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| − | __NOTOC__
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| − | <partinfo>BBa_K3076300 short</partinfo>
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| − | ==Description==
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| − | <p>This part is designed to use as double-stranded (ds) DNA substrate for knocking out <i>CopA</i> gene in <i>E. coli</i> by Lambda Red recombineering system.</p>
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| − | ==Usage and Biology==
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| − | <p>This part contains homology sequences of 50 bp flanking a double terminator and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the <i>CopA</i> gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the <i>CopA</i> gene and disrupting the expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. A double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html> was added at the 5’ end of this fragment to ensure the termination of gene expression.</p>
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| − | <p>To use this substrate, simply amplify this part by PCR. The PCR product is ready to be transformed and recombined. The <i>E. coli</i> strain used should express lambda red recombineering genes. On the other hand, the lambda red recombineering system can be introduced to the <i>E. coli</i> strain by transforming the <i>E. coli</i> with plasmids containing lambda red genes, such as pKD46.</p>
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| − | <p>Due to the time constraint, we obtained the knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.</p>
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