Device

Part:BBa_K5179102:Design

Designed by: Caden Sanko   Group: iGEM24_William-and-Mary   (2024-09-29)


dCas9 P4 Cosmid


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4825
    Illegal NotI site found at 662
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1185
    Illegal BamHI site found at 7104
    Illegal BamHI site found at 8113
    Illegal XhoI site found at 2867
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 8046
    Illegal BsaI.rc site found at 8023
    Illegal SapI site found at 3095


Design Notes

EcoRI cut site was removed from original dCas9 sequence after assembly of device via site directed mutagenesis at 5070 (t->a).

Source

Includes the Cos site and Sid operon from bacteriophage satellite P4. DNA sourced from P4 cosmid (Addgene plasmid # 196335), which was a gift from Baojun Wang. dCas9 cassette derived from S. pyogenes. DNA was sourced from plasmid pdCas9 (Addgene plasmid # 46569), which was a gift from Luciano Marraffini. DNA fragments were PCR amplified and assembled via Gibson Assembly.


References

Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12. PMID: 23761437; PMCID: PMC3753641.

Fa-Arun J, Huan YW, Darmon E, Wang B. Tail-Engineered Phage P2 Enables Delivery of Antimicrobials into Multiple Gut Pathogens. ACS Synth Biol. 2023 Feb 17;12(2):596-607. doi: 10.1021/acssynbio.2c00615. Epub 2023 Feb 2. PMID: 36731126; PMCID: PMC9942202.