Part:BBa_K510000

pUC18Sfi-miniTn7BB-Gm

pUC18Sfi-miniTn7BB-Gm is a vehicle vector for the minitransposon miniTn7BB-Gm. 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 genome.

Our [http://2011.igem.org/Team:UPO-Sevilla/Foundational_Advances/MiniTn7/Bioinformatics/attTn7_Insertion_Site Bioinformatic analysis] reveal that the insertion site of the miniTn7 is highly conserved along the philogeny. This means that the miniTn7 BioBrick tool kit is possibly useful non only in bacteria, but in other Archaea or Eukaryotic species. If the target site for the insertion of the miniTn7 is not present in your working organism, we also provide a portable attTn7 (BBa K510022).

The main features of miniTn7BB-Gm are: (i) wild-type Tn7R and Tn7L ends, enabling transposition in vivo when the Tn7 transposase is expressed in trans, (ii) prefix and suffix with unique EcoRI, XbaI, SpeI and PstI restriction sites, fully compatible with Assembly Standard 10, (iii) annealing sequences for standard primers to facilitate amplification and sequencing of cloned BioBricks, (iv) transcriptional terminators flanking the multi-cloning site to efficiently insulate cloned BioBricks from vector transcription, (v) a selectable gentamycin (Gm) resistance cassette flanked by duplicated SphI and NcoI sites to facilitate marker replacement, (vi) flipase recognition elements (FRTs) flanking the gentamycin resistance cassette, to allow clean excision of the marker in strains bearing transposon insertions, and (vii) SfiI sites flanking the complete transposon to facilitate cloning into the appropriate plasmid vectors. A schematic of miniTn7BB-Gm showcasing the aforementioned features is shown in the figure below.

pUC18Sfi is a pUC18-derived cloning vector harboring an ampicillin resistance marker, a high copy number mutant pMB1 replication origin and the pUC18 multi-cloning site flanked by two SfiI restriction sites. Because of its high copy number and its ability to replicate in any E. coli strain and in other enterobacteria, pUC18Sfi is suitable for maintenance of miniTn7 transposon derivatives, and for simple plasmid preparation and genetic manipulation. pUC18Sfi can be used to deliver transposons by transformation in bacteria in which it does not replicate (i.e., non-enteric bacteria), but not in the enterics.

As a BioBrick cloning vector, pUC18Sfi-miniTn7BB-Gm is fully compatible with Assembly Standard 10.

More information about this plasmid from the UPO-Sevilla 2011 team in the Experience section.

Sequence and Features

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