Promoter characterization part - R0040 without RiboJ
This part was created using Gibson Assembly methods. We used K2066035 as a template and an amplicon containing the relevant promoter sequence +/- RiboJ combination as the insert. RiboJ sequence sfGFP sequence from Lou et al., Supplement section V. sfGFP sequence derived and modified from Lou et al., Supplement section V. The UNS sequences are from Torella et al. 2013.
The part is flanked by UNS 2 and UNS 3 as per the WM UNS gibson cloning standard. This part is used to characterize the promoter part Bba_R0040 without riboJ insulation so that the effects of RiboJ insulation on gene expression can be characterized using the corresponding WM part K2066078. This part on 1C3 can be cotransformed into cells along with K2066016 (preferably on pSB3K3 or other low copy backbone for ideal induction behavior) and the resulting IPTG-induction curve can be compared to those of other parts (K2066059 - K20660108) with different promoter / RiboJ variations.
This part was designed to perform RiboJ characterization verifying and building on that performed in Lou et al. 2012 (???Ribozyme-based insulator parts buffer synthetic circuits from genetic context???). This part will be used in concert with WM16_015 (plLACO-1 Ribozyme Chracterization Part ??? cI GFP) to determine the influence of the RiboJ insulator sequence on gene expression.
Kalen Clifton, Christine Gao, Andrew Halleran, Ethan Jones, ￼Likhitha Kolla, Joseph Maniaci, John Marken, John Mitchell, Callan Monette, Adam Reiss
UNS 2 Sequence, from Torella et al., 2013
Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860.
This is Unique Nucleotide Sequence 2, (UNS 2), from Torella et al., 2013. The William and Mary iGEM team has adopted this as our standard prefix; as such, all of our parts will have this sequence immediately following the BioBrick prefix. We took this measure in order to allow easier Gibson Assembly cloning of our parts. Primer sequences which can be used to clone with the UNS 2/3 standard can be found on our wiki.
UNS 2 was chosen because it works well with UNS 3 and it is in accordance with the BioBrick standard.
Kalen Clifton, Christine Gao, Andrew Halleran, Ethan Jones, Likhitha Kolla, Joseph Maniaci, John Marken, John Mitchell, Callan Monette, Adam Reiss
UNS 3 Sequence, from Torella et al., 2013
This is Unique Nucleotide Sequence 3, (UNS 3), from Torella et al., 2013. The William and Mary iGEM team has adopted this as our standard prefix; as such, all of our parts will have this sequence immediately following the BioBrick prefix. We took this measure in order to allow easier Gibson Assembly cloning of our parts. Primer sequences which can be used to clone with the UNS 2/3 standard can be found on our wiki. The sequence for this part came from the following paper: Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, gkt860. A huge thanks to all the researchers involved in its original creation!
This UNS sequence was chosen to serve as the 3' primer in our standard because it works well with UNS 2 and it adheres to the BioBrick standards.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Double terminator consisting of BBa_B0010 and BBa_B0012
This is part B0034 with the scar region TACTAG added onto the part so that the correct scar region will be present in WM composite parts K2066059 - K20660108. This has no other purpose, but is just necessary do the system of the registry.
The sequence for this sfGFP reporter gene is modified from Lou et al. Supplement section V. This is the sequence of superfolder GFP BBa_I746916, but with four codon modifications to match WM16_015: at position 441, G->T. At 446, C->T. At 495, T->C. At 562, C->A.
The part is a reporter used for K2066014.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1