Last data update: 24 January 2024 16:39 CET
Plasmid name: pLPPsOmpArPDI (LMBP 3056)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3056.gb
(View with Genome Compiler) p3056.txt p3056.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Escherichia coli outer membrane protein A cDNA (ompA, tolG, GeneID 945571); signal sequence Rat protein disulfide isomerase cDNA (rPDI); mature sequence Escherichia coli lac repressor gene (lacI) |
| Promoter: | Escherichia coli lipoprotein promoter (lpp) Escherichia coli lac operon promoter; mutant (lacUV5) 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 lipoprotein terminator (lpp) |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pLPPHIs; pSE380rPDI |
| Further information: | The plasmid was constructed as follows: 1) pSE380rPDI was cleaved with BanI, filled in with Klenow DNA polymerase and further digested with BglII; 2) subsequently, the BanI (nucleotide position 5563) - BglII fragment encoding rat protein disulfide isomerase was ligated to the SacI (blunted with T4 DNA polymerase) - BamHI vector fragment of pLPPHIs. Expression of the fusion gene, under control of both the lpp and lacUV5 promoter, yields high levels of enzymatically active rat protein disulfide isomerase (from which the ompA signal peptide is precisely removed) in the bacterial periplasm. The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727428.1. In pLPPsOmpArPDI, the E. coli ompA signal sequence was fused in phase to the nucleotide sequence encoding the last two amino acids of the rat PDI signal sequence, followed by the rat PDI mature sequence. The nucleotide sequence at this fusion was confirmed by DNA sequence analysis. The nucleotide sequence of the rPDI cDNA corresponds with the EMBL Nucleotide Sequence Database accession number X02918.1. The nucleotide sequences of the 5' and 3' ends of the E. coli lacI insert, analysed by Roche Research USA, were kindly provided by Dr Yves Guisez (Roche Research Ghent). The unknown nucleotides in these analyses as well as some single nucleotides in other parts of the vector were replaced by the corresponding nucleotides of the E. coli K12 genome sequence obtained from the EMBL Nucleotide Sequence Database accession number U00096.2. However, the biological material deposited in the BCCM/GeneCorner collection seems not to correspond to the sequence analysis results of Roche Research USA (see info in the field 'Authenticity'). |
| EMBL Accession number: | X02918.1, view at EMBL, GenBank, DDBJ U00096.1, view at EMBL, GenBank, DDBJ LT727428.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 06/05/2008 |
| Sequence detail: | Nucleotide sequence at the fusion between the ompA signal sequence and the rPDI mature sequence:
--- ompA signal sequence --> -srPDI> --- rPDI mature sequence -->
5' ... TTC.GCT.ACC.GTA.GCG.CAG.GCC.GGC.GCC.GAC.GCT.CTG.GAG.GAG.GAG.GAC ... 3'
Phe Ala Thr Val Ala Gln Ala Gly Ala Asp Ala Leu Glu Glu Glu Asp
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AatII/KspAI, AccI, AdeI/PstI, AdeI/XbaI, Alw44I, Alw44I/KpnI, AvaI, BamHI/XbaI, BshTI, BshTI/EcoRI, CaiI/NdeI, Eco130I/Eco47III, EcoNI, HindII and KpnI/KspAI. The plasmid seems to be approx. 500 bp larger as compared to the compiled nucleotide sequence. The extra nucleotides are located in the vector part of the plasmid between the BshTI site at position 2375 and the KpnI site at position 2504. The restriction site analysis pattern revealed also the presence of an extra AccI, BshTI, EcoNI, HindII and KpnI site. These extra sites are located in the unknown nucleotide sequence of the approx. 500 bp fragment. |
| 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: | 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 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pLPPsOmpArPDI (LMBP 3056) 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.