Last data update: 24 January 2024 16:39 CET
Plasmid name: pJBkanT7POL2 (LMBP 3324)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3324.gb
(View with Genome Compiler) p3324.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Phage T7 RNA polymerase gene (T7g1) 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)) Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Phage fd terminator Phage λ early leftward terminator (λ L) |
| Selection marker: | Kanamycin (kan) |
| 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: | pJBkanT7POL1 |
| Further information: | This IncP, broad-host-range plasmid was derived from pJBkanT7POL1 by excising the BsaI (filled in with Klenow DNA polymerase) - ScaI fragment, containing part of the ampicillin resistance gene. As a result, this plasmid provides no longer resistance to ampicillin. The expression of the T7 RNA polymerase gene is controlled by the IPTG-inducible T5 N25/O2 promoter and is attenuated by a series of three tandemly arranged transcription terminators (two fd terminators and the λ early leftward terminator) placed between the T5 N25/O2 promoter and the coding sequence of the T7g1 gene. Upon induction, this attenuation can be overcome due to the incorporation of the phage λ nutL/N protein antitermination system. This provides a tighter control on gene expression. This expression cassette is very useful for the controlled induction (IPTG induction) of the attenuated T7g1 gene at lower temperatures. The promoter/operator element T5 N25/O2 consists of the highly efficient Escherichia coli phage T5 N25 promoter and two lac operator sequences (Bujard et al. (1987). The lacI(q) (lac repressor) cassette allows the use of any Gram-negative host strain, instead of only strains overproducing the E. coli lac repressor. 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. Introduce the plasmid by electroporation, or through conjugation, using plasmid RK2/RP4 transfer functions (provided in trans by RK2/RP4, RK2/RP4-derived vectors, other replicons carrying RK2/RP4 transfer functions (e.g. pRK2073) or strains expressing the tra genes of RK2/RP4). The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727464.1. Other names of the plasmid are pJBkanT7pol2 and pJBkanT7Amp(-). |
| EMBL Accession number: | LT727464.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 07/06/1996 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BamHI/NsiI, BglI, BglII, HindIII and NruI/SalI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by 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: | - |
| 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 + kanamycin (50 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pJBkanT7POL2 (LMBP 3324) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr E. Remaut. |
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.