Part:BBa_K864020

Low copy BioBrick temperature sensitive standard vector

pSB8A15(FRT) is a BioBrick standard vector with low copy pSC101ts replication origin BBa_K864051 (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30’C but not at at 42’C [1]. 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.

Usage and Biology

This part is available as pSB8A15(FRT)-pUCori-redq. This facilitates fast growth at 37° C, high plasmid yields and fast red color expression on transformation plates.

Origin switching

Easy ori replacement is possible in the pSB8x15 backbones. The plasmid can have the pSC101ts ori cut out with NheI and MluI and another ori ligated in. To remove any religated pSC101ts, the transformants can be grown at 42° C.

More reliable recombineering

A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones 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 3687
  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 3693
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3687
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3687
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3687
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3702
  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.