Part:BBa_K2361002:Design
hCas9 operon
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1124
Illegal NheI site found at 4774 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3403
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
To make hCas9 out of Cas9 the mutation I473F was introduced in the protein. To make the whole construct biobrick compatible a restriction site was removed and the biobrick prefix and suffix were introduced. The 2 restriction sites that were remove are the EcorI site in Cas9 and the XbaI site in Csn2. To create the biobrick compatible hCas9 operon the necessary parts were created individually. Two of the four parts were amplified from the the pWJ40 plasmid without any alterations. The other two parts were ordered as geneblocks. The different parts were assembled using Gibson assembly.
Source
The biobrick was constructed from a mixture of synthesized sequences designed by the team and specific pcr products. The template for the pcr amplifications was the pWJ40 plasmid provided by R. Heler from the Marraffini Laboratory at The Rockefeller University | 1230 York Avenue, New York, NY 10065.