Part:BBa_J119458

tClone Blue: Device for Testing Transciptional Terminators and Riboswitches via Golden Gate Assembly

tClone Red will allow users to clone and test new transcriptional terminators and riboswitches that function by antitermination without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new terminator or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert 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. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new terminator or riboswitch. Transcription will be initiated in the direction of the AmilCP Blue coding sequence. Whether or not transcription proceeds to the AmilCP Blue gene is determined by the strength of the terminator or whether termination or antitermination occurs at the riboswitch. 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
  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 1487
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 849
  Illegal BsaI.rc site found at 42