T7 promoter for expression of functional RNA
Two T7 promoters are reversely overlapping in five base pairs, with the third one mutated from A to G.
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.
The sequence of T7 promoter is TAATACGACTCACTATA (from 5' to 3').
T7 promoter with an A->T mutation at the -16 position of the consensus sequence
This primer anneals to the T7 Promoter region of any vector containing the T7 Promoter.
<biblio> #Wallace pmid=6282692 </biblio>
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.
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 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.
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.
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
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.
Two T7 promoters are reversely overlapping.
The sequence of T7 promoter is TAATACGACTCACTATA.
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)
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.
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.
Part composed of the T7 RNA Polymerase and the N-terminal degradation tag, but with no scar (no stop codon)
T7 promoter with an T->G mutation at the -17 position of the consensus sequence
T7 promoter with an A->C mutation at the -16 position of the consensus sequence
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.
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.
T7 promoter -> 434 cI N terminus with P22 repressor specificity
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.
"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.
T7 promoter with an A->T mutation at the -10 position of the consensus sequence
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.
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.
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.
Platform for terminators analysis under the control of T7 promoter
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.
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.
to be used qith t7 polymerase BBa_J34811
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)
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.
The T7 promoter should only produce PoPS when the T7 polymerase is also being expressed.
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.
Synthesized oligonucleotides as described on main page.