Plasmid

Part:BBa_K4761421

Designed by: Pinhan Pan   Group: iGEM23_HiZJU-China   (2023-09-30)


pTarget-pheA2

SgRNA(pheA)2 expression plasmid for pheA knockout. Created based on sgRNA(pheA)2 (BBa_K4761021) and pTarget (BBa_K4761107) using overlapping PCR for guide RNA to function in CRISPR-Cas9 double plasmid system.

Fig.1 The sgRNA(pheA)2 expression box was successfully constructed. DNA sequencing results showed that sgRNA(pheA)2 was successfully inserted into the pTarget plasmid.

We used pTarget-pheA1 paired with pCas plasmid and Donor DNA to obtain some ΔpheA mutant strains of Escherichia coli.

Fig.2 pheA gene knockout validation. Positive results should have a band around 2000bp.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2065
    Illegal XbaI site found at 2071
    Illegal SpeI site found at 1956
    Illegal PstI site found at 2083
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2065
    Illegal NheI site found at 1933
    Illegal SpeI site found at 1956
    Illegal PstI site found at 2083
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2065
    Illegal BglII site found at 2095
    Illegal BamHI site found at 1920
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2065
    Illegal XbaI site found at 2071
    Illegal SpeI site found at 1956
    Illegal PstI site found at 2083
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2065
    Illegal XbaI site found at 2071
    Illegal SpeI site found at 1956
    Illegal PstI site found at 2083
    Illegal NgoMIV site found at 771
  • 1000
    COMPATIBLE WITH RFC[1000]


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