Part:BBa_K510048

pUC18R6KT-miniTn7BB-Gm-attTn7

pUC18R6KT-miniTn7BB-Gm-attTn7 is a vehicle vector for the minitransposon miniTn7BB-Gm-attTn7. This transposon is a part of the [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Overview miniTn7 BioBrick toolkit], a set of plasmids harboring Tn7 derivatives that can be used for integration of BioBricks in single copy at a conserved target (the attTn7 site), in multiple bacterial genomes.

The mini-Tn7-Gm-attTn7 artificial transposon has been constructed based on pUC18R6KT-miniTn7-Gm (BBa_K510012) by inserting a portable attTn7 (BBa_K510022) in its BioBrick cloning site (BCS). This plasmid allows the integration of BioBricks into microbial chromosomes, using prefix or suffix cloning sites, without removing the attachment site of the Tn7 transposon (attTn7). This fact makes posible to integrate another BioBrick into the chromosome of a strain in which miniTn7BB-Gm-attTn7 transposition has occurred previously.

The structure of the mini-Tn7BB-Gm-attTn7 transposon is shown in the figure below. Tn7R and Tn7L are the right and left ends of Tn7 transposon, respectively, required for recognition of the transposase machinery and transposition. FRT is the Flp recombinase target site for excision of the resistance once the transposon is inserted in the genome of a living organism. The gentamycin resistance cassette (Gm) is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The portable attTn7 has been inserted in the BioBrick cloning site (BCS).

This plasmid is replicative only in E. coli strains expressing the pir gene thanks to its R6K replication origin. Also it has an origin for transference (oriT) for conjugation purposes. This plasmid can be used to maintain the miniTn7BB-Gm-attTn7 transposon in E. coli pir+ strains and may be used for transposition into the genomes of any bacteria without the pir gen, in which it is non-replicative. As a BioBrick cloning vector, pUC18R6KT-miniTn7BB-Gm-attTn7 is fully compatible with Assembly Standard 10. The complete transposon is flanked by SfiI restriction sites to facilitate subcloning in other vectors.

Sequence and Features

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