Part:BBa_K2813002
SaCas9 without gRNA
This part is a Cas9 nuclease enzyme derived from Streptococcus aureus (Sa) that is able to cleave DNA at highly specific sites based on a guide RNA sequence. In nature, it serves as a small part of the larger CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system which acts an immune system for the bacteria against bacteriophages. Specifically, Cas9 serves to seek out and cut the sequence that corresponds to the guide sequence given to it in the form of the guide RNA (gRNA). Additionally, it has a T7 RNA polymerase promoter (BBa_J64997), a rrnB T1 terminator (BBa_B0010), and a T7Te Terminator (BBa_B0012), to control the translational frame and protein expression. Upon addition of a guide RNA sequence and a gRNA Sa scaffold sequence, this plasmid can be used to perform CRISPR. Additionally, the Sa Cas9 has a his tag included for its purification through a nickel column.
The LacI repressor protein is used to inducibly control the expression of the Cas9 protein in the plasmid. The LacI repressor protein will bind to the LacI operator sequence, which is upstream of the Cas9 promoter allowing expression to occur in the presence of allolactose or IPTG.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1975
Illegal BglII site found at 2128
Illegal BglII site found at 2274 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1548
Illegal AgeI site found at 4104 - 1000COMPATIBLE WITH RFC[1000]
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