Difference between revisions of "Part:BBa K4361220"

 
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<partinfo>BBa_K4361220 short</partinfo>
 
<partinfo>BBa_K4361220 short</partinfo>
  
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TBD
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For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein ([[Part:BBa_K4361106]]) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as R<i>n</i> with <i>n</i> being the number of the set) and a varying number of forward primers (denoted as F<i>n</i>.<i>m</i> followed by the induced substitution). Every set covers a small range of amino acids in the DNA-binding domain of BlcR, with each position being mutated to different residues by different forward-facing primers. Do note, however, that while a large number of primers have been designed, sequencing showed that the mutagenesis protocol was not successful for all variants. Therefore, only the primers that resulted in the desired mutants will be described in our collection of BioBricks.
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<br>
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The primer sets are divided as follows:
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<ul>
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  <li><b>Set 1, residues 36 to 39</b>
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    <ul>
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      <li>[[Part:BBa_K4361200]]: R1</li>
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      <li>[[Part:BBa_K4361201]]: F1.1 D37R</li>
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      <li>[[Part:BBa_K4361202]]: F1.2 D37V</li>
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      <li>[[Part:BBa_K4361203]]: F1.3 L38V</li>
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    </ul>
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  </li>
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  <li><b>Set 4, residues 40 to 41</b>
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    <ul>
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      <li>[[Part:BBa_K4361204]]: R4</li>
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      <li>[[Part:BBa_K4361205]]: F4.4 A40V</li>
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    </ul>
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  </li>
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  <li><b>Set 5, residues 47 to 49</b>
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    <ul>
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      <li>[[Part:BBa_K4361206]]: R5</li>
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      <li>[[Part:BBa_K4361207]]: F5.1 T47S</li>
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      <li>[[Part:BBa_K4361208]]: F5.2 T47V</li>
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      <li>[[Part:BBa_K4361209]]: F5.3 A48V</li>
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    </ul>
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  </li>
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  <li><b>Set 7, residues 61 to 63</b>
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    <ul>
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      <li>[[Part:BBa_K4361210]]: R7</li>
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      <li>[[Part:BBa_K4361211]]: F7.1 S61V</li>
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      <li>[[Part:BBa_K4361212]]: F7.2 A62V</li>
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      <li>[[Part:BBa_K4361213]]: F7.3 A62I</li>
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      <li>[[Part:BBa_K4361214]]: F7.4 A62K</li>
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      <li>[[Part:BBa_K4361215]]: F7.5 A62T</li>
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      <li>[[Part:BBa_K4361216]]: F7.6 H63V</li>
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      <li>[[Part:BBa_K4361217]]: F7.7 H63Y</li>
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    </ul>
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  </li>
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  <li><b>Set 3, residues 65 to 68</b>
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    <ul>
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      <li>[[Part:BBa_K4361218]]: R3</li>
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      <li>[[Part:BBa_K4361219]]: F3.1 L66V</li>
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      <li><i><u>Part:BBa_K4361220</u>: F3.2 L66A</i></li>
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      <li>[[Part:BBa_K4361221]]: F3.3 L66I</li>
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      <li>[[Part:BBa_K4361222]]: F3.4 A67Q</li>
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      <li>[[Part:BBa_K4361223]]: F3.5 A67V</li>
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      <li>[[Part:BBa_K4361224]]: F3.6 A67H</li>
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      <li>[[Part:BBa_K4361225]]: F3.7 V68T</li>
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      <li>[[Part:BBa_K4361226]]: F3.8 V68K</li>
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      <li>[[Part:BBa_K4361227]]: F3.9 V68S</li>
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    </ul>
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  </li>
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</ul>
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This primer contains a mutation that changes the codon for leucine (L, CTC) in position 66 to that of valine (A, GCG).<br>
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<br>
  
 
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Revision as of 14:38, 14 September 2022


BlcR BTB primer F3.2 L66A

For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein (Part:BBa_K4361106) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as Rn with n being the number of the set) and a varying number of forward primers (denoted as Fn.m followed by the induced substitution). Every set covers a small range of amino acids in the DNA-binding domain of BlcR, with each position being mutated to different residues by different forward-facing primers. Do note, however, that while a large number of primers have been designed, sequencing showed that the mutagenesis protocol was not successful for all variants. Therefore, only the primers that resulted in the desired mutants will be described in our collection of BioBricks.
The primer sets are divided as follows:

This primer contains a mutation that changes the codon for leucine (L, CTC) in position 66 to that of valine (A, GCG).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]