Last data update: 24 January 2024 16:39 CET
Plasmid name: pSE380DsbA (LMBP 3059)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p3059.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Escherichia coli gene facilitating disulfide bond formation (dsbA) Escherichia coli lac repressor gene (lacI) |
| Promoter: | Escherichia coli hybrid tryptophan/lacUV5 promoter (trc) Escherichia coli lac repressor promoter; mutant (lacI(q)) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| Terminator: | Escherichia coli rrnB operon T1 terminator Escherichia coli rrnB operon T2 terminator |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pSE380; pT74DsbA |
| Further information: | The plasmid was constructed by ligating the BspHI (isoschizomer of RspXI) - XbaI fragment of pT74DsbA, containing the dsbA gene, to the NcoI-XbaI vector fragment of pSE380. Due to the favourable position of the ATG start codon of DsbA in the BspHI site and subsequent fusion to the ATG sequence of pSE380 exposed after NcoI digestion, the 5' flanking region of the dsbA gene has been completely eliminated. This plasmid was succesfully used for high-level production of enzymatically active DsbA under control of the IPTG-inducible trc promoter, followed by the lacZ ribosome binding site. This expression vector is further characterized by the presence of the rrnB 5S gene and the strong rrnB transcription terminators T1 and T2 downstream from the dsbA gene to prevent read-through transcription. The plasmid also contains the lacI(q) gene, leading to overproduction of the lac repressor (LacI), to repress the trc promoter in order to expand the host range of the vector to non-lacI(q) strains (e.g. MC1061). The nucleotide sequence of the dsbA coding region (starting at the ATG start codon) up to the SspI site was taken from the publication of Bardwell et al. Since most of the nucleotide sequence of the 3' flanking region of the dsbA gene is not known, there is uncertainty as to the cutting frequency of the restriction enzymes indicated. |
| EMBL Accession number: | - |
| Latest sequence update: | 24/06/1994 |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. De Sutter(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Related plasmid reference: | Brosius, DNA 8 (1989), 759-777 [PMID: 2558866] Bardwell et al., Cell 67 (1991), 581-589 [PMID: 1934062] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 MC1061 |
| Host reference: | Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Related host reference: | Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSE380DsbA (LMBP 3059) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter . |
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.