Part:BBa_K4727009

Low copy backbone for K. pneumoniae and A. baumannii

This plasmid backbone was developed by Wang et al [1], it is provided of two ORIs: the first one a p15a medium/low copy number ORI for E. coli, the second one is capable o maintaining the plasmid both in Acinetobacter baumannii and Klebsiella pneumoniae

This backbone has been made standard RFC[10] compatible and provided with the RFC[10] prefix and suffix. Flanked by prefix and suffix is located part BBa_J23119 that has been demonstrated able to promote mRNA synthesis in K. pneumoniae and A. baumannii

This part is the result of and engineering cycle applied on part BBa_K4727000 out of the necessity to express a dCas expression cassette. Our experiments, and the literature, suggests that plasmid backbone BBa_K4727000 has an ORI for E. coli that provides a high number of copies (500-700). This information, together with the knowledge that the Cas protein if present in large quantities results in cellular toxicity, made us hypothesize that cloning a dCas expression cassette in a high copy number plasmid as BBa_K4727000 can lead to no results. So, out of this necessity this new part was born. It maintains the same ORI for

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 4850
  Illegal suffix found at 4907
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4850
  Illegal NheI site found at 2155
  Illegal NheI site found at 4878
  Illegal NheI site found at 4901
  Illegal SpeI site found at 4908
  Illegal PstI site found at 4922
  Illegal NotI site found at 4856
  Illegal NotI site found at 4915
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4850
  Illegal BglII site found at 2348
  Illegal XhoI site found at 2961
  Illegal XhoI site found at 4966
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4850
  Illegal suffix found at 4908
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4850
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4865
  Illegal SpeI site found at 4908
  Illegal PstI site found at 4922
  Illegal AgeI site found at 5462
  Illegal AgeI site found at 5785
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SapI site found at 5194