Part:BBa_J100282
rClone Red Version 2 with RBS: Device for GGA Cloning and Testing RBS elements and Riboswitches
rClone Red Version 2 (V2) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS 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 RBS or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. The level of expression of RFP will depend on the efficiency of the newly cloned RBS or riboswitch. Hopefully by removing the hairpin the in the original rClone Red it will increase translation of RFP. By adding the RBS this should glow red with the rClone red RFP.
Note that base 33 (within promoter P5) is supposed to be a T but in our device, it is a C.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 612
Illegal AgeI site found at 724 - 1000COMPATIBLE WITH RFC[1000]
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