Difference between revisions of "Team:WHU-China/Description"

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              <h1 id="Brief">Brief introduction in RdhANP</h1>
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          <h1 id="Brief">Brief introduction in RdhANP</h1>
  
 
           <p>RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH. </p>
 
           <p>RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH. </p>
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         </div>
 
         </div>
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        <div>
 +
        <h1 id="Modularization">Modularization: Overview</h1>
 +
 +
        <img src="Modularization-Overview.png" width="850" height="893" alt=""/> </div>
 
        
 
        
 
         <h1 id="protocol">Our protocol</h1>
 
         <h1 id="protocol">Our protocol</h1>
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</tbody></table>
 
</tbody></table>
 
<br><br><br>
 
<br><br><br>
 +
<div>
 +
        <h1 id="Appendix">Appendix</h1>
 +
<strong>#Gene sequence (Without prefixes and suffixes)</strong>
 +
          <p>ATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGA </p>
  
 +
      <strong>#pSB1C3</strong>
 +
          <p>TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG </p>
 +
         
 +
          <strong>#sequencing primer</strong>
 +
<p>senCe
 +
5'  CAGATAAAAAAAATCCTTAG  3'
 +
AnTisenCe
 +
5'  TTCGGTTTTAAAGAAAAAGG  3'
 +
</p>
 +
        <strong>#recombinant plasmid</strong>
 +
<p>ATTCGCGGCCGCTTCTAGATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGATAATAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGG
 +
</p>
 +
     
 +
        </div>
 +
     
  
 +
     
 +
</div>
 
<!--<h1 id="Brief">Brief introduction in RdhANP</h1>
 
<!--<h1 id="Brief">Brief introduction in RdhANP</h1>
 
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Revision as of 06:32, 24 August 2017

Description

Brief introduction in RdhANP

RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH.

Description of our operating bacteria

Nitratireductor pacificus strain pht-3BT (MCCC 1A01024T, CCTCC AB 209302T, LMG 25541T) was isolated from deep sea sediments of the western Pacific Ocean enriched with pyrene as the sole carbon and energy source. Cells were Gram-negative, short rods that were motile by means of flagella. They were first grown on marine agar 2216 (BD) medium, which produces smooth grey non-pigmented colonies with regular edges and slightly raised in the center, 2–3 mm in diameter after 3 days at 28 ℃ .We use MB medium as alternative. The most important feature of the cell is its production of RdhANP.

Modularization: Overview

Our protocol

First, digest the vector pUC57 and the target fragment psb1C3 with two kinds of endonucleases, e.g. PstⅠand EcoRⅠ. Second, using agarose gel electrophoresis (AGE) to analyze the result of restriction endonuclease digestion. Third, recycle the agarose gel containing target bands to enrich the products. Forth, link the vector and target gene by T4 ligase. Fifth, using AGE again to evaluate the result. Finally, transform the recombination plasmids to the bacillus megatherium.

During the process, we encounter some problems. A typical one is that we failed in the transformation step several times. After debugging the experiment conditions in various aspects, we finally found the most appropriate condition. Another reason responsible for it is the threshold –below concentration of target segments contained in the recycled gel.



Appendix

#Gene sequence (Without prefixes and suffixes)

ATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGA

#pSB1C3

TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG

#sequencing primer

senCe 5' CAGATAAAAAAAATCCTTAG 3' AnTisenCe 5' TTCGGTTTTAAAGAAAAAGG 3'

#recombinant plasmid

ATTCGCGGCCGCTTCTAGATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGATAATAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGG