RBS + NpuDnaE C-intein RFC[105] non-slicing control construct (with His6)
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
This is the sequence of the SpeI/XbaI scar for RBS-CDS junctions in BioBricks standard assembly.
This is a shorter scar to ensure proper spacing between the RBS and CDS.
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.
N-terminal start overhang (T)(A)-(G)ATG=RBS+Start RFC[105] A
This standard overhang part was developed as part of the iGEM team Heidelberg 2014's Intein Toolbox [[#References[1]]]. All standard sequences can be reviewed in RFC[???] [[#References[2]]].
This is a standard overhang sequence for in-frame cloning of Proteins of Interest in front or behind an Intein. A detailed cloning strategy is found on the iGEM team Heidelberg 2014's wiki page as well as in RFC[???]. Specifically, this part contains the overhang A used to insert a protein behind any RFC[10] compatible RBS and in-frame with the start-codon. It lies within the four bases formed by the second last base of the XbaI/SpeI scar or the first base in front of start codon respectively and the start codon itself.
This part is only the sequence of a standard overhang. 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.
N-terminal GG-linker overhang GGTG=Gly+1/3Gly RFC[105] overhang CN
This standard overhang part was developed as part of the iGEM team Heidelberg 2014's Intein Toolbox [[#References|[1]]]. All standard sequences can be reviewed in RFC[???] [[#References|[2]]].
This is a standard overhang sequence for in-frame cloning of Proteins of Interest in front or behind an Intein. A detailed cloning strategy is found on the iGEM team Heidelberg 2014's [http://2014.igem.org/Team:Heidelberg/parts wiki page] as well as in RFC[???]. Specifically, this part contains the overhang D used to insert a protein-tag or similar in front of the N-terminal end of a C-Intein. It lies within the a double-glycine linker piece.
C-terminal non-splicing overhang AGGC=1/3Ala+Gly RFC[105] E
This is a standard overhang sequence for in-frame cloning of Proteins of Interest in front or behind an Intein. A detailed cloning strategy is found on the iGEM team Heidelberg 2014's [http://2014.igem.org/Team:Heidelberg/parts wiki page] as well as in RFC[???]. Specifically, this part contains the overhang E* used to insert a protein in front of a non-splicing N-Intein, which could often serve as control for protein splicing experiments. It lies within the four bases formed by the glycine and the first base of the subsequent Leucine/Isoleucine or similar of the mutated N-terminal splicing site.
An N-terminal hexahistidine-tag to be used as insert in RFC[???] cloning (Can be cloned in front of a C-Intein).
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
2bp sequence extending the terminal G in the RFC[???] overhang C to a Glycin codon.
mutated (c-terminal Ser ->Ala) NpuDnaE C-Intein cloning piece
This is a mutated version of the NpuDnaE C-intein. The C-terminal Histidine was mutated to an Alanine to prevent protein splicing. This part therefore usually serves as an experimental control. See all other specifications to this part in <partinfo>BBa_K1362400</partinfo>.
Design of a control construct for various assays.
C-terminal stop overhang TAAT=STOP+1/3Stop RFC[105] overhang Z
This is a standard overhang sequence for in-frame cloning of Proteins of Interest in front or behind an Intein. A detailed cloning strategy is found on the iGEM team Heidelberg 2014's [http://2014.igem.org/Team:Heidelberg/parts wiki page] as well as in RFC[???]. Specifically, this part contains the overhang F used to insert the last part of an intein-fused protein directly in front of an RFC[10] double stop-codon. It lies within the first for bases of the stopstop sequence.
mRFP selection marker with outward BsaI restriction sites
This selection marker part comes from the iGEM team Heidelberg 2014's Intein Toolbox [[#References|[1]]]. The associated cloning strateges can be reviewed in RFC[???]. The mRFP generator sequence was cloned from <partinfo>BBa_J04450 </partinfo>.
This part is an mRFP selection marker used in the cloning process described in RFC[???] [[#References|[2]]]. It was developed by the iGEM team Heidelberg 2014 as part of our toolbox constructs for intein cloning [[#References|[1]]].
This part will not be sent as physical DNA. It is however a useful part for golden gate cloning.
This part is used for the assembly of other parts. It has no function and will not be send in as physical DNA.
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
BsaI reverse 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.
BsaI restriction site for RFC[105] cloning
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