Composite

Part:BBa_K510021:Design

Designed by: David Caballero & Fernando Govantes   Group: iGEM11_UPO-Sevilla   (2011-09-19)
Revision as of 22:03, 19 September 2011 by Dcabpra (Talk | contribs) (→‎References)

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pUC18R6KT-miniTn7BB-Gm-Lux


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 NheI site found at 5762
    Illegal NheI site found at 8728
    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
    Illegal BglII site found at 7726
    Illegal BamHI site found at 5701
    Illegal XhoI site found at 8556
  • 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 5536
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 10115
    Illegal BsaI.rc site found at 1854
    Illegal BsaI.rc site found at 7124
    Illegal SapI site found at 518
    Illegal SapI site found at 5518
    Illegal SapI.rc site found at 10440


Design Notes

The BBa_K325909 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.