Part:BBa_K1321327

pTet02

This plasmid encodes for a TetR generator and an mRFP1 behind pTet promoter in pSEVA331Bb backbone, making mRFP1 inducible via tetracycline. See attached file for annotated sequence. This construct is a member of the Komagataeibacter genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332). File:IC14 PTet02.gb

Komagataeibacter toolkit was designed to ease genetic engineering of cellulose-based biomaterials using the cellulose synthesizing bacterium Komagataeibacter rhaeticus iGEM (parts Bba_K1321305 and BBa_K1321306). As no registry parts had been tested in K. rhaeticus, the aim of this toolkit was to determine the parts usable in K. rhaeticus and to characterize them in this host. pSEVA331-Bb is a non-standard broad host range plasmid capable of replication in K. rhaeticus and E. coli (a shuttle vector) and was selected because the registry's standard plasmid backbone pSB1C3 can not be used for K. rhaeticus engineering.

NOTE: Because the registry's standard plasmid backbone pSB1C3 is not capable of replication in Komagataeibacter or Gluconacetobacter species, the Komagataeibacter genetic engineering toolkit is housed mainly in pSEVA331-Bb. pSEVA331-Bb is a non-standard backbone, which therefore can't be quality controlled by and maintained in the Registry. However, in order to make the Komagataeibacter toolkit available for the synthetic biology community, Imperial iGEM 2014 team has made it freely available in AddGene or upon request, with quality control provided (see Experience).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4629
  Illegal SpeI site found at 890
  Illegal PstI site found at 1773
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4629
  Illegal SpeI site found at 890
  Illegal PstI site found at 1773
  Illegal NotI site found at 1766
  Illegal NotI site found at 4635
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4629
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4629
  Illegal SpeI site found at 890
  Illegal PstI site found at 1773
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4629
  Illegal XbaI site found at 4644
  Illegal SpeI site found at 890
  Illegal PstI site found at 1773
  Illegal NgoMIV site found at 2948
  Illegal AgeI site found at 1469
  Illegal AgeI site found at 1581
  
• 1000
  COMPATIBLE WITH RFC[1000]