Last data update: 24 January 2024 16:39 CET
Plasmid name: pET-3b (LMBP 1458)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
pet-3b.gb
(View with Genome Compiler) pet-3b.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Phage T7 gene 10 (T7g10); first 11 codons |
| Promoter: | Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10) |
| Terminator: | Phage T7 gene 10 terminator (T7g10) |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pBR322 |
| Further information: | The plasmid was constructed as follows: first the unique NdeI site of pBR322 was opened, filled in and religated. Then the terminator and the promoter sequences from bacteriophage T7 have been inserted into the unique BamHI site of pBR322 by means of BamHI linkers. New vectors can be created by moving the BglII-BamHI fragment (containing T7 gene 10 promoter, its ribosome binding site and part of its coding sequence), into any unique BamHI site. The BamHI site upstream to the T7 promoter was converted to a unique BglII site. This plasmid contains the T7 gene 10 promoter whose transcription is initiated by T7 RNA polymerase, making the vector useful for in vitro preparation of single-stranded RNAs from cloned DNA sequences. The plasmid can also be use for in vivo synthesis of proteins encoded by a gene (containing a ribosome binding site and a translation-initiation codon) that is cloned downstream from the T7 gene 10 promoter, provided that the cell contains a source of T7 RNA polymerase. The first 11 codons of T7 gene 10 are present and the promoter-distal BamHI site generates a GAT codon in the reading frame. Other reading frames are available in pET-3a and pET-3c. The T7 fragment can be moved as a BglII-EcoRV fragment to other vectors. For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3). Proceed as follows: first transform the expression strain with the auxiliary plasmid, make the transformants competent again, and then transform with the expression plasmid. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727575.1. Other name of the plasmid is pAR3039. |
| EMBL Accession number: | LT727575.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 19/12/1991 |
| Sequence detail: | Nucleotide sequence at the beginning of the T7 gene10:
1 2 3 4 5 6 7 8 9 10 11
5' TATACAT ATG.GCT.AGC.ATG.ACT.GGT.GGA.CAG.CAA.ATG.GGT.CGG.GAT.
Met Ala Ser Met Thr Gly Gly Gln Gln Met Arg BamHI
NdeI NheI
CC 3'
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BamHI and RsaI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr W. Studier(1). (1) Brookhaven National Laboratory, Upton, New York, USA |
| Plasmid reference: | Rosenberg et al., Gene 56 (1987), 125-135 [PMID: 3315856] |
| Related plasmid reference: | Studier et al., Methods Enzymol. 185 (1990), 60-89 [PMID: 2199796] Studier and Moffatt, J. Mol. Biol. 189 (1986), 113-130 [PMID: 3537305] [DOI: 10.1016/0022-2836(86)90385-2] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 HMS174 |
| Host reference: | Campbell et al., Proc. Natl. Acad. Sci. U.S.A. 75 (1978), 2276-2280 [PMID: 276868] [DOI: 10.1073/pnas.75.5.2276] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | NCCB 3276 PC-V 3276 |
Refer in your Materials and Methods: |
pET-3b (LMBP 1458) is available at BCCM/GeneCorner. This plasmid was deposited by Dr W. Studier and was published in Rosenberg et al., 1987. |
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.