Part:BBa_K592201:Design
Low to medium copy Lambda Red recombineering compatible plasmid
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1806
Illegal XbaI site found at 2736 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2946 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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).