Part:BBa_K864022

Low copy BioBrick temperature sensitive standard vector

pSB8K15(FRT) is a BioBrick standard vector with low copy pSC101ts replication origin BBa_K864051(~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30’C but stop replicating at 42’C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.

The resistance cassette is flanked by FRT sites (BBa_J61020) which allows for subsequent removal of the resistance cassette from the chromosome by the FLP recombinase. The pSB8X15 series are especially suitable for carrying the FLP recombinase, since they can be removed by incubation at 42°C.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3711
  Illegal NheI site found at 2425
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3717
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3711
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3711
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3711
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3726
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 2319
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.