Last data update: 24 January 2024 16:39 CET
Plasmid name: pSE380rPDIm (LMBP 3052)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3052.gb
(View with Genome Compiler) p3052.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Rat protein disulfide isomerase cDNA (rPDI); mutated sequence 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: | pSE380rPDI |
| Further information: | This plasmid was derived from pSE380rPDI via site-specific mutagenesis (Stanssens et al., Nucleic Acids Res. 17 (1989) 4441-4454) using the synthetic 32-mer 5'-CCCTGGAGGAAGAGCTCACCAAGTACAAACCG-3'. In this way, the internal ATG (Met, amino acid 306) of the mature rat PDI gene was substituted by CTC (Leu), and by this process a new SacI site was formed. This mutation was carried out to avoid the synthesis of a small peptide of about 21kDa (rPDIf) which accompanied the strongly regulated production of rat protein disulfide isomerase in E. coli from plasmid pSE380rPDI, due to internal translation initiation within the rat PDI gene. The mutated mature rPDI gene, preceded by its own signal sequence, is expressed under control of the strong, 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 rPDI 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). pSE380rPDIm was used for the bacterial production of a mutated, enzymatically active rat protein disulfide isomerase, which could be released from the periplasm by cold osmotic shock. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727425.1. The nucleotide sequence of the rPDI cDNA corresponds with the EMBL Nucleotide Sequence Database (Release 36; Accession number X02918). |
| EMBL Accession number: | X02918, view at EMBL, GenBank, DDBJ LT727425.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 16/06/1994 |
| Sequence detail: | Difference between the wild type rat PDI gene and the mutated gene.
wild type gene: 302 306 311
Leu Glu Glu Glu Met Thr Lys Tyr Lys Pro
5' CC.CTG.GAG.GAA.GAG.ATG.ACC.AAG.TAC.AAA.CCG. 3'
^
mutated gene: 302 306 311
Leu Glu Glu Glu Leu Thr Lys Tyr Lys Pro
5' CC.CTG.GAG.GAA.GAG.CTC.ACC.AAG.TAC.AAA.CCG. 3'
* *
-------
SacI
^: Internal translation-initiation codon within the mature rat
PDI gene.
*: Mutated nucleotide.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglI/BglII, EcoRI/PstI, NotI and SacI. |
| 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: | De Sutter et al., Gene 141 (1994), 163-170 [PMID: 8163184] |
| 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: |
pSE380rPDIm (LMBP 3052) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter and was published in De Sutter et al., 1994. |
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.