Plasmid_Backbone
K592202

Part:BBa_K592202:Design

Designed by: Antonio Ascue Avalos   Group: iGEM11_Uppsala-Sweden   (2011-09-21)

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 3199
    Illegal PstI site found at 2629
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3409
    Illegal NheI site found at 1403
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal PstI site found at 2629
    Illegal NotI site found at 9
    Illegal NotI site found at 3415
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3409
    Illegal BglII site found at 2842
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3409
    Illegal suffix found at 2
    Illegal XbaI site found at 1806
    Illegal XbaI site found at 3199
    Illegal PstI site found at 2629
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3409
    Plasmid lacks a suffix.
    Illegal XbaI site found at 1806
    Illegal XbaI site found at 3199
    Illegal XbaI site found at 3424
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal PstI site found at 2629
    Illegal NgoMIV site found at 1895
    Illegal NgoMIV site found at 2178
    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
    Illegal SapI site found at 2118
    Illegal SapI site found at 2328


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 was replaced with the late terminator of the Salmonella phage P22. To be able to remove the antibiotic resistance marker after chromosomal integration, the kanamycin marker from the pKD4 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 kanamycin resistance marker flanked by FRT sites comes from the pKD4 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.