Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Chris French
Date created: 2006-10-25 11:00:00
Date modified: 2015-08-31 04:08:46
hybrid lambda PR-lac promoter, repressed by lambda cI and lacI, plus lacZ
| Types | DnaRegion |
| Roles | sequence_feature Other |
| Sequences | BBa_J33205_sequence (Version 1) |
Description
This part is a hybrid promoter designed to be repressed by either lambda cI or LacI. It consists of the the PRM-PR promoter region of bacteriophage lambda, including cI binding sites OR1, OR2 and OR3 as well as the -35 and -10 sites of both PRM and PR, fused via an introduced XhoI site to the LacI binding site of the lac promoter. (In this version, the lacZ' gene encoding the N-terminal 76 amino acid residues of LacZ has also been included to facilitate testing.) This approximately preserves the relationship between the -10 promoter region and and the LacI binding site. Our hope is that this promoter will be effectively repressed in the presence of either lambda cI or LacI. However, this has not been properly tested at the time of writing. We can say that good LacZ activity is observed in the presence of IPTG, indicating that the basic promoter function of lambda PR appears to be intact. Our sequencing also suggests two single nucleotide mutations within OR2 as compared to published sequence, which may be natural variants or PCR-induced mutations; in the latter case, they may or may not have some effect on cI binding. Note also that this construct includes the PRM promoter, which ought to be functional in the reverse direction.Notes
This part includes the entire lambda PRM-PR region, with cI binding sites OR1, OR2 and OR3 and the -35 and -10 sequences of both promoters (PRM and PR). An XhoI site was introduced by PCR, and the DNA was joined to the region of the lac promoter beginning with the LacI binding site. The distance between the LacI binding site and the PR -10 site in the fusion is approximately the same as that between the LacI binding site and its native -10 site; thus we hope that LacI will be able to repress PR in our construct. However, this has yet to be properly tested.Source
The lambda-derived portion (bases 4 to 78) is derived from lambda DNA by PCR using primers based on published sequence (Genbank accession J02459, gi:215104). The lac portion (bases 84 to 353) is derived from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575).| Sequence Annotation | Location | Component / Role(s) |
| PRM -10 cI binding site OR3 PRM -35 cI binding site OR2 PR -35 cI binding site OR1 PR -10 XhoI fusion site LacI binding site rbs lacZ' | 9,14 13,29 32,37 36,52 50,55 60,76 73,78 79,84 85,105 112,115 123,353 | feature/promoter promoter feature/operator operator feature/promoter promoter operator feature/operator promoter feature/promoter operator feature/operator promoter feature/promoter feature/misc sequence_feature feature/operator operator ribosome_entry_site feature/rbs CDS feature/cds |