The promoter, RBS, and reporter sequences from Lou et. al 2012 "Ribozyme-based insulator parts buffer synthetic circuits from genetic context". UNS sequences from Torella et. al 2013 "Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly"
This part is used for 2016 WM iGEM Ribozyme Characterization project. Lou et. al showed that sequences in the 5' UTR of a gene can affect the input-output relationship (transfer function) of a circuit. The RiboJ serves as an insulator to generalize the transfer function of a circuit regardless of promoter. The part is used in combination with BBa_K2066016 (constitutive Lac Repressor) to look at input-output relationship without RiboJ.
We designed this part for our Ribozyme Characterization Project to look at how RiboJ affects a transfer function.
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.
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
Modified pLacO-1 Promoter (Lou et. al 2012)
Part sequence inspired by Lou et al. 2012 (???Ribozyme-based insulator parts buffer synthetic circuits from genetic context???)
This promoter sequence is modified from section V of Supplementary Material of Lou et al. The Supplementary sequence contains 98bp of the end of BioBrick backbone pSB1C3, followed by an EcoRI site and an XbaI site, then 20bp of the beginning of pTac (as described in fig. S1), before beginning the sequence of plLacO-1 (as described in fig. S1). Here we use only the 20bp of pTac followed by the plLacO-1 sequence as described in fig. S1. WM iGEM 2016 used this part for our Ribozyme Characterization project.
Design inspired by Lou et al. 2012 (???Ribozyme-based insulator parts buffer synthetic circuits from genetic context???) so that constitutive LacI repressor can bind to it.
Sequence is from from Lou et al. Supplement section V.
This part is a strong RBS from Lou et. al 2012 "Ribozyme-based insulator parts buffer synthetic circuits from genetic context". The RBS is used to make some of 2016 WM iGEM Ribozyme Characterization project parts.
The sequence is modified from Lou et al. Supplement section V "Ribozyme-based insulator parts buffer synthetic circuits from genetic context".
cI-GFP is used for our Ribozyme Characterization project. It is a reporter molecule fused with cI used to look at the input-output transfer functions of IPTG-cIGFP both with the RiboJ insulator and without (Lou et al 2012). The sequence is modified from Lou et al. Supplement section V. However, within this sequence the T at position 1161 used to be a G; the T at 1166 used to be a C; the C at 1215 used to be a T; the A at 1282 used to be a C. All single-nucleotide substitutions were designed to preserve amino acid sequence and result in a <1% change in codon usage frequency in E. coli.)
We wanted this part for our ribozyme characterization project.
ACTAGA, scar present in Lou et al. 2012 ???Ribozyme-based insulator parts buffer synthetic circuits from genetic context??? RiboJ sequence, Supplement Section V.
This is the scar as the end of the RiboJ sequence (Lou et al. 2012) which we conserved through all of our uses of RiboJ <partinfo>BBa_K2066506<\partinfo>. This exists as a parts page so that it can be inserted into sequences which do not have RiboJ but still have the scar for the sake of continuity.
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