Part:BBa_K5196000:Design

pJN105-NicA2 Plasmid

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2782
  Illegal XbaI site found at 4251
  Illegal PstI site found at 3021
  Illegal PstI site found at 3135
  Illegal PstI site found at 3417
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2782
  Illegal NheI site found at 2776
  Illegal PstI site found at 3021
  Illegal PstI site found at 3135
  Illegal PstI site found at 3417
  Illegal NotI site found at 4257
  Illegal NotI site found at 7170
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2782
  Illegal BglII site found at 476
  Illegal BamHI site found at 2715
  Illegal XhoI site found at 1462
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2782
  Illegal XbaI site found at 4251
  Illegal PstI site found at 3021
  Illegal PstI site found at 3135
  Illegal PstI site found at 3417
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2782
  Illegal XbaI site found at 4251
  Illegal PstI site found at 3021
  Illegal PstI site found at 3135
  Illegal PstI site found at 3417
  Illegal NgoMIV site found at 4898
  Illegal AgeI site found at 2550
  Illegal AgeI site found at 4738
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3462
  Illegal SapI site found at 2532
  

Design Notes

We had to consider the araBAD promoter site as well as the location of the restriction sites flanking the NicA2 gene. We decided to use the EcoRI and XbaI sites flanking this gene. However, the consensus Shine-Dalgarno sequence (AGGAGG) would be excised by this restriction digest, so we decided to regenerate this region up to the promoter via Gibson Assembly to ensure proper expression following arabinose induction.

Source

This plasmid was derived from Bordetella bronchiseptica; specifically, it is a derivative of the pBBR1MCS-5 plasmid which originates from this organism.