Difference between revisions of "Part:BBa K510000:Design"

(New page: The miniTn7BB-Gm synthetic minitransposon was digested at the flanking SfiI sites and cloned into SfiI-digested pUC18-Sfi. This results in the elimination of the complete pUC18-SfiI multi-...)
 
 
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The miniTn7BB-Gm synthetic minitransposon was digested at the flanking SfiI sites and cloned into SfiI-digested pUC18-Sfi. This results in the elimination of the complete pUC18-SfiI multi-cloning site, except for the duplicated SfiI sites that remain on both sides of the transposon, thus facilitating its transfer to other vectors.
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__NOTOC__
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<partinfo>BBa_K510000 short</partinfo>
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<partinfo>BBa_K510000 SequenceAndFeatures</partinfo>
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===Design Notes===
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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. The direction of miniTn7BB-Gm insertion was determined by digestion.
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More information about [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Experimental_Results/Design_and_Synthesis Design and Synthesis] and
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[http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Experimental_Results/Constructions_of_Vectors Construction of delivery vectors for miniTn7BB] in these links.
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===Source===
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The miniTn7BB-Gm minitransposon was synthesized commercially and pUC18SfiI is a commercial vector.
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===References===
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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.

Latest revision as of 17:56, 29 October 2011

pUC18Sfi-miniTn7BB-Gm


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 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
  • 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1681
    Illegal SapI.rc site found at 2763

Design Notes

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. The direction of miniTn7BB-Gm insertion was determined by digestion.

More information about [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Experimental_Results/Design_and_Synthesis Design and Synthesis] and [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Experimental_Results/Constructions_of_Vectors Construction of delivery vectors for miniTn7BB] in these links.

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.