Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Malcolm Campbell
Date created: 2016-06-20 11:00:00
Date modified: 2016-06-21 01:17:58
rClone Red Version 2: Device for GGA Cloning and Testing RBS elements and Riboswitches
| Types | DnaRegion |
| Roles | engineered_region Device |
| Sequences | BBa_J100272_sequence (Version 1) |
Description
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.Notes
When designing oligonucleotides for use with rClone Red V2, make sure they result in 5' overhang sticky ends that are CGAC (left) and GCGG (right). Also make sure the oligonucleotides do not contain binding sites for BsaI. Finally, make sure the RBS element ends immediately before the GCGG right sticky end. This will ensure a spacing of 6 bases between the RBS and the ATG start codon of RFP. Below is an example.Source
/| Sequence Annotation | Location | Component / Role(s) |
| P5 Promoter BsaI Site GFP RBS B0034 P2 Promoter BsaI Site Optimized E1010 RFP | 1,37 42,48 55,775 781,793 803,849 849,855 862,1542 | feature/promoter promoter sequence_feature feature/misc CDS feature/cds feature/rbs ribosome_entry_site feature/promoter promoter feature/misc sequence_feature feature/cds CDS |