RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). 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 Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
3' stabilizing mRNA designed by Christina Smolke and Jay Keasling for stabilizing gfp.
A single base pair change (A to T at position 25) from the original design was made in order to remove a PstI site occurring in the loop of the hairpin. This should have no effect on the secondary structure.
T3 promoter as annealed oligonucleotides
Synthesized oligonucleotides as described on main page.
This is a part used as a building block for T3 driven expression. However, this part uses a special cloning method with the following synthesized nucleotides: pT3sense: aattcgcggccgcttctagaaattaaccctcactaaagggagaa pT3antisense: ctagttctccctttagtgagggttaatttctagaagcggccgcg The following method can be used to insert pProt before a desired Biobrick part (which should contain an RBS at minimum and preferably a cds and terminator): 1. Cut the desired plasmid with EcoRI and XbaI. Purify the plasmid with a method of your choice (both gel purification and PCR purification kits from Qiagen serve to remove the short undesired oligonucleotide from the digestion). 2. Be sure that your oligos have 5' phosphorylation (treat unphosphorylated oligos with T4 Polynucleotide Kinase). Then, anneal the two oligos by slowly cooling from 98C to room temperature. 3. Finally, ligate the cut plasmid with the annealed oligos by using a 1:10 molar ratio of plasmid to insert. 4. Transform the ligation reaction into E. coli, and plate. 5. Pick colonies, and verify the success of the cloning with sequencing or PCR. (Doing a diagnostic digest is not recommended as the size of the original plasmid and the desired plasmid are very similar.) 6. Add a composite part to the iGEM registry by using this part with the original protein coding part, checking the box to create without a scar, as this part contains a modified scar.
When ligating with a cut plasmid, this leaves a modified Biobrick scar between the T7 promoter and original Biobrick part, which is included in the sequence of this part.
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The T3 promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part.
This part produces GFP in the presence of T3 RNA polymerase. It contains a T3 promoter, a strong RBS, the coding region for GFP, and a double terminator. The GFP is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040.
The part containing GFP was cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of cloning technique, a Biobrick scar site remains between the promoter and RBS.
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.
T7 promoter -> 434 cI N terminus with P22 repressor specificity
"A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli" (G. Di Lallo, L. Castagnoli, P. Ghelardini and L. Paolozzi; Microbiology (2001), 147, 1651???1656) described the use of this protein domain.
This part produces the DNA binding domain of the phage 434 cI repressor, with mutations that change the specificity of the DNA to which the protein binds. This protein domain was used as in "A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli" (G. Di Lallo, L. Castagnoli, P. Ghelardini and L. Paolozzi; Microbiology (2001), 147, 1651???1656). For more information on the uses of this part, visit 2015.igem.org/Team:Pitt/Protease/Project.
This part was designed with a T7 promoter so the protein could be overexpressed.
Platform for terminators analysis under the control of T7 promoter
It's made from a Roberta Lentini's construct (from Mansy lab) that consist of pET21b with the two proteins and a 20bp linker between them. That construct was mutated two times to eliminate illegal restriction sites; prefix-suffix linker was added by insertion PCR.
We used this part to test terminators: the two subsequent proteins allow to quantify the termination efficiency as the ratio between Venus's and Cherry's fluorescence. The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene. The two fluorescent proteins have partially overlapping emission spectra, thus the fluorimetric measures should be taken with these parameters, to avoid interference between the two: Venus excitation: 485nm, Venus emission: 528nm Cherry excitation: 528nm, Cherry emission: 615nm We inserted the prefix-suffix linker between the two proteins, to allow terminator characterization.
Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics. Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics.
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The T7 promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of technique, a Biobrick scar site remains between the promoter and RBS.
This part produces GFP in the presence of T7 RNA polymerase. It contains a T7 promoter, a strong RBS, the coding region for GFP, and a double terminator. The GFP is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040
The part containing GFP was cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of technique, a Biobrick scar site remains between the promoter and RBS.
This is the T7 gene 6 ORF, that codes for the T7 gene 6 5'-3' exonuclease. For properties of T7 gene 6 exonuclease see: Kerr and Sadowski "Gene 6 Exonuclease of Bacteriophage T7" The Journal of Biological Chemistry Vol. 247 No. 1 January 10, pp 311-318, 1972.
We needed to change the stop codon to the standard stop codon TAA. We later realized that we should have added tandem TAA. The Tandem TAA stop codon is present in the composite parts containing this ORF. The ORF alone has the single TAA stop codon.
The T3 promoter comes from the T3 bacteriophage. The RBS site comes from...
This is the T3 promoter with an RBS site added. Other groups should use this part to regulate their part by adding it in front.
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that includes this gene (DE3).
There are three portions to the T7 promoter: the recognition site, the melting box (or TATA box) and the initation site. Mutations in these sequences caused differential behavior from the wild type.
Wild-type found in T3 bacteriophage. Synthesized by IDT.
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -16.
A mutant version of the T7lac promoter designed by Dubendorff and Studier. The promoter is based on the strong ??10 T7 promoter but incorporates an A->C mutation at -16. The LacI operator site is a partially symmetric 25bp sequence.
The -16 mutation was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
The part was generated by PCR amplification of part E0040 with the SapI site incorported into the forward promoter.
GFP (mut3) part number E0040 with a unpstream SapI site. Cleave with SapI results in an overhang of 3'-TAC - 5' from the left end of the GFP coding region, making the coding sequence compatible with Heather Keller's alternative assembly method for blunt cloning of RBS sequences upstream of a coding sequence.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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
T7 promoter with an A->C mutation at the -16 position of the consensus sequence
The sequence of T7 promoter is TAATACGACTCACTATA (from 5' to 3').
Two T7 promoters are reversely overlapping in two base pairs while a point mutation occurs at the 12th base pair, from A-T to T-A.
It is difficult to make this short DNA by PCR as both sides are T7 promoters, which are very similar. It is highly possible that they form loops before they can be synthesized by PCR. Thus we order two single stranded DNA and then anneal them together by touchdown PCR.
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -9.
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -9 base to an A, which should make it less than 3% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
The -9 mutation from consensus was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
This primer anneals to the T7 Promoter region of any vector containing the T7 Promoter.
<biblio> #Wallace pmid=6282692 </biblio>
T7 promoter with an A->T mutation at the -16 position of the consensus sequence
mCherry part number J06504 with an upstream SapI site. Cleave with SapI results in an overhang of 3'-TAC - 5' from the left end of the mCherry coding region, making the coding sequence compatible with Heather Keller's alternative assembly method for blunt cloning of RBS sequences upstream of a coding sequence.
This part was generated by PCR amplification. The upstream biobricks cut sites (EcoRI and XbaI) as well as the R0011 promoter were incorporated into the forward promoter recognizing the ompA region in the MG1655 genome. The AvrII cut site, as well as the downstream biobricks cut sites (SpeI and PstI) were incorporated into the reverse promoter.
This part is an intermediate assembly product that contains the R0011 promoter (lambda cI promoter with lacI binding sites), the OmpaA 5' UTR containing a double hairpin for transcript stability, and an AvrII restriction site to facilitate later downstream cloning of an RBS via Heather Keller's RBS characterization scheme. These parts are cloned bluntly, without generating biobricks mixed sites in between.
This part was generated by PCR assembly in order to minimize the number of digestions and ligations necessary to generate a larger construct. Especially given the short sequence of the three individual parts (and the difficulting in purifying small parts) it was much simpler to amplify the product in this way rather than generate the 3 parts individually and clone them together.
Two T7 promoters are reversely overlapping in five base pairs, with the third one mutated from A to G.
T7 promoter with an A->T mutation at the -10 position of the consensus sequence
T7 promoter for expression of functional RNA
to be used qith t7 polymerase BBa_J34811
Had to create primers to amplify just the T3
T4 holin, complete CDS, berkeley standard
T4 holin gene, with start and stop codons. This part is actually in BBb format.
This part is just for upload test. It is meaningless
The T7 promoter should only produce PoPS when the T7 polymerase is also being expressed.
T7 promoter (strong promoter from T7 bacteriophage)
T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
The ccd operon having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. This part is used to aid the process of moving a BioBrick part to a new plasmid. A target plasmid carrying P1010 is cut and mixed with the cut insert. Plasmids that recombine are selected against because they kill the cell that picks them up. P1010 is only provided as plasmid or in DB3.1 which is ccdB resistant.
This is a part used as a building block for T7 driven expression. However, this part uses a special method with the following synthesized nucleotides: pT7sense: aattcgcggccgcttctagataatacgactcactatagggagaa pT7antisense: ctagttctccctatagtgagtcgtattatctagaagcggccgcg The following method can be used to insert a T7 promoter before a desired Biobrick part: 1. Cut the desired plasmid (which should contain an RBS and cds at minimum) with EcoRI and XbaI. Purify the plasmid with a method of your choice (both gel purification and PCR purification kits from Qiagen serve to remove the short undesired oligonucleotide from the digestion). 2. Be sure that your oligos have 5' phosphorylation (treat unphosphorylated oligos with T4 Polynucleotide Kinase). Then, anneal the two oligos by slowly cooling from 98C to room temperature. 3. Finally, ligate the cut plasmid with the annealed oligos by using a 1:10 molar ratio of plasmid to insert. 4. Transform the ligation reaction into E. coli, and plate. 5. Pick colonies, and verify the success of the cloning with sequencing or PCR. (Doing a diagnostic digest is not recommended as the size of the original plasmid and the desired plasmid are very similar.) 6. Add a composite part to the iGEM registry by using this part with the original protein coding part.
When ligating with a cut plasmid, this leaves a Biobrick scar between the T7 promoter and original Biobrick part.
T7 promoter with an T->G mutation at the -17 position of the consensus sequence
Transcriptional terminator consisting of a 64 bp stem-loop.
The sequence of T7 promoter is TAATACGACTCACTATA.
Two T7 promoters are reversely overlapping.
RBS(B0032)+T3 DNA-directed RNA polymerase
This part is the coding region of MerR, which is a mercury responsive regulator from Tn21.
We cloned this part from plasmid NR1, which was a gift from Anne O. Summers.
Part composed of the T7 RNA Polymerase and the N-terminal degradation tag, but with no scar (no stop codon)
T7 RNA Polymerase with N-terminal UmuD degradation tag.
This part is different from the simple composition of the tag and the polymerase, as there cannot be a stop codon in the scar.
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -10.
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -10 base to a G, which should make it about 4% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
The -10 mutation from consensus was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -9 base to an G, which should make it less than 3% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
Conversion of the iGEM parts registry to SBOL2.1