Part:BBa_K3044026:Design
sgRNA/Cas9 system targeting mRFP
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 478
Illegal BglII site found at 1552 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The Cas9 is an active version of part No part name specified with partinfo tag. with catalytically active domains. The catalytically domains RuvC and HNH were activated by inducing A10D and A840H mutations. The mutations were made with point mutations ensuring the optimal codon for E. coli K12 TOP10. The point mutations are as follows; Point mutations at c.29C>A, c.30G>T for RuvC and at c.2518C>G, c.2519C>A, c.2520G>T for HNH.
The sgRNA was designed according to the design protocol "CRISPR interference (CRISPRi) for sequence-specific control of gene expression". [1] An important thing to keep ind mind when designing a sgRNA is off-targets. Therefore, the sgRNA sequence was aligned to the core genome of our chassis E coli K12 TOP10 and KG22 and no off-targets were found. Though, off-targets may appear in other bacteria strains.
Source
The Cas9 protein is an active version of part No part name specified with partinfo tag. with mutations A10D and A840H. The point mutations are made according to a codon frequency table [2]
The sequence of the sgRNA is designed on the basis of the mRFP sequence. The target sequence is determined according to the location of PAM sequences in the target sequence, which the dCas9 protein recognizes.