Part:BBa_K4761422

pTarget-pykA1

SgRNA(pykA)1 expression plasmid for pykA knockout. Created based on sgRNA(pykA)1 (BBa_K4761022) and pTarget (BBa_K4761107) using overlapping PCR for guide RNA to function in CRISPR-Cas9 double plasmid system.

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

We used pTarget-pykA1 paired with pCas plasmid and Donor DNA to obtain some ΔpykA mutant strains of Escherichia coli.

Fig.2 pykA 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 2064
  Illegal XbaI site found at 2070
  Illegal SpeI site found at 1955
  Illegal PstI site found at 2082
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2064
  Illegal NheI site found at 1932
  Illegal SpeI site found at 1955
  Illegal PstI site found at 2082
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2064
  Illegal BglII site found at 2094
  Illegal BamHI site found at 1920
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2064
  Illegal XbaI site found at 2070
  Illegal SpeI site found at 1955
  Illegal PstI site found at 2082
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2064
  Illegal XbaI site found at 2070
  Illegal SpeI site found at 1955
  Illegal PstI site found at 2082
  Illegal NgoMIV site found at 771
  
• 1000
  COMPATIBLE WITH RFC[1000]