Device

Part:BBa_K5179101:Design

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


KanR Targeting 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 8102
    Illegal XhoI site found at 2867
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 3095
    Illegal SapI.rc site found at 8031


Design Notes

Contains a single base pair substitution at 5070 (t->a) compared to original P4 cosmid (Fa-Arun et al., 2023) to remove EcoRI cut site. Please note: characterization work by William-and-Mary iGEM was done prior to induction of this synonymous mutation via site directed mutagenesis, however the mutation is silent so the results of characterization work done for this part applies readily to either variant of the part. We recomend using the sequence listed in this registry entry for de novo synthesis, especially if being used in standard assembly.

Source

Includes the Cos site and Sid operon from bacteriophage satellite P4. Cas9 cassette derived from S. pyogenes. DNA sourced from P4 cosmid (Addgene plasmid # 196335), which was a gift from Baojun Wang. Neomycin phosphotransferase crRNA spacer was synthesized by Integrated DNA Technologies. Fragments were assembled in a golden gate reaction using BsaI.

References

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.