Part:BBa_J119139

Device for Testing New Promoters via Golden Gate Assembly

This part will allow users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly. The left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. Successful GGA assembly replaces the reverse ampil (Blue) gene expression casette with the new promoter. The new promoter will drive RFP expression in the forward direction or GFP expression if it has reverse promoter function. The part incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 750
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 750
  Illegal NotI site found at 756
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 750
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 750
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 750
  Illegal XbaI site found at 765
  Illegal AgeI site found at 796
  Illegal AgeI site found at 904
  Illegal AgeI site found at 2233
  Illegal AgeI site found at 2345
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 72
  Illegal BsaI site found at 1583
  Illegal BsaI.rc site found at 777