Composite

Part:BBa_K510015:Design

Designed by: David Caballero   Group: iGEM11_UPO-Sevilla   (2011-09-19)

pUC18R6KT-miniTn7BB-Gm-RBS+RFP


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4528
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4528
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4534
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4528
    Illegal BglII site found at 3212
    Illegal BglII site found at 3483
    Illegal BglII site found at 3769
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4528
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4528
    Illegal XbaI site found at 4543
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 5130
    Illegal AgeI site found at 5242
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1854
    Illegal SapI site found at 518


Design Notes

The BBa_K093005 BioBrick was inserted within the BCS of pUC18R6KT-miniTn7BB-Gm by EcoRI and PstI clonning.

Source

The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg. The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products.

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.