Abstract:
The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant  Bacillus subtilis  that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from  Streptococcus zooepidemicus  and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L −1 . Moreover, HAs with a broad range of molecular weights (10 3  Da to 10 6  MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

Description:
CROSS-REFERENCES AND RELATED APPLICATIONS 
     This application claims the benefit of priority to Chinese Application No. 201510573851.X, entitled “A method of constructing a recombinant  Bacillus subtilis  that can produce specific-molecular-weight hyaluronic acids”, filed Sep. 10, 2015, which is herein incorporated by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention relates to the field of biotechnology engineering, and more particularly relates to a method of constructing a recombinant  Bacillus subtilis  that can produce specific-molecular-weight hyaluronic acids. 
     Description of the Related Art 
     Hyaluronic Acid (HA or hyaluronan), a highly viscous polysaccharide, was first isolated from bovine vitreous and it is the best moisturizing substances found in nature. The unique rheological, the viscoelastic and hygroscopic properties along with the biocompatibility and non-immunogenicity has enabled HA to be widely used in cosmetics, food and pharmaceutics. Recent studies have found that HAs with low molecular weight (less than 1×10 4  Da) and HA oligosaccharides have unique biological functions, for example, HA oligosaccharides with molecular weight less than 1×10 4  Da are involved in wound healing and tumor cell apoptosis. In addition, the HA oligosaccharides can be easily absorbed by human body and act as precursors for the body&#39;s own synthesis of polysaccharides. Therefore, HA oligosaccharides have important applications in the areas of food, health care and medicine. 
     HA is widely distributed in animal tissues, such as comb, synovial fluid, cartilage and vitreous. And it is also distributed in bacteria, such as  Bacillus aerogenes, Pseudomonas aeruginosa  and  Hemolytic streptococcus . Due to disadvantages (such as the risk of cross-species viral infection) of the traditional HA extraction methods from animal tissues, the commercial HA production is mainly relied on fermentation of certain attenuated strains of group C  Streptococcus  (such as  S. equi  and  S. zooepidemicus ). The HA produced by microbial fermentation was mostly high molecular weight HA. With increasing health and safety requirements, it becomes more and more urgent to find a safe and reliable microbial host for HA production. 
     Although de novo synthesis methods for preparing HA oligosaccharides have been reported, it is difficult to achieve large-scale production due to high cost of substrates, complex synthesis steps and low yields. Currently, physical and chemical degradation are the main methods used for low-molecular weight HA production, which have many disadvantages, such as generation of HA with a wide range of molecular weight distribution, poor product stability, high cost of purification, high energy consumption and high pollution. Compared to physical or chemical degradation, enzymatic catalytic synthesis of HA oligosaccharides is a promising approach that offers great industrial potential. However, the enzymatic method requires preparation of large amount of hyaluronidase (HAase) and precise control of the enzymatic catalytic reaction conditions. Therefore, constructing a single microbial strain that simultaneously produces both HA and HAase can offer great benefits in HA research and industrial applications. 
     HAase is a class of hyaluronic acid-degrading enzymes widely distributed in nature. According to sources, structures and function mechanisms of the enzyme, HAase are divided into three categories: endo-β-N-acetyl-glucosaminidase (EC 3.2.1.35, mainly exist in mammals and venom of bees, snakes and spiders), endo-β-glucuronidase enzyme (EC 3.2.1.36, mainly in the leech) and hyaluronic acid lyase (EC 4.2.2.1, mainly exist in bacteria, bacteriophages and fungi). As a “scatter factor”, HAase is widely used as auxiliaries for drug diffusion in clinical applications. However, the commercial HAase obtained from bovine testicular tissue is usually of poor quality and expensive to produce, and has the risk of Animal foci infection. 
     In the present invention, a HA biosynthetic pathway was constructed in  Bacillus subtilis  ( B. subtilis ) and high yield of HA was achieved by regulating the expression of important genes for synthesis of HA precursors, UDP-GlcNAc and UDP-GlcA, in the engineered strain. In addition, Leech HAase was co-expressed in the engineered strain with a HA biosynthetic pathway to achieve synchronous production of HA and HAase. Production of HA with specific molecular weights was achieved by precise regulation of the expression levels of HAase. The problem of fermentation stagnation caused by high viscosity of HA was solved by coupling the production of HA and HAase, thus greatly increasing the production efficiency. The present invention for the first time achieved efficient synthesis of HA with specific molecular weights, which has potential for bringing great economic gains in industrial applications. 
     DETAILED DESCRIPTION 
     The present invention provides a recombinant  B. subtilis  that can produce HA having molecular weights within specific ranges (specific-molecular-weight HA). A HA synthetic pathway is constructed and a HAase is coexpressed in the recombinant strain; and the RBS (ribosome binding sites) optimization strategy is performed to regulate the expression levels of HAase at the translational level, which in turn regulates the average molecular weight of HA products. 
     In one embodiment, the present invention provides a recombinant  B. subtilis  that the molecular weight of HA products in the recombinant strain is controlled by the expression level of HAase, wherein the higher is the expression of HAase, the lower is the molecular weight of the HA product and the higher is the yield of HA production. 
     In one embodiment of the present invention, a regulatory DNA fragment and a HAase gene are integrated into the genome of a  B. subtilis  containing a HA synthetic pathway. The said regulatory DNA fragment contains a constitutive promoter P lepA , a RBS sequence and a signal peptide. In one embodiment, the nucleotide sequences of the regulatory DNA fragment is set forth in SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13. The DNA integration in the recombinant strain is mediated by plasmid pBlueScript SK (+). 
     In one embodiment of the present invention, the  B. subtilis  host is  B. subtilis  168. 
     In one embodiment of the present invention, the nucleotide sequence of the HAase is SEQ ID NO: 7. 
     In one embodiment of the present invention, genes of the HA synthetic pathway contains hasA, which encodes a hyaluronan synthase. The hasA is derived from  Streptococcus zooepidemicus, Streptococcus equi  or  Streptococcus equissp.    
     In one embodiment of the present invention, the hasA is derived from  Streptococcus zooepidemicus  with a nucleotide sequence of SEQ ID NO: 1. 
     In one embodiment of the present invention, the HA biosynthetic pathway is obtained by further constructing a biosynthetic pathway for HA precursors, UPD-N-acetylglucosamine (UDP-GlcNAc) and UDP-D-glucuronide (UDP-GlcA), in a recombinant  B. subtilis  containing a hyaluronan synthase. 
     In one embodiment of the present invention, genes of UDP-GlcA and UDP-GlcNAc biosynthetic pathway are derived from  Streptococcus  species,  Escherichia coli  or  Bacillus . In one embodiment of the present invention, the genes are derived from  B. subtilis , containing tuaD (the nucleotide sequence is SEQ ID NO: 2) which encodes a UDP-glucose dehydrogenase, glmU (the nucleotide sequence is SEQ ID NO: 3) which encodes a UDP-N-acetylglucosamine pyrophosphorylase, gtaB (the nucleotide sequence is SEQ ID NO: 4) which encodes a UDP-glucose pyrophosphorylase, glmM (the nucleotide sequence is SEQ ID NO: 5) which encodes a mutase and glmS (the nucleotide sequence is SEQ ID NO: 6) which encodes an amino transferase. 
     In one embodiment of the present invention, the HAase gene deriving from leeches is fused with a signal peptide and a promoter, and then integrated into a recombinant  B. subtilis  with a HA biosynthetic pathway. 
     The present invention provides a method of constructing a recombinant  B. subtilis  that produces specific-molecular-weight HA. The method comprises the following steps:
         (1) construction of a HA biosynthetic pathway: a hyaluronan synthase hasA gene is inserted into plasmid pAX01, and the obtained recombinant plasmid is transformed into  B. subtilis , resulting in the hasA gene integrated into the genome of  B. subtilis  under the control of Pxyl promoter. The recombinant  B. subtilis  strain is designated as E168T. The tuaD and glmU are respectively fused with strong ribosome binding site, P43. The gtaB was fused with promoter Pveg and P43 RBS, the glmM and glmS were respectively fused with P43 RBS. The five fused fragments are connected in series and inserted into pP43NMK vector. The resulting recombinant plasmid is transformed into E168T. A recombinant  B. subtilis  with a HA biosynthetic pathway is thus obtained.   (2) The hyaluronidase gene is fused with PlepA promoter, a RBS and a signal peptide, and integrated into the genome of the recombinant  B. subtilis  obtained in step (1), resulting in a recombinant  B. subtilis  containing a HA biosynthetic pathway and HAase coexpression.   (3) Expression levels of the HAase in step (2) are precisely controlled by the translational strength of different RBS sequences, the higher the translational strength of the RBS, the lower the molecular weight of HA produced by the recombinant  B. subtilis.          

     In one embodiment of the present invention, hasA gene is integrated at the lacA (β-galactosidase gene) locus of  B. subtilis  chromosome in the step (1). 
     In one embodiment of the present invention, hyaluronidase gene is integrated at the glucosamine-6-phosphate deaminase 1 (nagA-nagBA) locus of  B. subtilis  chromosome in the step (2). 
     In one embodiment of the present invention, the expression of HAase in step (2) is controlled by the wild-type RBS with a nucleotide sequence of aggaggaa (contained in the regulatory DNA fragment shown in SEQ ID NO:8). The average molecular weight of HA produced by the recombinant is 6628 dalton, the HAase activity reaches 1.62×10 6  U/mL and the yield of HA reaches 19.38 g/L. 
     In another embodiment of the present invention, the expression of HAase in step (2) is controlled by RBS mutant R1 with a nucleotide sequence of aagaggag (contained in the regulatory DNA fragment shown in SEQ ID NO:12). The average molecular weight of HA produced by the recombinant is 18,000 dalton, the HAase activity reaches 8.8×10 5  U/mL and the yield of HA reaches 9.18 g/L. 
     In another embodiment of the present invention, the expression of HAase in step (2) is controlled by RBS mutant R2 with a nucleotide sequence of acgtagac (contained in the regulatory DNA fragment shown in SEQ ID NO:13). The average molecular weight of HA produced by the recombinant is 49,600 dalton, the HAase activity reaches 6.4×10 4  U/mL and the yield of HA reaches 7.13 g/L. 
     In another embodiment of the present invention, the average molecular weight of HA produced by the recombinant strain obtained in step (1), which has no expression of HAse, is 1,420,000 dalton and the yield of HA is 5.96 g/L. 
     The present invention also provides a method of producing specific-molecular-weight HA (10 3  Da&lt;Mr&lt;10 6  Da) by using the recombinant  B. subtilis  strain. The method is performed by cultivating the recombinant in a 3 L fermenter with a fed-batch fermentation strategy at 37° C. for 56-96 hours, and specific-molecular-weight HA ranging from 6628 Da to 1420000 Da can be obtained. The medium for cultivation contains 5% yeast extract, 2% sucrose, 15.6 g/L sodium dihydrogen phosphate and 3.9 g/L potassium sulfate. In addition, the main products are low-molecular-weight HA (LMW-HA) or HA oligosaccharides (HA-4, HA-6, HA-8, HA-10, and so on) if secreted HAase is allowed to continue digesting the HA product after the end of fermentation. 
     In one embodiment, the carbon source in the cultivation medium is sucrose. 
     In one embodiment, the fermentation temperature is 37° C. 
     In one embodiment of the present invention, the fermentation time is set differently according to different recombinant strains. 
     After separation and purification, the HAase in the fermentation broth can be used in food, medical or clinical applications. 
     Compared with other engineered strains, the HA producing recombinant of the present invention has many advantages. Firstly, the present invention uses the food grade host of the recombinant strain that meets the requirements of health and food safety, and does not have any infection risk of endotoxins or pathogens. Secondly, the high molecular weight HA produced by the recombinant strain is degraded into LMW-HA by an extracellularly secreted HAase. The biosynthesis of HA is coupled with secretory expression of HAase in the present invention to reduce the viscosity of the fermentation broth, thus increasing the dissolved oxygen and enhance the yield of HA. Additionally, the molecular weight of HA in fermentation broth could be precisely controlled with a broad range from 10 3  to 10 6  Da, and purification and recovery of final products is very simple and easy to operate. Therefore, the method of the present invention has great value for large-scale production of specific low-molecular-weight hyaluronic acids. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1 . Construction schematic of the regulatory DNA fragment for controlling HAase expression. 
         FIG. 2 . High throughput screening of the different expression levels of HAase activity using a standard plate. 
         FIG. 3 . The curve of dissolved oxygen (DO) in recombinant strains with different expression levels of HAase cultured in a 3 L fermentor. 
         FIG. 4 . The HAase activity of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor. 
         FIG. 5 . The production of HA of recombinant strains with different expression levels of HAase cultured in a 3 L fermentor. 
         FIG. 6 . The average molecular weight of HA produced in different recombinant strains. 
     
    
    
     EXAMPLES 
     Materials and Methods: 
     Information of related nucleotide sequences:
         (1) SEQ ID NO: 1 is the nucleotide sequence of hyaluronic acid synthase gene hasA from  Streptococcus pneumoniae.      (2) SEQ ID NO: 2 is the nucleotide sequence of UDP-glucose dehydrogenase gene tuaD from  B. subtilis.      (3) SEQ ID NO: 3 is the nucleotide sequence of UDP-N-acetylglucosamine pyrophosphorylase gene glmU from  B. subtilis.      (4) SEQ ID NO: 4 is the nucleotide sequence of UDP-glucose pyrophosphorylase gene gtaB from  B. subtilis.      (5) SEQ ID NO: 5 is the nucleotide sequence of a mutase gene glmM from  B. subtilis.      (6) SEQ ID NO: 6 is the nucleotide sequence of an amino transferase gene glmS from  B. subtilis.      (7) SEQ ID NO: 7 is the nucleotide sequence of a Leech hyaluronidase gene.   (8) SEQ ID NO: 8 is the nucleotide sequence of a regulatory DNA fragment P lepA -RBS-yewA.   (9) SEQ ID NO: 9 is the nucleotide sequence of a bleomycin resistant gene.   (10) SEQ ID NO: 10 is the nucleotide sequence of constitutive promoter P43.   (11) SEQ ID NO: 11 is the nucleotide sequence of inducible promoter Pveg.   (12) SEQ ID NO: 12 is the nucleotide sequence of a regulatory DNA fragment P lepA -RBS1-yewA.   (13) SEQ ID NO:13 is the nucleotide sequence of a regulatory DNA fragment P lepA -RBS2-yewA.       

     The HA titers were routinely estimated by the modified carbazole assay. The HA titer is assumed to be 2.067 times the glucuronic acid titer. 
     Leech hyaluronidase (LHAse or LHyal) activity was quantified by measuring the amount of reducing sugar liberated from HA using the 3,5-dinitrosalicylic acid (DNS) colorimetric spectrophotometric method. One unit of enzymatic activity is defined as equal to the reducing power of glucuronic acid (glucose equivalents in micrograms) liberated per hour from HA at 38° C., pH 5.5. Specific activity is defined as units of enzyme per ml of culture supernatant. The standard enzymatic reaction contained appropriate volumes of fermentation supernatant and 1.6 mg·ml −1  of HA as the substrate was incubated in 50 mM citrate-disodium hydrogen phosphate buffer at 38° C., pH 5.5 for 10 min in a total volume of 1 ml. The reaction was stopped by immersing in boiling water for 2 min and the enzyme activity was examined using the DNS method. Controls with fermentation supernatant of  B. subtilis  168 were prepared and analyzed in the same manner 
     The average molecular weight of HA was measured by high performance gel filtration chromatography (HPGFC) with a multi-angle laser light scattering detector (MALLS). The mobile phase was 0.1 mol·L −1  NaNO 3  and the temperature of the column was maintained at 40° C. The sample size was 40 μL and elution time for each sample was 25 min. Dextran produced from Chinese Institute of food and drug testing was used as a standard and GPC software was used to calculate the average molecular weight. 
     Example 1 
     Construction of the Recombinant Plasmid pAX01-hasA 
     Hyaluronan synthase hasA was cloned from  S. zooepidemicus  ATCC 35246 with primers hasA-F/hasA-R to amplify the hasA gene by polymerase chain reaction (PCR). The  S. zooepidemicus  strain was incubated in 5 mL M17 media at 37° C., 200 rpm for 16 hours, and the chromosome of  S. zooepidemicus  was extracted by a bacterial genome extraction kit. 
     The nucleotide sequences of primers hasA-F and hasA-R were as follows (from 5′ to 3′): 
     
       
         
               
               
             
           
               
                   
                 hasA-F: 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 CGCGGATCCATGAGAACATTAAAAAACCTCATAAC 
               
               
                   
                   
               
               
                   
                 hasA-R: 
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 TGCATGCATTTATAATAATTTTTTACGTGTTCC 
               
             
          
         
       
     
     Gene fragment of hasA amplified by PCR and pAX01 plasmid were digested with restriction enzymes BamHI and SacII, respectively. The digested fragments were recovered for ligation. Then the ligation products were used to transform to JM109 competent cells and positive recombinant plasmid pAX01-hasA was verified by sequencing. Then, the pAX01-hasA was transformed into  B. subtilis  168, resulting in the hasA gene integrated into the genome of  B. subtilis  168 under the control of Pxyl promoter. The recombinant strain was designated as E168T. 
     Example 2 
     Construction of the Recombinant Plasmid pP43NMK/pP43-DU-PBMS 
     tuaD gene and glmU gene were amplified from  B. subtilis  168 by PCR using primers tuaD-F/tuaD-R and glmU-F/glmU-R, respectively. KpnI restriction site and P43 RBS sequence (shown in SEQ ID NO:14) were introduced to the 5′ of tuaD-F. SacI restriction site was introduced to the 5′ of tuaD-R. SacI restriction site and P43 RBS sequence were introduced to the 5′ of glmU-F. XhoI and XbaI restriction sites were introduced to the 5′ of glmU-R. The resulting tuaD fragment and glmU fragment were digested with KpnI/SacI and SacI/XhoI, respectively. The digested fragments were purified and ligated together with digested pP43NMK (KpnI/XhoI) fragment. Then, the obtained ligation product was transformed into JM109 competent cells. The positive recombinant cells was verified by sequencing and the recombinant plasmid was designated as pP43-DU. 
     The Pveg promoter fragment amplified with the primer pair Pveg-F/Pveg-R was fused with the gtaB gene amplified with the primer pair Pveg-gtaB-F(containing a P43 RBS) and gtaB-R. SpeI and XbaI-XhoI restriction sites were introduced to the 5′ and the 3′ of the fusion fragment, respectively. The fusion product was digested with SpeI and XhoI, and ligated with digested pP43-DU fragment (XbaI and XhoI), resulting in a recombinant plasmid designated as pP43-DU-PB. 
     By use of the same isocaudarner SpeI/XbaI, glmM and glmS genes were amplified with primers glmM-F/R and glmS-F/R, respectively. The glmM and glmS fragments were inserted into plasmid pP43-DU-PB in order, generating the recombinant plasmid pP43-DU-PBMS. pP43-DU-PBMS was transformed into E168T competent cells and a recombinant strain E168T/pP43-DU-PBMS with high yield of HA was obtained. 
     The primers used were as follows: 
     
       
         
               
             
           
               
                 tuaD-F: 
               
               
                 (SEQ ID NO: 17) 
               
               
                 CGGGGTACCAAGAGAGGAATGTACACATGAAAAAAATAGCTGTCATTGG 
               
               
                   
               
               
                 tuaD-R: 
               
               
                 (SEQ ID NO: 18) 
               
               
                 CCGGAGCTCTTATAAATTGACGCTTCCCAAG 
               
               
                   
               
               
                 glmU-F: 
               
               
                 (SEQ ID NO: 19) 
               
               
                 CGGGAGCTCAAGAGAGGAATGTACACATGGATAAGCGGTTTGCAGTTG 
               
               
                   
               
               
                 glmU-R: 
               
               
                 (SEQ ID NO: 20) 
               
               
                 CCGCTCGAGCGGACTCTAGTCTAGATTATTTTTTATGAATATTTTTCAC 
               
               
                   
               
               
                 Pveg-F: 
               
               
                 (SEQ ID NO: 21) 
               
               
                 GGACTAGTGGAGTTCTGAGAATTGGTATGC 
               
               
                   
               
               
                 Pveg-R: 
               
               
                 (SEQ ID NO: 22) 
               
               
                 ATGTAAATCGCTCCTTTTTAACTAC 
               
               
                   
               
               
                 Pveg-gtaB-F: 
               
               
                 (SEQ ID NO: 23) 
               
               
                 GTAGTTAAAAAGGAGCGATTTACATATGAAAAAAGTACGTAAAGC 
               
               
                   
               
               
                 glmM-F: 
               
               
                 (SEQ ID NO: 24) 
               
               
                 GGACTAGTAAGAGAGGAATGTACACATGGGCAAGTATTTTGGAACAG 
               
               
                 ACGG 
               
               
                   
               
               
                 glmM-R: 
               
               
                 (SEQ ID NO: 25) 
               
               
                 CCGCTCGAGCGGACTCTAGTCTAGATTACTCTAATCCCATTTCTGAC 
               
               
                 CGGAC 
               
               
                   
               
               
                 glmS-F: 
               
               
                 (SEQ ID NO: 26) 
               
               
                 GGACTAGTAAGAGAGGAATGTACACATGTGTGGAATCGTAGGTTATA 
               
               
                 TCGG 
               
               
                   
               
               
                 glmS-R: 
               
               
                 (SEQ ID NO: 27) 
               
               
                 CCGCTCGAGCGGACTCTAGTCTAGATTACTCCACAGTAACACTCTTCGC 
               
             
          
         
       
     
     Example 3 
     Construction of the Integrated Gene Fragment of LHyal 
     The gene encoding hyaluronidase was integrated at the glucosamine-6-phosphate deaminase 1 (nagA-nagBA) locus of  B. subtilis  168 using Zeocin gene as the selection marker. The integrated fragment (shown in  FIG. 1 ) was obtained by homologous recombination technique. 
     The primers used were as follows: 
     
       
         
               
               
             
           
               
                   
                 H6LHyal-F: 
               
               
                   
                 (SEQ ID NO: 28) 
               
               
                   
                 ATGCACAGTCTGCAGAATTCCACCACCACCACCACCACATG 
               
               
                   
                   
               
               
                   
                 H6LHyal-R: 
               
               
                   
                 (SEQ ID NO: 29) 
               
               
                   
                 TTACTTTTTGCACGCTTCAACAT 
               
               
                   
                   
               
               
                   
                 ZHLHPlepA-F: 
               
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
                 CGCAGCCAAAGGAGTGGATTGCCTCAATCCTAGGAGAAACAG 
               
               
                   
                   
               
               
                   
                 ZHLHPlepA-R: 
               
               
                   
                 (SEQ ID NO: 31) 
               
               
                   
                 GAATTCTGCAGACTGTGCATGAGC 
               
               
                   
                   
               
               
                   
                 ZHLH-front-F: 
               
               
                   
                 (SEQ ID NO: 32) 
               
               
                   
                 TCAGCTGGTCTAGATCACTAGTC 
               
               
                   
                   
               
               
                   
                 ZHLH-front-R: 
               
               
                   
                 (SEQ ID NO: 33) 
               
               
                   
                 AATCCACTCCTTTGGCTGCGCTC 
               
               
                   
                   
               
               
                   
                 ZHLH-zeocin-F: 
               
               
                   
                 (SEQ ID NO: 34) 
               
               
                   
                 TTGAAGCGTGCAAAAAGTAAGAGCTCGGTACCCGGGGATCC 
               
               
                   
                   
               
               
                   
                 ZHLH-zeocin-R: 
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                 GCTTGCATGCCTGCAGGTCGAC 
               
               
                   
                   
               
               
                   
                 ZHLH-back-F: 
               
               
                   
                 (SEQ ID NO: 36) 
               
               
                   
                 CGACCTGCAGGCATGCAAGCCACTTCTTTCAGACGGAACCCTTGC 
               
               
                   
                   
               
               
                   
                 ZHLH-back-R: 
               
               
                   
                 (SEQ ID NO: 37) 
               
               
                   
                 CGGTCGTTCATATAGAAGTGATAG 
               
               
                   
                   
               
               
                   
                 ZHLH-pSK-F: 
               
               
                   
                 (SEQ ID NO: 38) 
               
               
                   
                 CACTTCTATATGAACGACCGCCTGTGTGAAATTGTTATCCGCTC 
               
               
                   
                   
               
               
                   
                 ZHLH-pSK-R: 
               
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                 TAGTGATCTAGACCAGCTGAGTGACTGGGAAAACCCTGGCGTTAC 
               
             
          
         
       
     
     The LHyal gene encoding a leech hyaluronidase (LHyal) was amplified with primers H6LHyal-F/H6LHyal-R and the Zeocin gene was amplified with primers ZHLH-zeocin-F/ZHLH-zeocin-R. The regulatory DNA fragment containing the promoter PlepA, the RBS P43 and the signal peptide yweA was amplified with primers ZHLHPlepA-F/R. The front and back flanking fragments of the target for integration were amplified with primers ZHLH-front-F/R and ZHLH-back-F/R, respectively. A recombinant vector was amplified with primers ZHLH-pSK-F/ZHLH-pSK-R using the plasmid pBlueScript SK(+) as template. The five DNA fragments and the recombinant vector described above were assembled using homologous recombination technology, and the assembled products were transformed into  E. coli  JM109 competent cells. The recombinant plasmid containing the regulatory DNA fragment and leech hyaluronidase gene was designated as pSKZHLH. 
     pSKZHLH was transformed into the competent cells of HA producing strain E168T/pP43-DU-PBMS and the recombinant strain was screened with 25 ug/ml Zeocin. The positive recombinant strain expressing HAase was designated as E168TH/pP43-DU-PBMS. 
     Example 4 
     Construction of RBS Mutant Library for Controlling the Expression of HAase 
     A RBS mutant library with a wide range of translational strength was constructed by genetic engineering at the ribosome regulation level. The degenerate primer JB/lepA-RBS-R, which includes the RBS region, and reverse primer ZHLH-H6F were used to amplify the RBS mutant library using the pSKZHLH as the template. KpnI restriction site was added to the 5′ of both primers. The primers used were as follows: 
     
       
         
               
             
           
               
                 JB/lepA-RBS-R: 
               
               
                 (SEQ ID NO: 40) 
               
               
                 ACGGGGTACCACTNTNYNHBYACTATTAAACGCAAAATACACTAGCTTAG 
               
               
                   
               
               
                 ZHLH-H6F: 
               
               
                 (SEQ ID NO: 41) 
               
               
                 ACGGGGTACCATGCTAAAAAGAACTTCATTCG 
               
             
          
         
       
     
     The PCR product was first digested with DpnI, and then further digested with the restriction endonuclease KpnI, which was used for ligation. The ligation products were transformed into E168T/pP43-DU-PBMS competent cells. Five hundred transformants were picked from LB agar plates with 25 ug/ml Zeocin and then grown in 96-well microtiter at 37° C., 200 rpm for 60 hours. The culture medium contains 2% yeast powder, 7% sucrose, 15.6 g/L sodium dihydrogen phosphate, 3.9 g/L potassium sulfate. 
     The quantitation of hyaluronidase activity of culture supernatants was performed with high throughput screening by transparent ring colorimetric plate assay. 2 mg/ml HA was dissolved in citric acid buffer (pH 5.5) to make a HA buffer and 1.5% agarose was melted by heat in the same citric buffer. Equal volume of HA buffer and heated agarose buffer was mixed and poured into a plastic plate to allow solidification. Multiple holes were drilled in the agarose plate as shown in  FIG. 2 . After centrifuged at 4000 rpm for 5 min, 150 μL supernatant of the fermentation broth of RBS mutant strains was added to the holes in the agarose plate and cultivated at 37° C. for 10 hours. After that, 2.5 g/L cetyltrimethyl ammonium bromide was added and incubated for 30 min. Results ( FIG. 2 ) demonstrated that the mutant strains with RBS modifications exhibit significantly different levels of HAse expression. E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS were two mutant strains with different RBS translational strengths. 
     Example 5 
     Fed-batch Fermentation of the Recombinant Strains in a 3-L Fermentor 
     Recombinant strains E168T/pP43-DU-PBMS, E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 were fermented, respectively. 
     The recombinant strains were grown in a LB medium with 50 μg/ml kanamycin at 37° C. and 200 rpm for 12 hours. The 3-L fermentor contained an initial 1.35 L of fermentation medium (2% Yeast extract, 1.5% sucrose, 15.6 g/L sodium dihydrogen phosphate and 3.9 g/L potassium sulfate, pH 7.0). The seed cultures were transferred into the fermentor with a 10% inoculation volume. Xylose with a final concentration of 20 g/L was used to induce the expression of hasA at 2 hours after the inoculation. 
     Feed started at about 8 hours after inoculation with a simple sucrose solution at index-fed-batch feed rates of 7.5, 7.5, 15.0, 10.0 g·h − ·L −1  for the first 4 hours. The constant feed rate was maintained at 5 g·h − ·L −1  until the end of fermentation. Samples were periodically withdrawn to determine the HA production and HAase activity of the fermentation. After centrifugation at 10000 rpm for 10 min, the fermentation supernatant was transferred to another tube, and 2 volumes ethanol was added to precipitate HA and incubated for 1 hour. The precipitate was collected by centrifugation (10000 rpm for 20 min) and redissolved in equal volume 1 mol·L −1  NaCl solution. The suspension was used for further determination of yield and molecular weight. 
     Due to the viscoelastic properties of HA, the fermentation of engineered strain E168T/pP43-DU-PBMS became very viscous after 15 h and concomitantly resulting in the dramatic decline of dissolved oxygen (DO), which seriously affected the growth of cells and the accumulation of HA.  FIG. 3  showed that the fermentation DO of E168T/pP43-DU-PBMS was almost reduced to 0 at 40 hours, while the fermentation DO of other engineered strains which had different expression levels of HAase were maintained at a higher level. The HAase activities of E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 reached high values of 1.62×10 6 U/mL, 8.8×10 5 U/mL and 6.4×10 4  U/mL, respectively. The HA yield of E168T/pP43-DU-PBMS reached the maximal HA titer of 5.96 g·L −1  due to viscous fermentation. However, the HA yield of the highest HAase expression strain, E168TH/pP43-DU-PBMS, reached 19.38 g·L −1 , and the HA yield of the other two strains, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4, with lower HAase expression reached 9.18 g·L −1  and 7.13 g·L −1 , respectively. These results demonstrated that the higher is the HAase production, the higher is the HA yield. 
     There was a significant difference between the average molecular weight of HA of engineered strains with different HAase expression levels (shown in  FIG. 6 ). The average molecular weight of HA from strain E168T/pP43-DU-PBMS which did not express the HAase was 1.42×10 6  Da, while those of strains E168TH/pP43-DU-PBMS, E168THR1/pP43-DU-PBMS and E168THR2/pP43-DU-PBMS4 were 6628 Da, 18000 Da and 49600 Da, respectively. 
     The results showed that the molecular weight of HA could be precisely controlled within a range from 10 3  to 10 6  Da through controlling the expression level of HAase. Additionally, HA10, HA8, HA6, HA4 and other oligosaccharides could be obtained by allowing the supernatant of the fermentation broth to incubate at room temperature for additional 1-3 hours. 
     * * * 
     While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference. 
     
       
         
               
             
               
             
               
               
             
           
               
                   
               
               
                 Sequence Listing 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 &lt;210&gt; 1 
               
               
                 &lt;211&gt; 1254 
               
               
                 &lt;212&gt; DNA 
               
               
                 &lt;213&gt;  Streptococcus zooepidemicus   
               
               
                 &lt;400&gt; 1 
               
             
          
           
               
                 atgagaacat taaaaaacct cataactgtt gtggccttta gtattttttg ggtactgttg 
                 60 
               
               
                   
               
               
                 atttacgtca atgtttatct ctttggtgct aaaggaagct tgtcaattta tggctttttg 
                 120 
               
               
                   
               
               
                 ctgatagctt acctattagt caaaatgtcc ttatcctttt tttacaagcc atttaaggga 
                 180 
               
               
                   
               
               
                 agggctgggc aatataaggt tgcagccatt attccctctt ataacgaaga tgctgagtca 
                 240 
               
               
                   
               
               
                 ttgctagaga ccttaaaaag tgttcagcag caaacctatc ccctagcaga aatttatgtt 
                 300 
               
               
                   
               
               
                 gttgacgatg gaagtgctga tgagacaggt attaagcgca ttgaagacta tgtgcgtgac 
                 360 
               
               
                   
               
               
                 actggtgacc tatcaagcaa tgtcattgtt caccggtcag aaaaaaatca aggaaagcgt 
                 420 
               
               
                   
               
               
                 catgcacagg cctgggcctt tgaaagatca gacgctgatg tctttttgac cgttgactca 
                 480 
               
               
                   
               
               
                 gatacttata tctaccctga tgctttagag gagttgttaa aaacctttaa tgacccaact 
                 540 
               
               
                   
               
               
                 gtttttgctg cgacgggtca ccttaatgtc agaaatagac aaaccaatct cttaacacgc 
                 600 
               
               
                   
               
               
                 ttgacagata ttcgctatga taatgctttt ggcgttgaac gagctgccca atccgttaca 
                 660 
               
               
                   
               
               
                 ggtaatattc tcgtttgctc aggcccgctt agcgtttaca gacgcgaggt ggttgttcct 
                 720 
               
               
                   
               
               
                 aacatagata gatacatcaa ccagaccttc ctgggtattc ctgtaagtat cggtgatgac 
                 780 
               
               
                   
               
               
                 aggtgcttga ccaactatgc aactgattta ggaaagactg tttatcaatc cactgctaaa 
                 840 
               
               
                   
               
               
                 tgtattacag atgttcctga caagatgtct acttacttga agcagcaaaa ccgctggaac 
                 900 
               
               
                   
               
               
                 aagtccttct ttagagagtc cattatttct gttaagaaaa tcatgaacaa tccttttgta 
                 960 
               
               
                   
               
               
                 gccctatgga ccatacttga ggtgtctatg tttatgatgc ttgtttattc tgtggtggat 
                 1020 
               
               
                   
               
               
                 ttctttgtag gcaatgtcag agaatttgat tggctcaggg ttttggcctt tctggtgatt 
                 1080 
               
               
                   
               
               
                 atcttcattg ttgctctttg tcgtaatatt cactatatgc ttaagcaccc gctgtccttc 
                 1140 
               
               
                   
               
               
                 ttgttatctc cgttttatgg ggtactgcat ttgtttgtcc tacagccctt gaaattgtat 
                 1200 
               
               
                   
               
               
                 tctcttttta ctattagaaa tgctgactgg ggaacacgta aaaaattatt ataa 
                 1254 
               
               
                   
               
               
                 &lt;211&gt; 1386 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 2 
                   
               
               
                 atgaaaaaaa tagctgtcat tggaacaggt tatgtaggac tcgtatcagg cacttgcttt 
                 60 
               
               
                   
               
               
                 gcggagatcg gcaataaagt tgtttgctgt gatatcgatg aatcaaaaat cagaagcctg 
                 120 
               
               
                   
               
               
                 aaaaatgggg taatcccaat ctatgaacca gggcttgcag acttagttga aaaaaatgtg 
                 180 
               
               
                   
               
               
                 ctggatcagc gcctgacctt tacgaacgat atcccgtctg ccattcgggc ctcagatatt 
                 240 
               
               
                   
               
               
                 atttatattg cagtcggaac gcctatgtcc aaaacaggtg aagctgattt aacgtacgtc 
                 300 
               
               
                   
               
               
                 aaagcggcgg cgaaaacaat cggtgagcat cttaacggct acaaagtgat cgtaaataaa 
                 360 
               
               
                   
               
               
                 agcacagtcc cggttggaac agggaaactg gtgcaatcta tcgttcaaaa agcctcaaag 
                 420 
               
               
                   
               
               
                 gggagatact catttgatgt tgtatctaac cctgaattcc ttcgggaagg gtcagcgatt 
                 480 
               
               
                   
               
               
                 catgacacga tgaatatgga gcgtgccgtg attggttcaa caagtcataa agccgctgcc 
                 540 
               
               
                   
               
               
                 atcattgagg aacttcatca gccattccat gctcctgtca ttaaaacaaa cctagaaagt 
                 600 
               
               
                   
               
               
                 gcagaaatga ttaaatacgc cgcgaatgca tttctggcga caaagatttc ctttatcaac 
                 660 
               
               
                   
               
               
                 gatatcgcaa acatttgtga gcgagtcggc gcagacgttt caaaagttgc tgatggtgtt 
                 720 
               
               
                   
               
               
                 ggtcttgaca gccgtatcgg cagaaagttc cttaaagctg gtattggatt cggcggttca 
                 780 
               
               
                   
               
               
                 tgttttccaa aggatacaac cgcgctgctt caaatcgcaa aatcggcagg ctatccattc 
                 840 
               
               
                   
               
               
                 aagctcatcg aagctgtcat tgaaacgaac gaaaagcagc gtgttcatat tgtagataaa 
                 900 
               
               
                   
               
               
                 cttttgactg ttatgggaag cgtcaaaggg agaaccattt cagtcctggg attagccttc 
                 960 
               
               
                   
               
               
                 aaaccgaata cgaacgatgt gagatccgct ccagcgcttg atattatccc aatgctgcag 
                 1020 
               
               
                   
               
               
                 cagctgggcg cccatgtaaa agcatacgat ccgattgcta ttcctgaagc ttcagcgatc 
                 1080 
               
               
                   
               
               
                 cttggcgaac aggtcgagta ttacacagat gtgtatgctg cgatggaaga cactgatgca 
                 1140 
               
               
                   
               
               
                 tgcctgattt taacggattg gccggaagtg aaagaaatgg agcttgtaaa agtgaaaacc 
                 1200 
               
               
                   
               
               
                 ctcttaaaac agccagtcat cattgacggc agaaatttat tttcacttga agagatgcag 
                 1260 
               
               
                   
               
               
                 gcagccggat acatttatca ctctatcggc cgtcccgctg ttcggggaac ggaaccctct 
                 1320 
               
               
                   
               
               
                 gacaagtatt ttccgggctt gccgcttgaa gaattggcta aagacttggg aagcgtcaat 
                 1380 
               
               
                   
               
               
                 ttataa 
                 1386 
               
               
                   
               
               
                 &lt;210&gt; 3 
                   
               
               
                 &lt;211&gt; 1371 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 3 
                   
               
               
                 atggataagc ggtttgcagt tgttttagcg gctggacaag gaacgagaat gaaatcgaag 
                 60 
               
               
                   
               
               
                 ctttataaag tccttcatcc agtttgcggt aagcctatgg tagagcacgt cgtggacgaa 
                 120 
               
               
                   
               
               
                 gccttaaaat tatctttatc aaagcttgtc acgattgtcg gacatggtgc ggaagaagtg 
                 180 
               
               
                   
               
               
                 aaaaagcagc ttggtgataa aagcgagtac gcgcttcaag caaaacagct tggcactgct 
                 240 
               
               
                   
               
               
                 catgctgtaa aacaggcaca gccatttctt gctgacgaaa aaggcgtcac aattgtcatt 
                 300 
               
               
                   
               
               
                 tgcggagata cgccgctttt gacagcagag acgatggaac agatgctgaa agaacataca 
                 360 
               
               
                   
               
               
                 caaagagaag cgaaagctac gattttaact gcggttgcag aagatccaac tggatacggc 
                 420 
               
               
                   
               
               
                 cgcattattc gcagcgaaaa cggagcggtt caaaaaatag ttgagcataa ggacgcctct 
                 480 
               
               
                   
               
               
                 gaagaagaac gtcttgtaac tgagatcaac accggtacgt attgttttga caatgaagcg 
                 540 
               
               
                   
               
               
                 ctatttcggg ctattgatca ggtgtctaat gataatgcac aaggcgagta ttatttgccg 
                 600 
               
               
                   
               
               
                 gatgtcatag agattcttaa aaatgaaggc gaaactgttg ccgcttacca gactggtaat 
                 660 
               
               
                   
               
               
                 ttccaagaaa cgctcggagt taatgataga gttgctcttt ctcaggcaga acaatttatg 
                 720 
               
               
                   
               
               
                 aaagagcgca ttaataaacg gcatatgcaa aatggcgtga cgttgattga cccgatgaat 
                 780 
               
               
                   
               
               
                 acgtatattt ctcctgacgc tgttatcgga agcgatactg tgatttaccc tggaactgtg 
                 840 
               
               
                   
               
               
                 attaaaggtg aggtgcaaat cggagaagat acgattattg gccctcatac ggagattatg 
                 900 
               
               
                   
               
               
                 aatagtgcca ttggcagccg tacggttatt aaacaatcgg tagtcaatca cagtaaagtg 
                 960 
               
               
                   
               
               
                 gggaatgatg taaacatagg accttttgct cacatcagac ctgattctgt catcgggaat 
                 1020 
               
               
                   
               
               
                 gaagtgaaga tcgggaattt tgtagaaatt aaaaagactc aattcggaga ccgaagcaag 
                 1080 
               
               
                   
               
               
                 gcatctcatc taagctatgt cggcgatgct gaggtaggca ctgatgtaaa cctgggctgc 
                 1140 
               
               
                   
               
               
                 ggttcaatta ctgtcaatta tgatggaaag aataagtatt tgacaaaaat tgaagatggc 
                 1200 
               
               
                   
               
               
                 gcgtttatcg gctgcaattc caacttggtt gcccctgtca cagtcggaga aggcgcttat 
                 1260 
               
               
                   
               
               
                 gtggcggcag gttcaactgt tacggaagat gtacctggaa aagcacttgc tattgccaga 
                 1320 
               
               
                   
               
               
                 gcgagacaag taaataaaga cgattatgtg aaaaatattc ataaaaaata a 
                 1371 
               
               
                   
               
               
                 &lt;210&gt; 4 
                   
               
               
                 &lt;211&gt; 879 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus   subtilis   
                   
               
               
                 &lt;400&gt; 4 
                   
               
               
                 atgaaaaaag tacgtaaagc cataattcca gcagcaggct taggaacacg ttttcttccg 
                 60 
               
               
                   
               
               
                 gctacgaaag caatgccgaa agaaatgctt cctatcgttg ataaacctac cattcaatac 
                 120 
               
               
                   
               
               
                 ataattgaag aagctgttga agccggtatt gaagatatta ttatcgtaac aggaaaaagc 
                 180 
               
               
                   
               
               
                 aagcgtgcga ttgaggatca ttttgattac tctcctgagc ttgaaagaaa cctagaagaa 
                 240 
               
               
                   
               
               
                 aaaggaaaaa ctgagctgct tgaaaaagtg aaaaaggctt ctaacctggc tgacattcac 
                 300 
               
               
                   
               
               
                 tatatccgcc aaaaagaacc taaaggtctc ggacatgctg tctggtgcgc acgcaacttt 
                 360 
               
               
                   
               
               
                 atcggcgatg agccgtttgc ggtactgctt ggtgacgata ttgttcaggc tgaaactcca 
                 420 
               
               
                   
               
               
                 gggttgcgcc aattaatgga tgaatatgaa aaaacacttt cttctattat cggtgttcag 
                 480 
               
               
                   
               
               
                 caggtgcccg aagaagaaac acaccgctac ggcattattg acccgctgac aagtgaaggc 
                 540 
               
               
                   
               
               
                 cgccgttatc aggtgaaaaa cttcgttgaa aaaccgccta aaggcacagc accttctaat 
                 600 
               
               
                   
               
               
                 cttgccatct taggccgtta cgtattcacg cctgagatct tcatgtattt agaagagcag 
                 660 
               
               
                   
               
               
                 caggttggcg ccggcggaga aattcagctc acagacgcca ttcaaaagct gaatgaaatt 
                 720 
               
               
                   
               
               
                 caaagagtgt ttgcttacga ttttgaaggc aagcgttatg atgttggtga aaagctcggc 
                 780 
               
               
                   
               
               
                 tttatcacaa caactcttga atttgcgatg caggataaag agcttcgcga tcagctcgtt 
                 840 
               
               
                   
               
               
                 ccatttatgg aaggtttact aaacaaagaa gaaatctaa 
                 879 
               
               
                   
               
               
                 &lt;210&gt; 5 
                   
               
               
                 &lt;211&gt; 1347 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 5 
                   
               
               
                 atgggcaagt attttggaac agacggtgta agaggtgtcg ccaatagtga gcttacacct 
                 60 
               
               
                   
               
               
                 gagctggcct ttaaagtcgg acgtttcggc ggttatgtgc tgacaaaaga caaacaacgt 
                 120 
               
               
                   
               
               
                 ccaaaagtgc tgataggccg cgatacacgc atctccggcc atatgctgga gggagccctt 
                 180 
               
               
                   
               
               
                 gtcgccggac ttttatccat tggcgcagaa gtcatgcgcc tgggtgtcat ttctacacca 
                 240 
               
               
                   
               
               
                 ggtgtatctt atttgacaaa agcgatggat gcagaggcgg gcgtcatgat ttccgcttct 
                 300 
               
               
                   
               
               
                 cataacccag tgcaggataa cggcatcaaa ttctttgggg gagatggatt taagctttct 
                 360 
               
               
                   
               
               
                 gatgaacagg aggctgaaat tgagcgcctg atggacgaac ctgaggataa gctgccaaga 
                 420 
               
               
                   
               
               
                 cctgtcggag cagaccttgg acttgtaaac gattattttg aaggcggaca aaaatatctg 
                 480 
               
               
                   
               
               
                 caattcttaa aacagacagc tgatgaagat ttcacaggca ttcatgtggc attggactgt 
                 540 
               
               
                   
               
               
                 gccaatggcg caacgtcatc cttggcgaca cacctgtttg ctgatttaga tgcagatgtt 
                 600 
               
               
                   
               
               
                 tctacaatgg ggacttcccc gaacggatta aacattaatg acggcgtcgg ttcgactcat 
                 660 
               
               
                   
               
               
                 cccgaagcgc tcagcgcgtt tgtcaaagag aaaaacgcgg atctcggtct tgcgttcgac 
                 720 
               
               
                   
               
               
                 ggtgacggcg accgcctgat tgctgtcgat gaaaaaggaa atattgtaga cggcgaccaa 
                 780 
               
               
                   
               
               
                 atcatgtaca tatgctcaaa acatctgaaa tcagagggcc gtttaaagga tgatacagtg 
                 840 
               
               
                   
               
               
                 gtttcaaccg tgatgagcaa cctcggcttc tataaggcgc tcgaaaaaga aggcatcaaa 
                 900 
               
               
                   
               
               
                 agcgtgcaga cagctgtcgg cgaccgctac gtagtagaag caatgaaaaa agacggctac 
                 960 
               
               
                   
               
               
                 aacgtcggcg gagagcagtc aggacatctt attttccttg attacaacac gacaggggac 
                 1020 
               
               
                   
               
               
                 ggattattgt ctgctattat gctgatgaac actttaaaag caacaggcaa gccgctgtca 
                 1080 
               
               
                   
               
               
                 gagcttgcag ctgaaatgca gaagttcccg cagctgttag tcaatgtgag agtgactgat 
                 1140 
               
               
                   
               
               
                 aaatataaag ttgaagaaaa tgaaaaagta aaagcagtta tttctgaagt tgaaaaagaa 
                 1200 
               
               
                   
               
               
                 atgaacggcg acggccggat tttggtgcgc ccttcaggaa ctgaaccgct cgtccgtgtc 
                 1260 
               
               
                   
               
               
                 atggctgaag cgaagacgaa agagctgtgc gatgagtatg tcaatcgcat tgttgaagtc 
                 1320 
               
               
                   
               
               
                 gtccggtcag aaatgggatt agagtaa 
                 1347 
               
               
                   
               
               
                 &lt;210&gt; 6 
                   
               
               
                 &lt;211&gt; 1803 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 6 
                   
               
               
                 atgtgtggaa tcgtaggtta tatcggtcag cttgatgcga aggaaatttt attaaaaggg 
                 60 
               
               
                   
               
               
                 ttagagaagc ttgagtatcg cggttatgac tctgctggta ttgctgttgc caacgaacag 
                 120 
               
               
                   
               
               
                 ggaatccatg tgttcaaaga aaaaggacgc attgcagatc ttcgtgaagt tgtggatgcc 
                 180 
               
               
                   
               
               
                 aatgtagaag cgaaagccgg aattgggcat actcgctggg cgacacacgg cgaaccaagc 
                 240 
               
               
                   
               
               
                 tatctgaacg ctcacccgca tcaaagcgca ctgggccgct ttacacttgt tcacaacggc 
                 300 
               
               
                   
               
               
                 gtgatcgaga actatgttca gctgaagcaa gagtatttgc aagatgtaga gctcaaaagt 
                 360 
               
               
                   
               
               
                 gacaccgata cagaagtagt cgttcaagta atcgagcaat tcgtcaatgg aggacttgag 
                 420 
               
               
                   
               
               
                 acagaagaag cgttccgcaa aacacttaca ctgttaaaag gctcttatgc aattgcttta 
                 480 
               
               
                   
               
               
                 ttcgataacg acaacagaga aacgattttt gtagcgaaaa acaaaagccc tctattagta 
                 540 
               
               
                   
               
               
                 ggtcttggag atacattcaa cgtcgtagca tctgatgcga tggcgatgct tcaagtaacc 
                 600 
               
               
                   
               
               
                 aacgaatacg tagagctgat ggataaagaa atggttatcg tcactgatga ccaagttgtc 
                 660 
               
               
                   
               
               
                 atcaaaaacc ttgatggtga cgtgattaca cgtgcgtctt atattgctga gcttgatgcc 
                 720 
               
               
                   
               
               
                 agtgatatcg aaaaaggcac gtaccctcac tacatgttga aagaaacgga tgagcagcct 
                 780 
               
               
                   
               
               
                 gttgttatgc gcaaaatcat ccaaacgtat caagatgaaa acggcaagct gtctgtgcct 
                 840 
               
               
                   
               
               
                 ggcgatatcg ctgccgctgt agcggaagcg gaccgcatct atatcattgg ctgcggaaca 
                 900 
               
               
                   
               
               
                 agctaccatg caggacttgt cggtaaacaa tatattgaaa tgtgggcaaa cgtgccggtt 
                 960 
               
               
                   
               
               
                 gaagtgcatg tagcgagtga attctcctac aacatgccgc ttctgtctaa gaaaccgctc 
                 1020 
               
               
                   
               
               
                 ttcattttcc tttctcaaag cggagaaaca gcagacagcc gcgcggtact cgttcaagtc 
                 1080 
               
               
                   
               
               
                 aaagcgctcg gacacaaagc cctgacaatc acaaacgtac ctggatcaac gctttctcgt 
                 1140 
               
               
                   
               
               
                 gaagctgact atacattgct gcttcatgca ggccctgaga tcgctgttgc gtcaacgaaa 
                 1200 
               
               
                   
               
               
                 gcatacactg cacaaatcgc agttctggcg gttcttgctt ctgtggctgc tgacaaaaat 
                 1260 
               
               
                   
               
               
                 ggcatcaata tcggatttga cctcgtcaaa gaactcggta tcgctgcaaa cgcaatggaa 
                 1320 
               
               
                   
               
               
                 gctctatgcg accagaaaga cgaaatggaa atgatcgctc gtgaatacct gactgtatcc 
                 1380 
               
               
                   
               
               
                 agaaatgctt tcttcatcgg acgcggcctt gactacttcg tatgtgtcga aggcgcactg 
                 1440 
               
               
                   
               
               
                 aagctgaaag agatttctta catccaggca gaaggttttg ccggcggtga gctaaagcac 
                 1500 
               
               
                   
               
               
                 ggaacgattg ccttgatcga acaaggaaca ccagtattcg cactggcaac tcaagagcat 
                 1560 
               
               
                   
               
               
                 gtaaacctaa gcatccgcgg aaacgtcaaa gaagttgctg ctcgcggagc aaacacatgc 
                 1620 
               
               
                   
               
               
                 atcatctcac tgaaaggcct agacgatgcg gatgacagat tcgtattgcc ggaagtaaac 
                 1680 
               
               
                   
               
               
                 ccagcgcttg ctccgttggt atctgttgtt ccattgcagc tgatcgctta ctatgctgca 
                 1740 
               
               
                   
               
               
                 ctgcatcgcg gctgtgatgt ggataaacct cgtaaccttg cgaagagtgt tactgtggag 
                 1800 
               
               
                   
               
               
                 taa 
                 1803 
               
               
                   
               
               
                 &lt;210&gt; 7 
                   
               
               
                 &lt;211&gt; 1470 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Leech 
                   
               
               
                 &lt;400&gt; 7 
                   
               
               
                 atgaaagaga tcgcggtgac aattgacgat aagaacgtta ttgcctctgt cagcgagtca 
                 60 
               
               
                   
               
               
                 ttccatggtg ttgcctttga tgcgtcgtta ttttcaccga aggggttgtg gagctttgtt 
                 120 
               
               
                   
               
               
                 gacattacct caccgaaatt gtttaaactc ttggagggtc tctctcctgg ttacttcagg 
                 180 
               
               
                   
               
               
                 gttggaggaa cgtttgctaa ctggctgttc tttgacttag atgaaaataa taagtggaaa 
                 240 
               
               
                   
               
               
                 gactattggg cttttaaaga taaaacaccc gagactgcaa caatcacaag gaggtggctg 
                 300 
               
               
                   
               
               
                 tttcgaaaac aaaacaacct gaaaaaagag acttttgacg acttagtcaa actaaccaaa 
                 360 
               
               
                   
               
               
                 ggaagcaaaa tgagactgtt atttgattta aacgctgaag tgagaactgg ttatgaaatt 
                 420 
               
               
                   
               
               
                 ggaaagaaaa tgacatccac ttgggatagc tcggaagctg aaaaattatt caaatactgt 
                 480 
               
               
                   
               
               
                 gtgtcaaaag gttatggaga taatattgat tgggaacttg gtaatgaacc ggaccatacc 
                 540 
               
               
                   
               
               
                 tccgcacaca atcttactga aaagcaagtt ggagaggact ttaaagccct gcataaagtg 
                 600 
               
               
                   
               
               
                 ctagagaaat atccgacgtt gaataaagga tcgcttgttg gacctgacgt tggatggatg 
                 660 
               
               
                   
               
               
                 ggagtctctt atgtgaaagg attagcagac ggggctggtg atcacgtaac cgcttttact 
                 720 
               
               
                   
               
               
                 cttcatcagt attattttga cggcaatacc tcagatgtgt caacatacct tgacgctact 
                 780 
               
               
                   
               
               
                 tattttaaaa aacttcaaca gctgtttgac aaagttaagg atgtcttgaa aaattctcca 
                 840 
               
               
                   
               
               
                 cataaagata aaccgctctg gcttggagaa acaagttctg gatacaacag cggcacaaaa 
                 900 
               
               
                   
               
               
                 gatgtatccg atcgatatgt tagcggattt ctaacattgg acaagttggg actcagtgca 
                 960 
               
               
                   
               
               
                 gcgaacaatg tgaaagttgt gataagacaa acgatctata atggatacta cggacttctt 
                 1020 
               
               
                   
               
               
                 gataaaaata ctctagagcc aaatccggat tattggctaa tgcatgttca caattctctg 
                 1080 
               
               
                   
               
               
                 gttggaaata cggtttttaa agttgacgtt agtgacccta caaataaagc tagagtttat 
                 1140 
               
               
                   
               
               
                 gcacagtgca ccaaaacaaa tagcaaacat actcagagta gatactacaa gggctcattg 
                 1200 
               
               
                   
               
               
                 acgatctttg ctcttaatgt tggagatgaa gatgtgacgt tgaagattga tcaatacagt 
                 1260 
               
               
                   
               
               
                 ggaaaaaaga tttattcata tattctgacc ccagaaggcg gccaacttac atcacaaaaa 
                 1320 
               
               
                   
               
               
                 gttcttttga atggaaaaga attaaaatta gtgtcggatc aattgccaga actgaatgca 
                 1380 
               
               
                   
               
               
                 gacgagtcga aaacctcttt cactctgtct ccaaagacat ttggattttt tgttgttagc 
                 1440 
               
               
                   
               
               
                 gatgctaacg ttgaagcctg caaaaaataa 
                 1470 
               
               
                   
               
               
                 &lt;210&gt; 8 
                   
               
               
                 &lt;211&gt; 403 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 8 
                   
               
               
                 gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg 
                 60 
               
               
                   
               
               
                 gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta 
                 120 
               
               
                   
               
               
                 accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa 
                 180 
               
               
                   
               
               
                 agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct 
                 240 
               
               
                   
               
               
                 ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt aggaggaaag 
                 300 
               
               
                   
               
               
                 tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt 
                 360 
               
               
                   
               
               
                 actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc 
                 403 
               
               
                   
               
               
                 &lt;210&gt; 9 
                   
               
               
                 &lt;211&gt; 556 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 9 
                   
               
               
                 gagctcggta cccggggatc ctctagagat tctaccgttc gtatagcata cattatacga 
                 60 
               
               
                   
               
               
                 agttatcttg atatggcttt ttatatgtgt tactctacat acagaaagga ggaactaaat 
                 120 
               
               
                   
               
               
                 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 
                 180 
               
               
                   
               
               
                 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 
                 240 
               
               
                   
               
               
                 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 
                 300 
               
               
                   
               
               
                 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 
                 360 
               
               
                   
               
               
                 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 
                 420 
               
               
                   
               
               
                 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 
                 480 
               
               
                   
               
               
                 gaggagcagg actgaataac ttcgtatagc atacattata cgaacggtag aatcgtcgac 
                 540 
               
               
                   
               
               
                 ctgcaggcat gcaagc 
                 556 
               
               
                   
               
               
                 &lt;210&gt; 10 
                   
               
               
                 &lt;211&gt; 268 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 10 
                   
               
               
                 tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 
                 60 
               
               
                   
               
               
                 taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 
                 120 
               
               
                   
               
               
                 tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 
                 180 
               
               
                   
               
               
                 aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 
                 240 
               
               
                   
               
               
                 gcgattatgt aaaatataaa gtgatagc 
                 268 
               
               
                   
               
               
                 &lt;210&gt; 11 
                   
               
               
                 &lt;211&gt; 257 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt;  Bacillus subtilis   
                   
               
               
                 &lt;400&gt; 11 
                   
               
               
                 ggagttctga gaattggtat gccttataag tccaattaac agttgaaaac ctgcatagga 
                 60 
               
               
                   
               
               
                 gagctatgcg ggttttttat tttacataat gatacataat ttaccgaaac ttgcggaaca 
                 120 
               
               
                   
               
               
                 taattgagga atcatagaat tttgtcaaaa taattttatt gacaacgtct tattaacgtt 
                 180 
               
               
                   
               
               
                 gatataattt aaattttatt tgacaaaaat gggctcgtgt tgtacaataa atgtagttaa 
                 240 
               
               
                   
               
               
                 aaaggagcga tttacat 
                 257 
               
               
                   
               
               
                 &lt;210&gt; 12 
                   
               
               
                 &lt;211&gt; 403 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 12 
                   
               
               
                 gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg 
                 60 
               
               
                   
               
               
                 gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta 
                 120 
               
               
                   
               
               
                 accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa 
                 180 
               
               
                   
               
               
                 agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct 
                 240 
               
               
                   
               
               
                 ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt aagaggagag 
                 300 
               
               
                   
               
               
                 tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt 
                 360 
               
               
                   
               
               
                 actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc 
                 403 
               
               
                   
               
               
                 &lt;210&gt; 13 
                   
               
               
                 &lt;211&gt; 403 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 13 
                   
               
               
                 gcctcaatcc taggagaaac agtcacggca aaagatttag tagaaaaaca aaaagagctg 
                 60 
               
               
                   
               
               
                 gaaaaggtgg agacattcaa tatgttttca aaagccggaa aagcgctttc ggacaccgta 
                 120 
               
               
                   
               
               
                 accaatactg cccagtcaat gtatgaatgg atacgggata tgaatcaata agtacgtgaa 
                 180 
               
               
                   
               
               
                 agagaaaagc aacccagata tgatagggaa cttttctctt tcttgtttta cattgaatct 
                 240 
               
               
                   
               
               
                 ttacaatcct attgatataa tctaagctag tgtattttgc gtttaatagt acgtagacag 
                 300 
               
               
                   
               
               
                 tggtaccatg ctaaaaagaa cttcattcgt atcttcatta ttcatcagtt cagctgtttt 
                 360 
               
               
                   
               
               
                 actatcaatc ttacttcctt cgggccaagc tcatgcagaa ttc 
                 403 
               
               
                   
               
               
                 &lt;210&gt; 14 
                   
               
               
                 &lt;211&gt; 17 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 14 
                   
               
               
                 aagagaggaa tgtacac 
                 17 
               
               
                   
               
               
                 &lt;210&gt; 15 
                   
               
               
                 &lt;211&gt; 35 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; DNA 
                   
               
               
                 &lt;400&gt; 15 
                   
               
               
                 cgcggatcca tgagaacatt aaaaaacctc ataac 
                 35 
               
               
                   
               
               
                 &lt;210&gt; 16 
                   
               
               
                 &lt;211&gt; 33 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 16 
                   
               
               
                 tgcatgcatt tataataatt ttttacgtgt tcc 
                 33 
               
               
                   
               
               
                 &lt;210&gt; 17 
                   
               
               
                 &lt;211&gt; 49 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 17 
                   
               
               
                 cggggtacca agagaggaat gtacacatga aaaaaatagc tgtcattgg 
                 49 
               
               
                   
               
               
                 &lt;210&gt; 18 
                   
               
               
                 &lt;211&gt; 31 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 18 
                   
               
               
                 ccggagctct tataaattga cgcttcccaa g 
                 31 
               
               
                   
               
               
                 &lt;210&gt; 19 
                   
               
               
                 &lt;211&gt; 48 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 19 
                   
               
               
                 cgggagctca agagaggaat gtacacatgg ataagcggtt tgcagttg 
                 48 
               
               
                   
               
               
                 &lt;210&gt; 20 
                   
               
               
                 &lt;211&gt; 49 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 20 
                   
               
               
                 ccgctcgagc ggactctagt ctagattatt ttttatgaat atttttcac 
                 49 
               
               
                   
               
               
                 &lt;210&gt; 21 
                   
               
               
                 &lt;211&gt; 30 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 21 
                   
               
               
                 ggactagtgg agttctgaga attggtatgc 
                 30 
               
               
                   
               
               
                 &lt;210&gt; 22 
                   
               
               
                 &lt;211&gt; 25 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 22 
                   
               
               
                 atgtaaatcg ctccttttta actac 
                 25 
               
               
                   
               
               
                 &lt;210&gt; 23 
                   
               
               
                 &lt;211&gt; 45 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 23 
                   
               
               
                 gtagttaaaa aggagcgatt tacatatgaa aaaagtacgt aaagc 
                 45 
               
               
                   
               
               
                 &lt;210&gt; 24 
                   
               
               
                 &lt;211&gt; 51 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 24 
                   
               
               
                 ggactagtaa gagaggaatg tacacatggg caagtatttt ggaacagacg g 
                 51 
               
               
                   
               
               
                 &lt;210&gt; 25 
                   
               
               
                 &lt;211&gt; 52 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 25 
                   
               
               
                 ccgctcgagc ggactctagt ctagattact ctaatcccat ttctgaccgg ac 
                 52 
               
               
                   
               
               
                 &lt;210&gt; 26 
                   
               
               
                 &lt;211&gt; 51 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 26 
                   
               
               
                 ggactagtaa gagaggaatg tacacatgtg tggaatcgta ggttatatcg g 
                 51 
               
               
                   
               
               
                 &lt;210&gt; 27 
                   
               
               
                 &lt;211&gt; 49 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 27 
                   
               
               
                 ccgctcgagc ggactctagt ctagattact ccacagtaac actcttcgc 
                 49 
               
               
                   
               
               
                 &lt;210&gt; 28 
                   
               
               
                 &lt;211&gt; 41 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 28 
                   
               
               
                 atgcacagtc tgcagaattc caccaccacc accaccacat g 
                 41 
               
               
                   
               
               
                 &lt;210&gt; 29 
                   
               
               
                 &lt;211&gt; 23 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 29 
                   
               
               
                 ttactttttg cacgcttcaa cat 
                 23 
               
               
                   
               
               
                 &lt;210&gt; 30 
                   
               
               
                 &lt;211&gt; 42 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 30 
                   
               
               
                 cgcagccaaa ggagtggatt gcctcaatcc taggagaaac ag 
                 42 
               
               
                   
               
               
                 &lt;210&gt; 31 
                   
               
               
                 &lt;211&gt; 24 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 31 
                   
               
               
                 gaattctgca gactgtgcat gagc 
                 24 
               
               
                   
               
               
                 &lt;210&gt; 32 
                   
               
               
                 &lt;211&gt; 23 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 32 
                   
               
               
                 tcagctggtc tagatcacta gtc 
                 23 
               
               
                   
               
               
                 &lt;210&gt; 33 
                   
               
               
                 &lt;211&gt; 23 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 33 
                   
               
               
                 aatccactcc tttggctgcg ctc 
                 23 
               
               
                   
               
               
                 &lt;210&gt; 34 
                   
               
               
                 &lt;211&gt; 41 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 34 
                   
               
               
                 ttgaagcgtg caaaaagtaa gagctcggta cccggggatc c 
                 41 
               
               
                   
               
               
                 &lt;210&gt; 35 
                   
               
               
                 &lt;211&gt; 22 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 35 
                   
               
               
                 gcttgcatgc ctgcaggtcg ac 
                 22 
               
               
                   
               
               
                 &lt;210&gt; 36 
                   
               
               
                 &lt;211&gt; 45 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 36 
                   
               
               
                 cgacctgcag gcatgcaagc cacttctttc agacggaacc cttgc 
                 45 
               
               
                   
               
               
                 &lt;210&gt; 37 
                   
               
               
                 &lt;211&gt; 24 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 37 
                   
               
               
                 cggtcgttca tatagaagtg atag 
                 24 
               
               
                 &lt;210&gt; 38 
                   
               
               
                 &lt;211&gt; 44 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 38 
                   
               
               
                 cacttctata tgaacgaccg cctgtgtgaa attgttatcc gctc 
                 44 
               
               
                   
               
               
                 &lt;210&gt; 39 
                   
               
               
                 &lt;211&gt; 45 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 39 
                   
               
               
                 tagtgatcta gaccagctga gtgactggga aaaccctggc gttac 
                 45 
               
               
                   
               
               
                 &lt;210&gt; 40 
                   
               
               
                 &lt;211&gt; 50 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;221&gt; misc_feature 
                   
               
               
                 &lt;222&gt; (14)..(14) 
                   
               
               
                 &lt;223&gt; n is a, c, g, t or u 
                   
               
               
                   
               
               
                 &lt;221&gt; misc_feature 
                   
               
               
                 &lt;222&gt; (16)..(16) 
                   
               
               
                 &lt;223&gt; n is a, c, g, t or u 
                   
               
               
                   
               
               
                 &lt;221&gt; misc_feature 
                   
               
               
                 &lt;222&gt; (18)..(18) 
                   
               
               
                 &lt;223&gt; n is a, c, g, t or u 
                   
               
               
                   
               
               
                 &lt;400&gt; 40 
                   
               
               
                 acggggtacc actntnynhb yactattaaa cgcaaaatac actagcttag 
                 50 
               
               
                   
               
               
                 &lt;210&gt; 41 
                   
               
               
                 &lt;211&gt; 32 
                   
               
               
                 &lt;212&gt; DNA 
                   
               
               
                 &lt;213&gt; Artificial sequence 
                   
               
               
                 &lt;400&gt; 41 
                   
               
               
                 acggggtacc atgctaaaaa gaacttcatt cg 
                 32