Part:BBa_K2306012:Design
Cas13a with purification tags
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 70
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 260
Illegal BglII site found at 845
Illegal BglII site found at 2006
Illegal BglII site found at 2480
Illegal BamHI site found at 1
Illegal BamHI site found at 451 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We codon optimized the sequence for E. Coli K12 using the online codon optimization tool from IDT. Finally, to make it a valid BioBrick some forbidden restriction sites were excluded by manually introducing silent mutations with the same tool. To check whether the translation of this gene will still be the same we did a protein blast of our gene and the gene from Gootenberg et al. 2017 with the online Translate tool from Expasy and the online Blast tool from NCBI. A last modification was made: the TGA stop codon was changed into a double TAATAA stop codon.
Before the gene sequence a His tag, a thrombin site, a strep tag and a SUMO cleavage site were added to enable purification (see figure tags).These tags were also used by Gootenberg et al. 2017 (plasmid pC013). The His tag is used to purify the Cas13a protein. The thrombin site is a cleavage site that ensures the removal of the His tag once it is no longer necessary. If Cas13a is not pure enough after a His tag purification, the strep tag can be used for a second purification step. Finally, the SUMO cleavage site can be used to remove all tags if desired. We included BamHI restriction sites flanking the sequence for the tags, so that it can be removed if desired.
Source
The source was based on the gene LwCas13a from plasmid pC013 (Gootenberg et al. 2017).
References
Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Koonin, E. V. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2, 9321(April). https://doi.org/10.1126/science.aam9321