Part:BBa_K510014

pUC18R6KT-miniTn7BB-Km

pUC18R6KT-miniTn7BB-Km is a vehicle vector for the minitransposon miniTn7BB-Km. 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 structure of the mini-Tn7BB-Gm 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 kanamycin resistance cassette (Km) is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The BioBrick cloning site (BCS) includes the prefix and suffix restrictions sites used in assembly standard 10, and it is the place where BioBricks are inserted for genome integration.

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-Km 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-Km 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 4799
  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4799
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4805
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4799
  Illegal BglII site found at 3212
  Illegal BglII site found at 3483
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4799
  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4799
  Illegal XbaI site found at 4814
  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