Part:BBa_K592201:Design

Low to medium copy Lambda Red recombineering compatible plasmid

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1806
  Illegal XbaI site found at 2736
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2946
  Illegal NheI site found at 1403
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2952
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2946
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2946
  Illegal suffix found at 2
  Illegal XbaI site found at 1806
  Illegal XbaI site found at 2736
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2946
  Plasmid lacks a suffix.
  Illegal XbaI site found at 1806
  Illegal XbaI site found at 2736
  Illegal XbaI site found at 2961
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 994
  Illegal AgeI site found at 1317
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 289
  

Design Notes

To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. To be able to remove the antibiotic resistance marker after chromosomal integration, the resistance marker from the pKD3 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind.

Source

The P22 late terminator come from the genome of the Salmonella phage P22 (GenBank: AF217253.1, 19662..19702), and the chloramphenicol resistance marker flanked by FRT sites comes from the pKD3 plasmid (Datsenko and Wanner, 2000).

References

Datsenko, K. A. and B. L. Wanner (2000). "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products." Proc Natl Acad Sci U S A 97(12): 6640-5.