Coding

Part:BBa_K3636000:Design

Designed by: Daria Smolyarova, Nadezhda Korostyleva, Anastasia Kislova   Group: iGEM20_Moscow   (2020-10-21)


CasX (Cas12e)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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:

  1. noEcoR1-F-58
    acgaaaaggaattTtatgcctgtgaga
  2. noEcoR1-R-58
    tctcacaggcataAaattccttttcgt
  3. noSpe1-F-59
    ttacgaggggcttacAagtaaaacgtattt
  4. noSpe1-R-59
    aaatacgttttactTgtaagcccctcgtaa
  5. no1Ng04-F-60
    gaagaagccCgccaaacgt
  6. no1Ng04-R-60
    acgtttggcGggcttcttc
  7. no2Ng04-F-59
    gatgagccCgccctgttt
  8. no2Ng04-R-59
    aaacagggcGggctcatc

Source

Addgene plasmid # 126418 ; http://n2t.net/addgene:126418 ; RRID:Addgene_126418

References

  1. 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