-31” (Table 1, B).  
                                           TABLE 1                           Screened clone and colony size (mm)                Screened clone   pHM1 (control)                        A:  E. coli  LE392 *1     1.77 ± 0.33   2.65 ± 0.47       B:  E. coil  with barstar   1.39 ± 0.10   1.55 ± 0.08       gene expression *2                                    
 
       EXAMPLE 2  
       [0040]    Determination of Nucleotide Sequence of Mutant Barnase Gene  
         [0041]    From the  E. coli  screened in Example 1, a cloned mutant barnase gene appropriate for the object of the present invention was prepared. First, the clone #4-31 screened in Example 1 was propagated. Next, the mutant barnase gene fragment in the clone #4-31 was excised with KpnI and XbaI and ligated into the KpnI, XbaI-site of pUC119 having been cleaved similarly with KpnI and XbaI. After amplifying the obtained plasmid by using  E. coli,  a reaction was performed by the cycle sequence method with the use of Taq polymerase (Taq Dye Terminator Cycle Sequencing Kit, manufactured by Applied Biosystems Inc.) in accordance with the manufacturer&#39;s protocol. Subsequently, the sequence was analyzed with a DNA Sequencer (Model 373A, manufactured by Applied Biosytems Inc.), thereby yielding the sequence represented by SEQ ID NO: 3. Compared with the DNA sequence of the wild type barnase gene, this sequence had an insertion of T at the 15-position from A of the initiation codon ATG and a deletion of A at the 333-position.  
       EXAMPLE 3  
       [0042]    Construction of Male Sterile Rice by using Attenuated Mutant Barnase Gene  
         [0043]    The attenuated mutant barnase gene integrated into pUC119 as described above was cleaved with restriction enzymes XbaI and KpnI and thus the plasmid vector pTS431 represented by SEQ ID NO: 5 was constructed. Although this plasmid pTS431 differs from the known plasmid pVE108 (PCT International Publication WO92/13956) in the following points, it is substantially equivalent thereto except for the moieties of the anther-specific promoter and the mutant barnase.  
         [0044]    (1) In the plasmid vector pTS431 according to the present invention, the barnase gene (SEQ ID NO: 1) in the known plasmid pVE108 has been converted into the mutant barnase gene (SEQ ID NO: 3).  
         [0045]    (2) A tobacco-origin anther specific promoter is used in the known plasmid pVE108, while the anther specific promoter of rice E1 gene (PCT International Publication WO92/13956) is used in the plasmid vector pTS431 according to the present invention.  
         [0046]    (3) In the present invention, a 35S3 promoter (EP 0344029) of 1376 bp, which is not used in the plasmid pVE108, is employed upstream of the barnase gene.  
         [0047]    (4) In the present invention, a sequence originating in the downstream of Agrobacterium T-DNA gene 7 excised from pJD884 (PCT International Publication WO93/09218) is employed whereas such a sequence is not used in the plasmid pVE108.  
         [0048]    (5) In the present invention, the region corresponding to lacZ has been deleted from the pUC19-origin moiety. In addition a plasmid (pTS172) having the wild type barnase gene integrated therein is represented by SEQ ID NO: 4.  
         [0049]    The fragment of about 4.5 kbp was excised with a restriction enzyme EcoRI respectively from pTS431 (a plasmid having the mutant barnase gene transferred thereinto; SEQ ID NO: 5) and pTS172 (a plasmid having the barnase gene transferred thereinto; SEQ ID NO: 4) and inserted into the EcoRI-site of the intermediate vector pSB11 (T. Komari et al., Plant J. 10(1), 165-174:1996), and further, the T-DNA region thereof was integrated into the acceptor vector pSB1 (T. Komari et al., Plant J. 10(1), 165-174:1996) by way of homologous recombination. By using  Agrobacterium tumefaciens  LBA4404 having the obtained recombinant plasmids (pSB1431 and pSB1172 respectively), rice (variety: Asanohikari) was transformed. While the transformation was performed basically in accordance with the method of Hiei et al. (Plant J. 6(2), 271-282:1994), phosphinothricine (concentration: 10 mg/L) was employed for screening transformants since the thus constructed male sterility genes contained bar gene encoding phosphinithricine acetyl transferase as a selective marker. Phosphinothricine facilitates selection of calluses having the gene transferred thereinto.  
         [0050]    A comparison of the rice transformant obtained with the use of the wild type barnase gene with the one obtained with the use of the mutant barnase gene indicates that the transfer of the mutant barnase gene gave rise to a remarkable improvement in the transformation efficiency and an increase in the rate of morphologically normal male sterility transformants, as shown in Table 2.  
                                                           TABLE 2                           Transformation efficiency                        No. of   No. of           No. of   No. of   PCR-   morphologically           infected   regenerated   positive   normal male           calluses   calluses   lines *1     sterile lines                        pSB1172   2838   83   52/83           9/52(17.3%)         (control)       pSB1431   787   69   43/45 *2     27/28 *3 (96.4%)       (present       invention)                                          
 
       EFFECTS OF THE INVENTION  
       [0051]    In the present invention, a barnase gene having a weakened effect is constructed via mutation. A male sterility gene comprising the mutant barnase gene can be introduced into a plant to successfully produce a male sterile plant free from any unfavorable characteristic at a high efficiency with the use of a single gene without resort to the use of a barstar gene.  
     
       
       
         1 
         
           
             7  
           
           
             1  
             343  
             DNA  
             Bacillus amyloliquefaciens  
             
               misc_feature  
               wild type barnase gene  
             
           
            1 

atg gta ccg gtt atc aac acg ttt gac ggg gtt gcg gat tat ctt cag       48 
Met Val Pro Val Ile Asn Thr Phe Asp Gly Val Ala Asp Tyr Leu Gln 
1               5                   10                  15 

aca tat cat aag cta cct gat aat tac att aca aaa tca gaa gca caa       96 
Thr Tyr His Lys Leu Pro Asp Asn Tyr Ile Thr Lys Ser Glu Ala Gln 
            20                  25                  30 

gcc ctc ggc tgg gtg gca tca aaa ggg aac ctt gca gac gtc gct ccg      144 
Ala Leu Gly Trp Val Ala Ser Lys Gly Asn Leu Ala Asp Val Ala Pro 
        35                  40                  45 

ggg aaa agc atc ggc gga gac atc ttc tca aac agg gaa ggc aaa ctc      192 
Gly Lys Ser Ile Gly Gly Asp Ile Phe Ser Asn Arg Glu Gly Lys Leu 
    50                  55                  60 

ccg ggc aaa agc gga cga aca tgg cgt gaa gcg gat att aac tat aca      240 
Pro Gly Lys Ser Gly Arg Thr Trp Arg Glu Ala Asp Ile Asn Tyr Thr 
65                  70                  75                  80 

tca ggc ttc aga aat tca gac cgg att ctt tac tca agc gac tgg ctg      288 
Ser Gly Phe Arg Asn Ser Asp Arg Ile Leu Tyr Ser Ser Asp Trp Leu 
                85                  90                  95 

att tac aaa aca acg gac cat tat cag acc ttt aca aaa atc aga taa      336 
Ile Tyr Lys Thr Thr Asp His Tyr Gln Thr Phe Thr Lys Ile Arg 
            100                 105                 110 

ggtaacc                                                              343 

 
           
             2  
             111  
             PRT  
             Bacillus amyloliquefaciens  
             
               misc_feature  
               wild type barnase gene  
             
           
            2 

Met Val Pro Val Ile Asn Thr Phe Asp Gly Val Ala Asp Tyr Leu Gln 
1               5                   10                  15 

Thr Tyr His Lys Leu Pro Asp Asn Tyr Ile Thr Lys Ser Glu Ala Gln 
            20                  25                  30 

Ala Leu Gly Trp Val Ala Ser Lys Gly Asn Leu Ala Asp Val Ala Pro 
        35                  40                  45 

Gly Lys Ser Ile Gly Gly Asp Ile Phe Ser Asn Arg Glu Gly Lys Leu 
    50                  55                  60 

Pro Gly Lys Ser Gly Arg Thr Trp Arg Glu Ala Asp Ile Asn Tyr Thr 
65                  70                  75                  80 

Ser Gly Phe Arg Asn Ser Asp Arg Ile Leu Tyr Ser Ser Asp Trp Leu 
                85                  90                  95 

Ile Tyr Lys Thr Thr Asp His Tyr Gln Thr Phe Thr Lys Ile Arg 
            100                 105                 110 

 
           
             3  
             342  
             DNA  
             Artificial Sequence  
             
               A mutant barnase gene derived from Bacillus 
      amyloliquefaciens  
             
           
            3 

atggtaccgg ttattcaaca cgtttgacgg ggttgcggat tatcttcaga catatcataa     60 

gctacctgat aattacatta caaaatcaga agcacaagcc ctcggctggg tggcatcaaa    120 

agggaacctt gcagacgtcg ctccggggaa aagcatcggc ggagacatct tctcaaacag    180 

ggaaggcaaa ctcccgggca aaagcggacg aacatggcgt gaagcggata ttaactatac    240 

atcaggcttc agaaattcag accggattct ttactcaagc gactggctga tttacaaaac    300 

aacggaccat tatcagacct ttacaaaaat cagtaatcta ga                       342 

 
           
             4  
             6548  
             DNA  
             Escherichia coli LE392  
             
               misc_feature  
               Clone pTS172  
             
           
            4 

aattcaagct tgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt     60 

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg    120 

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt    180 

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta    240 

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc    300 

ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa    360 

gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc    420 

cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt    480 

acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact    540 

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac    600 

aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata    660 

ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta    720 

ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg    780 

gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat    840 

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt    900 

aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga    960 

aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa   1020 

gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag   1080 

gtgaagatcc tttttggctc gagtctcatg accaaaatcc cttaacgtga gttttcgttc   1140 

cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg   1200 

cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg   1260 

gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca   1320 

aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg   1380 

cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg   1440 

tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga   1500 

acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac   1560 

ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat   1620 

ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc   1680 

tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga   1740 

tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc   1800 

ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg   1860 

gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag   1920 

cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc   1980 

gcgcgttggc ctgatcagaa ttcatatgca cgtgttcccg atctagtaac atagatgaca   2040 

ccgcgcgcga taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa   2100 

tgtataattg cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca   2160 

tgttaattat tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac   2220 

cggcaacagg attcaatctt aagaaacttt attgccaaat gtttgaacga tctgcttcgg   2280 

aggttacctt atctgatttt tgtaaaggtc tgataatggt ccgttgtttt gtaaatcagc   2340 

cagtcgcttg agtaaagaat ccggtctgaa tttctgaagc ctgatgtata gttaatatcc   2400 

gcttcacgcc atgttcgtcc gcttttgccc gggagtttgc cttccctgtt tgagaagatg   2460 

tctccgccga tgcttttccc cggagcgacg tctgcaaggt tcccttttga tgccacccag   2520 

ccgagggctt gtgcttctga ttttgtaatg taattatcag gtagcttatg atatgtctga   2580 

agataatccg caaccccgtc aaacgtgttg ataaccggta ccatcgcgac ggcttgatgg   2640 

atctcttgct ggacaccggg atgctaggat gggttatcgt ggccggcgtg cgtgtgtggc   2700 

ttttgtaggc gccggcgacg gcgggggcaa tgtggcaggt gagtcacggt gcaagcgtgc   2760 

gcaagtgact gcaacaacca aggacggtca tggcgaaagc acctcacgcg tccaccgtct   2820 

acaggatgta gcagtagcac ggtgaaagaa gtgttgtccc gtccattagg tgcattctca   2880 

ccgttggcca gaacaggacc gttcaacagt taggttgagt gtaggacttt tacgtggtta   2940 

atgtatggca aatagtagta aattttgccc ccattggtct ggctgagata gaacatattc   3000 

tggaaagcct ctagcatatc ttttttgaca gctaaacttt gcttcttgcc ttcttggtct   3060 

agcaatgacg ttgcccatgt cgtggcaaac atctggtaag gtaactgtat tcgtttgttc   3120 

ccttcaacgg ctcaatcccc acaggccaag ctatcctttc cttggcagta taggctcctt   3180 

gagagattat actaccattt ttaagtgctt ataaagacga tgctctctaa ccagatcgat   3240 

cagaaacaca aagttttagc agcgtaatat cccacacaca tacacacacg aagctatgcc   3300 

tcctcatttt ccgagagatt ctgacagtga ccagaatgtc agaatgccat ttcatgggca   3360 

caagtcgatc cacaagcttc ttggtggagg tcaaggtgtg ctattattat tcgctttcta   3420 

ggaaattatt cagaattagt gccttttatc ataacttctc tctgagccga tgtggttttg   3480 

gatttcattg ttgggagcta tgcagttgcg gatattctgc tgtggaagaa caggaactta   3540 

tctgcggggg tccttgctgg ggcaacattg atatggttcc tgttcgatgt agtagaatac   3600 

aatataattc cgctcctttg ccagattgcc attcttgcca tgcttgtgat cttcatttgg   3660 

tcaaatgccg caccactctt ggacaggtat tagctttatt tcctgtggag atggtagaaa   3720 

actcagctta cagaaatggc atttcacgta gtataacgca agacattagg tactaaaact   3780 

caactaactg tttccgaatt tcagggcccc tccaaggatc ccagaaatca tcatctctga   3840 

acatgccttc agagaaatgg cattgaccgt ccattacaaa ctaacgtaca ctgtatctgt   3900 

tctttacgac attgcatgtg gaaaggatct gaagagattt ctcctggtac ataataatct   3960 

actcctttgc tacgttaata agagatgtaa aaacatgcaa cagttccagt gccaacattg   4020 

tccaaggatt gtgcaattct ttctggagcg ctaaaattga ccagattaga cgcatcagaa   4080 

tattgaattg cagagttagc caataatcct cataatgtta atgtgctatt gttgttcact   4140 

actcaatata gttctggact aacaatcaga ttgtttatga tattaaggtg gttggatctc   4200 

tattggtatt gtcggcgatt ggaagttctt gcagcttgac aagtctacta tatattggta   4260 

ggtattccag ataaatatta aattttaata aaacaatcac acagaaggat ctgcggccgc   4320 

tagcctaggc ccgggcccac aaaaatctga gcttaacagc acagttgctc ctctcagagc   4380 

agaatcgggt attcaacacc ctcatatcaa ctactacgtt gtgtataacg gtccacatgc   4440 

cggtatatac gatgactggg gttgtacaaa ggcggcaaca aacggcgttc ccggagttgc   4500 

acacaagaaa tttgccacta ttacagaggc aagagcagca gctgacgcgt acacaacaag   4560 

tcagcaaaca gacaggttga acttcatccc caaaggagaa gctcaactca agcccaagag   4620 

ctttgctaag gccctaacaa gcccaccaaa gcaaaaagcc cactggctca cgctaggaac   4680 

caaaaggccc agcagtgatc cagccccaaa agagatctcc tttgccccgg agattacaat   4740 

ggacgatttc ctctatcttt acgatctagg aaggaagttc gaaggtgaag gtgacgacac   4800 

tatgttcacc actgataatg agaaggttag cctcttcaat ttcagaaaga atgctgaccc   4860 

acagatggtt agagaggcct acgcagcagg tctcatcaag acgatctacc cgagtaacaa   4920 

tctccaggag atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac   4980 

taattgcatc aagaacacag agaaagacat atttctcaag atcagaagta ctattccagt   5040 

atggacgatt caaggcttgc ttcataaacc aaggcaagta atagagattg gagtctctaa   5100 

aaaggtagtt cctactgaat ctaaggccat gcatggagtc taagattcaa atcgaggatc   5160 

taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg   5220 

acaagaagaa aatcttcgtc aacatggtgg agcacgacac tctggtctac tccaaaaatg   5280 

tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa aggataattt   5340 

cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag   5400 

aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggct atcattcaag   5460 

atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa   5520 

aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgacatc tccactgacg   5580 

taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt   5640 

catttcattt ggagaggaca cgctgaaatc accagtctct ctctataaat ctatctctct   5700 

ctctataacc atggacccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga   5760 

catgccggcg gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg   5820 

taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta   5880 

tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg   5940 

gaaggcacgc aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca   6000 

ccagcggacg ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca   6060 

gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca   6120 

cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa   6180 

ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt   6240 

cctgcccgtc accgagatct gagatcacgc gttctaggat cccccgatga gctaagctag   6300 

ctatatcatc aatttatgta ttacacataa tatcgcactc agtctttcat ctacggcaat   6360 

gtaccagctg atataatcag ttattgaaat atttctgaat ttaaacttgc atcaataaat   6420 

ttatgttttt gcttggacta taatacctga cttgttattt tatcaataaa tatttaaact   6480 

atatttcttt caagatggga attaacatct acaaattgcc ttttcttatc gaccatgtac   6540 

gtatcgcg                                                            6548 

 
           
             5  
             6539  
             DNA  
             Escherichia coli LE392  
             
               misc_feature  
               Clone pTS431  
             
           
            5 

aattcaagct tgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt     60 

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg    120 

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt    180 

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta    240 

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc    300 

ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa    360 

gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc    420 

cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt    480 

acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact    540 

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac    600 

aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata    660 

ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta    720 

ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg    780 

gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat    840 

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt    900 

aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga    960 

aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa   1020 

gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag   1080 

gtgaagatcc tttttggctc gagtctcatg accaaaatcc cttaacgtga gttttcgttc   1140 

cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg   1200 

cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg   1260 

gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca   1320 

aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg   1380 

cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg   1440 

tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga   1500 

acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac   1560 

ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat   1620 

ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc   1680 

tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga   1740 

tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc   1800 

ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg   1860 

gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag   1920 

cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc   1980 

gcgcgttggc ctgatcagaa ttcttcccga tctagtaaca tagatgacac cgcgcgcgat   2040 

aatttatcct agtttgcgcg ctatattttg ttttctatcg cgtattaaat gtataattgc   2100 

gggactctaa tcataaaaac ccatctcata aataacgtca tgcattacat gttaattatt   2160 

acatgcttaa cgtaattcaa cagaaattat atgataatca tcgcaagacc ggcaacagga   2220 

ttcaatctta agaaacttta ttgccaaatg tttgaacgat ctgcttcgga tcctctagat   2280 

tactgatttt tgtaaaggtc tgataatggt ccgttgtttt gtaaatcagc cagtcgcttg   2340 

agtaaagaat ccggtctgaa tttctgaagc ctgatgtata gttaatatcc gcttcacgcc   2400 

atgttcgtcc gcttttgccc gggagtttgc cttccctgtt tgagaagatg tctccgccga   2460 

tgcttttccc cggagcgacg tctgcaaggt tcccttttga tgccacccag ccgagggctt   2520 

gtgcttctga ttttgtaatg taattatcag gtagcttatg atatgtctga agataatccg   2580 

caaccccgtc aaacgtgttg aataaccggt accatcgcga cggcttgatg gatctcttgc   2640 

tggacaccgg gatgctagga tgggttatcg tggccggcgt gcgtgtgtgg cttttgtagg   2700 

cgccggcgac ggcgggggca atgtggcagg tgagtcacgg tgcaagcgtg cgcaagtgac   2760 

tgcaacaacc aaggacggtc atggcgaaag cacctcacgc gtccaccgtc tacaggatgt   2820 

agcagtagca cggtgaaaga agtgttgtcc cgtccattag gtgcattctc accgttggcc   2880 

agaacaggac cgttcaacag ttaggttgag tgtaggactt ttacgtggtt aatgtatggc   2940 

aaatagtagt aaattttgcc cccattggtc tggctgagat agaacatatt ctggaaagcc   3000 

tctagcatat cttttttgac agctaaactt tgcttcttgc cttcttggtc tagcaatgac   3060 

gttgcccatg tcgtggcaaa catctggtaa ggtaactgta ttcgtttgtt cccttcaacg   3120 

gctcaatccc cacaggccaa gctatccttt ccttggcagt ataggctcct tgagagatta   3180 

tactaccatt tttaagtgct tataaagacg atgctctcta accagatcga tcagaaacac   3240 

aaagttttag cagcgtaata tcccacacac atacacacac gaagctatgc ctcctcattt   3300 

tccgagagat tctgacagtg accagaatgt cagaatgcca tttcatgggc acaagtcgat   3360 

ccacaagctt cttggtggag gtcaaggtgt gctattatta ttcgctttct aggaaattat   3420 

tcagaattag tgccttttat cataacttct ctctgagccg atgtggtttt ggatttcatt   3480 

gttgggagct atgcagttgc ggatattctg ctgtggaaga acaggaactt atctgcgggg   3540 

gtccttgctg gggcaacatt gatatggttc ctgttcgatg tagtagaata caatataatt   3600 

ccgctccttt gccagattgc cattcttgcc atgcttgtga tcttcatttg gtcaaatgcc   3660 

gcaccactct tggacaggta ttagctttat ttcctgtgga gatggtagaa aactcagctt   3720 

acagaaatgg catttcacgt agtataacgc aagacattag gtactaaaac tcaactaact   3780 

gtttccgaat ttcagggccc ctccaaggat cccagaaatc atcatctctg aacatgcctt   3840 

cagagaaatg gcattgaccg tccattacaa actaacgtac actgtatctg ttctttacga   3900 

cattgcatgt ggaaaggatc tgaagagatt tctcctggta cataataatc tactcctttg   3960 

ctacgttaat aagagatgta aaaacatgca acagttccag tgccaacatt gtccaaggat   4020 

tgtgcaattc tttctggagc gctaaaattg accagattag acgcatcaga atattgaatt   4080 

gcagagttag ccaataatcc tcataatgtt aatgtgctat tgttgttcac tactcaatat   4140 

agttctggac taacaatcag attgtttatg atattaaggt ggttggatct ctattggtat   4200 

tgtcggcgat tggaagttct tgcagcttga caagtctact atatattggt aggtattcca   4260 

gataaatatt aaattttaat aaaacaatca cacagaagga tctgcggccg ctagcctagg   4320 

cccgggccca caaaaatctg agcttaacag cacagttgct cctctcagag cagaatcggg   4380 

tattcaacac cctcatatca actactacgt tgtgtataac ggtccacatg ccggtatata   4440 

cgatgactgg ggttgtacaa aggcggcaac aaacggcgtt cccggagttg cacacaagaa   4500 

atttgccact attacagagg caagagcagc agctgacgcg tacacaacaa gtcagcaaac   4560 

agacaggttg aacttcatcc ccaaaggaga agctcaactc aagcccaaga gctttgctaa   4620 

ggccctaaca agcccaccaa agcaaaaagc ccactggctc acgctaggaa ccaaaaggcc   4680 

cagcagtgat ccagccccaa aagagatctc ctttgccccg gagattacaa tggacgattt   4740 

cctctatctt tacgatctag gaaggaagtt cgaaggtgaa ggtgacgaca ctatgttcac   4800 

cactgataat gagaaggtta gcctcttcaa tttcagaaag aatgctgacc cacagatggt   4860 

tagagaggcc tacgcagcag gtctcatcaa gacgatctac ccgagtaaca atctccagga   4920 

gatcaaatac cttcccaaga aggttaaaga tgcagtcaaa agattcagga ctaattgcat   4980 

caagaacaca gagaaagaca tatttctcaa gatcagaagt actattccag tatggacgat   5040 

tcaaggcttg cttcataaac caaggcaagt aatagagatt ggagtctcta aaaaggtagt   5100 

tcctactgaa tctaaggcca tgcatggagt ctaagattca aatcgaggat ctaacagaac   5160 

tcgccgtgaa gactggcgaa cagttcatac agagtctttt acgactcaat gacaagaaga   5220 

aaatcttcgt caacatggtg gagcacgaca ctctggtcta ctccaaaaat gtcaaagata   5280 

cagtctcaga agaccaaagg gctattgaga cttttcaaca aaggataatt tcgggaaacc   5340 

tcctcggatt ccattgccca gctatctgtc acttcatcga aaggacagta gaaaaggaag   5400 

gtggctccta caaatgccat cattgcgata aaggaaaggc tatcattcaa gatgcctctg   5460 

ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg   5520 

ttccaaccac gtcttcaaag caagtggatt gatgtgacat ctccactgac gtaagggatg   5580 

acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt   5640 

tggagaggac acgctgaaat caccagtctc tctctataaa tctatctctc tctctataac   5700 

catggaccca gaacgacgcc cggccgacat ccgccgtgcc accgaggcgg acatgccggc   5760 

ggtctgcacc atcgtcaacc actacatcga gacaagcacg gtcaacttcc gtaccgagcc   5820 

gcaggaaccg caggagtgga cggacgacct cgtccgtctg cgggagcgct atccctggct   5880 

cgtcgccgag gtggacggcg aggtcgccgg catcgcctac gcgggcccct ggaaggcacg   5940 

caacgcctac gactggacgg ccgagtcgac cgtgtacgtc tccccccgcc accagcggac   6000 

gggactgggc tccacgctct acacccacct gctgaagtcc ctggaggcac agggcttcaa   6060 

gagcgtggtc gctgtcatcg ggctgcccaa cgacccgagc gtgcgcatgc acgaggcgct   6120 

cggatatgcc ccccgcggca tgctgcgggc ggccggcttc aagcacggga actggcatga   6180 

cgtgggtttc tggcagctgg acttcagcct gccggtaccg ccccgtccgg tcctgcccgt   6240 

caccgagatc tgagatcacg cgttctagga tcccccgatg agctaagcta gctatatcat   6300 

caatttatgt attacacata atatcgcact cagtctttca tctacggcaa tgtaccagct   6360 

gatataatca gttattgaaa tatttctgaa tttaaacttg catcaataaa tttatgtttt   6420 

tgcttggact ataatacctg acttgttatt ttatcaataa atatttaaac tatatttctt   6480 

tcaagatggg aattaacatc tacaaattgc cttttcttat cgaccatgta cgtatcgcg    6539 

 
           
             6  
             37  
             DNA  
             Artificial Sequence  
             
               Pimer 1 targeted to Bacillus amyloliquefaciens  
             
           
            6 

cgttcggctc gatggtaccg gttatcaaca cgtttga                              37 

 
           
             7  
             38  
             DNA  
             Artificial Sequence  
             
               Primer 2 targeted to Bacillus amyloliquefaciens  
             
           
            7 

cctctagatt atctgatttt tgtaaaggtc tgataatg                             38