Part:BBa_K510048:Design

pUC18R6KT-miniTn7BB-Gm-attTn7

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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1854
  Illegal SapI site found at 518
  

Design Notes

The portable attTn7 (BBa_K510022) BioBrick was inserted within the BCS of pUC18R6KT-miniTn7BB-Gm (BBa_K510012) 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.

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.