Sortase A circularization construct (with Smt3 and His6)
synthesized as found in the T7 genome and several commercial expression plasmids.
RFC10 compatible strong RBS derived from the T7 phage gene 10a (major capsid protein)[1]. When assembled to a coding part with the A of the start codon being part of the XbaI site, the RBS will be shifted one bp downstream compared to the native sequence. The sequence was successfully used by the iGEM team Heidelberg 2014 for the expression of many proteins in E.coli. 1. Olinss, P. & Rangwala, S. H. Derived from Bacteriophage T7 mRNA Acts ELS an Enhancer of Translation of the lac2 Gene in. 16973???16976 (1989).
The 18 bp including the XbaI that can be found upstream of the presumably important part of the RBS were included into the sequence just to make sure it works. However to fully comply with RFC10 a G was inserted behind the XbaI site.
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
Sortase A circularization construct (with Smt3 and His6; without RBS)
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
BsaI restriction site for RFC[105] cloning
This standard restriction site sequence is used as part of the iGEM team Heidelberg 2014's Intein Toolbox [[#References|[1]]]. All associated standard sequences can be reviewed in RFC[???]
This is the reverse complement of a BsaI restriction site headed by an Adenine as a spacer-base to separate the recognition sequence from the outward-lying cutting sequence. It was used by us for scarless golden-gate cloning to fuse inteins to other proteins and thereby implement a variety of possible port-translational modifications.
This part is only the sequence of a restriction site. It will not be sent in as physical DNA and was merely created to easily compose new parts in the RFC[???] standard. In the actual cloning process this sequence was and is recommended to be inserted with the according PCR primers.
This part codes for the amino acid sequence ENLYFQG. This site is recognized by TEV protease (catalytic domain of the Tobacco Etch Virus nuclear inclusion a (NIa) protein), which will cleave the Gln-Gly peptide bond [[{{PAGENAME}}:Design#References|[1]]]. The final four nucleotides of this sequence are GGGT, which will be the overhang produced wenn a reverse-facing BsaI site (<partinfo>BBa_K1362423</partinfo>, <partinfo>BBa_K1362427</partinfo>, <partinfo>BBa_K1362447</partinfo>) is directly following this part, as in the Sortase A circularization constructs (<partinfo>BBa_K1362202</partinfo>, <partinfo>BBa_K1362203</partinfo>, <partinfo>BBa_K1362204</partinfo>, <partinfo>BBa_K1362205</partinfo>).
BsaI reverse restriction site for RFC[???] cloning (shortened by one C)
This Sequence starts with a part of the reverse-complemented BsaI recognition site. The missing C of the recognition site must be included in the downstream part. It contains a spacer thymidine so that BsaI will cut the bottom strand directly upstream and the top strand 4 nucleotides upstream.
This part codes for the amino acid sequence LPETGG. This site is recognized by Sortase A, which will cleave the Thr-Gly peptide bond and subsequently act as a transpeptidase to attach any peptide with an N-terminal glycine [[{{PAGENAME}}:Design#References|[1]]]. The first four nucleotides of this sequence are CTTC, which will be the overhang produced wenn a forward-facing BsaI site (<partinfo>BBa_K1362424</partinfo>, <partinfo>BBa_K1362425</partinfo>) is directly following this part, as in the Sortase A circularization constructs (<partinfo>BBa_K1362202</partinfo>, <partinfo>BBa_K1362203</partinfo>, <partinfo>BBa_K1362204</partinfo>, <partinfo>BBa_K1362205</partinfo>).
This part codes for a hexahistidine tag, which can be used in protein purification by affinity chromatography.
A RFC12 compatible part that simply codes for two stop codons. This part does not have any degradation tag.
Made with synthetic oligos: 5' AATTC GCGGCGC T ACTAGT TAATAA GCTAGC A GCGGCCG CTGCA 3' 5' GCGGCCGCTGCTAGC TTATTA ACTAGTAGCGCCGC G 3' Note that both primers were ordered phosphorylated. An alternative is to phosphorylate the primers yourself with a kinase.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
Transcriptional terminator consisting of a 64 bp stem-loop.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Conversion of the iGEM parts registry to SBOL2.1