Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Todd Eckdahl
Date created: 2014-08-26 11:00:00
Date modified: 2015-08-31 04:08:28
tClone: Device for Testing Transciptional Terminators and Riboswitches via Golden Gate Assembly
| Types | DnaRegion |
| Roles | Intermediate engineered_region |
| Sequences | BBa_J119367_sequence (Version 1) |
Description
tClone is a construction intermediate that can be used to build devices that will allow users to clone and test new transcriptional terminators and riboswitches that function by antitermination without gel purification or other preparation of DNA. Use of BioBrick cloning to add the RFP gene into the suffix of tClone produce tClone Red, which can be used as a reporter for cloning and testing new transcriptional terminators and riboswitches. Similarly, use of BioBrick cloning to insert the TetA gene into the suffix of tClone produces tClone TetA, which can be used to measure new transcriptional terminators and riboswitches. tClone vectors can be used as destination vectors 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 RFP coding sequence. Whether or not transcription proceeds to the RFP gene is determined by the strength of the terminator or whether termination or antitermination occurs at the riboswitch. The part incorporates the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.Notes
NoneSource
IDT G block| Sequence Annotation | Location | Component / Role(s) |
| P5 Promoter BsaI Left L3SP22 Txn Terminator GFP B0030 RBS rev P2 Promoter rev LS1P53 Txn Terminator BsaI Right | 1,36 42,47 48,104 112,831 838,852 860,905 906,957 958,963 | promoter feature/promoter feature/dna sequence_feature feature/stem_loop stem_loop CDS feature/cds feature/rbs ribosome_entry_site promoter feature/promoter feature/stem_loop stem_loop feature/dna sequence_feature |