Part:BBa_J119137

pClone Red - Device for Testing New Promoters via Golden Gate Assembly

pClone Red (previously called pClone Green) 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. For proper assembly, the new promoter must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end which means the insert will have a sticky end on the left side of the top strand, and a sticky end on the right side of the bottom strand. Successful GGA assembly replaces the reverse promoter driving GFP expression 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. Related parts are pClone Blue and pClone Basic . Development of the pClone plasmids and their use in courses are described in [http://www.lifescied.org/content/13/2/285.full?sid=d90271de-0445-493c-b663-d4e8df014e54 Campbell et al. 2014].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1499
  Illegal AgeI site found at 1611
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 837
  Illegal BsaI.rc site found at 760