Part:BBa_K2019000:Design
Histidine Tagged WT Staphylococcus Aureus Cas9 Protein
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 627
Illegal BglII site found at 780
Illegal BglII site found at 926 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 200
Illegal AgeI site found at 2756 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
We linearized a backbone with the tet promoter and then used gibson assembly to attach two gblocks with the saCas9 sequence codon optimized for E. Coli. Then, we used to PCR to amplify out saCas9 with overhangs that matched the homology region on the pET28a backbone. We then performed a 2 piece Gibson assembly of saCas9 with homology to pET28 and the linearized pET28a backbone. Finally, we PCR'd saCas9 out of the tet backbone with overhangs that attached the biobricks prefix and suffix to the ends of saCas9. With this piece, we performed a restriction digestion of our saCas9 PCR product into the linearized PSB1C3 backbone.
Source
SaCas9 was extracted from the genomic sequence of staphylococcus aureus and then codon optimized for E. Coli.
References
F. Ann Ran, Le Cong, Winston X. Yan, David A. Scott, Jonathan S. Gootenberg, Andrea J. Kriz, Bernd Zetsche, Ophir Shalem, Xuebing Wu, Kira S. Makarova, Eugene V. Koonin, Phillip A. Sharp, Feng Zhang. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520.
Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, Marco E, Gotta GM, Maeder ML, Kennedy EM, Kornepati AVR, Sousa A, Collins MA, Jayaram H, Cullen BR, Bumcrot D. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 2015;16(257).