Last data update: 24 January 2024 16:39 CET
Plasmid name: pJBpn25LUC (LMBP 3519)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p3519.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Photinus pyralis (firefly) luciferase gene (LUC) Escherichia coli lac repressor gene (lacI) Broad-host-range plasmid RK2 trans-acting replication initiator protein DNA (trfA) |
| Promoter: | Phage T5 N25 promoter with two lac operator sequences (N25/O2) Transposon Tn5 neomycin resistance gene promoter (neo) Escherichia coli lac repressor promoter; mutant (lacI(q)) Phage T3 promoter |
| Ribosome binding site: |
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10) |
| Terminator: | Phage fd terminator Phage T7 gene 10 terminator (T7g10) |
| Selection marker: | Tetracycline (tet) |
| Replicon: | Broad-host-range plasmid RK2 vegetative replication origin Broad-host-range plasmid RK2/RP4 origin of transfer (oriT) |
| Host range: | Escherichia coli Gram-negative bacterial strains |
| Parental clone: | pJB3tet2; pLT10LUCT3; pUC18lacIq; pDS12-RBSII |
| Further information: | This IncP, broad-host-range plasmid was constructed as follows: (i) the AatII-XbaI fragment from pUC18lacIq (containing the lacI gene with the I(q) promoter) was ligated to the AatII-XhoI fragment from pDS12-RBSII (containing the PN25 promoter and RBS II) (ii) the the EcoRI-XbaI fragment of the intermediate construct (containing the RBS) was replaced by the BsaI-AlwNI fragment of pLT10LUCT3 (containing the RBS of T7 gene10, followed by the luciferase gene and fd and T7 terminators) (iii) then the AatII-NaeI fragment (containing the lacI gene and the pN25-luciferase cassette) was inserted into the ScaI opened pJB3tet2 vector. All restriction sites used for this construction were filled-in/blunted with Klenow DNA polymerase. The expression of the luciferase gene is controlled by the IPTG inducible T5 N25/O2 promoter. The promoter/operator element T5 N25/O2 consists of the highly efficient E. coli phage T5 N25 promoter and two lac operator sequences (Bujard et al. (1987)). Due to the presence of the lacI(q) (repressor)-N25/O2 (promoter/operator) cassette on the plasmid, regulated (IPTG inducible) expression should be possible in a wide range of Gram-negative hosts. The plasmid RK2 trfA gene encodes two polypeptide products P382 and P285 of 382 and 285 amino acids respectively, using a different initiation codon within the same reading frame. The resulting protein is essential for vegetative replication. 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. |
| EMBL Accession number: | - |
| Latest sequence update: | 16/11/1998 |
| 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 K. Keymeulen(1) (2) and Prof. Dr E. Remaut(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Remaut et al., in 'Biotechnological Applications of Cold-Adapted Organisms', Springer (1999), 1-16 [ISSN/ISBN: 3642636632] [DOI: 10.1007/978-3-642-58607-1] |
| Related plasmid reference: | Bujard et al., Methods Enzymol. 155 (1987), 416-433 [PMID: 2828874] |
| 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 + tetracycline (10 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | LMBP 03891 |
Refer in your Materials and Methods: |
pJBpn25LUC (LMBP 3519) is available at BCCM/GeneCorner. This plasmid was deposited by K. Keymeulen and Prof. Dr E. Remaut and was published in Remaut et al., 1999. |
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.