Composite

Part:BBa_K510042:Design

Designed by: David Caballero, Fernando Govantes   Group: iGEM11_UPO-Sevilla   (2011-10-19)

pUC18Sfi-miniTn7BB-Gm-Lux


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4367
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4367
    Illegal NheI site found at 5601
    Illegal NheI site found at 8567
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4373
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4367
    Illegal BglII site found at 3051
    Illegal BglII site found at 3322
    Illegal BglII site found at 3608
    Illegal BglII site found at 7565
    Illegal BamHI site found at 5540
    Illegal XhoI site found at 8395
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4367
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4367
    Illegal XbaI site found at 4382
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 5375
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1681
    Illegal BsaI site found at 9954
    Illegal BsaI.rc site found at 6963
    Illegal SapI site found at 5357
    Illegal SapI.rc site found at 2763
    Illegal SapI.rc site found at 10279


Design Notes

The BBa_K325909 BioBrick was inserted within the BCS of pUC18Sfi-miniTn7BB-Gm by EcoRI and PstI clonning. In order to construct the pUC18Sfi-miniTn7BB-Gm delivery plasmid, the miniTn7BB-Gm transposon was cleaved from the commercial plasmid pMA (Mr. Gene) with SfiI and ligated to SfiI-digested pUC18Sfi. This strategy removes all multi-cloning restriction sites from pUC18Sfi, except for the flanking SfiI, thus guaranteeing that the unique sites in the transposon are not duplicated. The remove of the multi-cloning restriction sites was verified by analytic digestions.

Source

The miniTn7BB-Gm minitransposon was synthesized commercially and pUC18SfiI is a commercial vector.

References

Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.