Difference between revisions of "Part:BBa K510047:Design"

 
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===Design Notes===
 
===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.
 
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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===Source===
 
===Source===
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===References===
 
===References===
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Ella Gringauz, Karina A. Orle, Candace S. Waddell, and Nancy L. Craig (1988). Recognition of Escherichia coli attTn7 by Transposon Tn7: Lack of Specific Sequence Requirements at the Point of Tn7 Insertion. Journal of Bacteriology, June 1988, p. 2832-2840.
 +
Candace S. Waddellt and Nancy L. Craig (1989). Tn7 transposition: Recognition of the attTn7 target sequence. Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3958-3%2, June 1989, Biochemistry. Joseph E. Peters and Nancy L. Craig (2001). Tn7: smarter than we thought. Nature reviews, November 2001, Vol. 2, 806.

Revision as of 15:40, 22 October 2011

pUC18Sfi-miniTn7BB-Gm-attTn7


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.

Source

The miniTn7BB-Gm minitransposon was synthesized commercially and pUC18SfiI is a commercial vector. The attTn7 was obtained from the purified genome of E. coli MC4100 by PCR (using a high accuracy Pfu polymerase) with the follow primers: gaattcgcggccgcttctagagTGCAGCTGCTGGCTTACCATG and ctgcagcggccgctactagtaACATGGAGTTGGCAGGATGTTTG.

References

Ella Gringauz, Karina A. Orle, Candace S. Waddell, and Nancy L. Craig (1988). Recognition of Escherichia coli attTn7 by Transposon Tn7: Lack of Specific Sequence Requirements at the Point of Tn7 Insertion. Journal of Bacteriology, June 1988, p. 2832-2840. Candace S. Waddellt and Nancy L. Craig (1989). Tn7 transposition: Recognition of the attTn7 target sequence. Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3958-3%2, June 1989, Biochemistry. Joseph E. Peters and Nancy L. Craig (2001). Tn7: smarter than we thought. Nature reviews, November 2001, Vol. 2, 806.