Difference between revisions of "Part:BBa K1723000:Design"
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===Source=== | ===Source=== | ||
| − | + | The omega subunit was extracted from the plasmid pWJ66 given to the iGEM15_EPFL team by David Bikard. | |
The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249) | The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249) | ||
Latest revision as of 21:25, 17 September 2015
dCas9-ω
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1099
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3378
Illegal BamHI site found at 4212 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
dCas9 is a Cas9 double mutant, with mutations at amino acid positions D10A and H840A. These mutations inactivate Cas9 nuclease and nickase activities. Cas9-ω contains an EcoRI restriction site which was removed via site-directed mutagenesis. Fusion of the omega subunit (rpoZ) to dCas9 was achieved by Gibson assembly.
Source
The omega subunit was extracted from the plasmid pWJ66 given to the iGEM15_EPFL team by David Bikard. The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249)