Coding
Part:BBa_K3636000:Design
Designed by: Daria Smolyarova, Nadezhda Korostyleva, Anastasia Kislova Group: iGEM20_Moscow (2020-10-21)
CasX (Cas12e)
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We conducted a bioinformatical analysis that showed that there are four restriction sites in the CasX sequence that are incompatible with the Assembly Standards. In future work, we will need to include four silent point mutations to exclude those restrictions sites. We designed eight primers that will allow us to conduct this work.
Primers for 4 point mutations:
- noEcoR1-F-58
acgaaaaggaattTtatgcctgtgaga - noEcoR1-R-58
tctcacaggcataAaattccttttcgt - noSpe1-F-59
ttacgaggggcttacAagtaaaacgtattt - noSpe1-R-59
aaatacgttttactTgtaagcccctcgtaa - no1Ng04-F-60
gaagaagccCgccaaacgt - no1Ng04-R-60
acgtttggcGggcttcttc - no2Ng04-F-59
gatgagccCgccctgttt - no2Ng04-R-59
aaacagggcGggctcatc
Source
Addgene plasmid # 126418 ; http://n2t.net/addgene:126418 ; RRID:Addgene_126418
References
- CasX enzymes comprise a distinct family of RNA-guided genome editors. Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brotzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA. Nature. 2019 Feb 4. pii: 10.1038/s41586-019-0908-x. doi: 10.1038/s41586-019-0908-x. 10.1038/s41586-019-0908-x PubMed 30718774