Part:BBa_K510047:Design

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.