Part:BBa_K2813003
SaCas9 with 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.
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.
The gRNA target Sequence corresponds to the DNA sequence to be cut. In our case, we included a guide sequence that corresponds to a reporter plasmid containing CFP but this gene can be modified with a double digest reaction. The gRNA Sa scaffold will connect to the gRNA and serves to stabilize the RNA within the Sa Cas9 enzyme. The gRNA Sa scaffold sequence is specific to the enzyme and will remain constant regardless of target sequence.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1255
Illegal NheI site found at 1278 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2305
Illegal BglII site found at 2458
Illegal BglII site found at 2604
Illegal BamHI site found at 1333 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1878
Illegal AgeI site found at 4434 - 1000COMPATIBLE WITH RFC[1000]
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