Last data update: 24 January 2024 16:39 CET
Plasmid name: pRH016 (LMBP 5427)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5427.gb
(View with Genome Compiler) p5427.txt p5427.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene) Bordetella bronchiseptica S87 plasmid pBBR1 mobilisation gene (mob); with modified 3' end Influenza HA epitope encoding the haemagglutinin tagging peptide; C-terminal |
| Promoter: | Escherichia coli lac operon promoter Escherichia coli lac operon promoter; mutant (lacUV5) Phage T3 promoter Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| Terminator: | Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1); fragment |
| Selection marker: | Chloramphenicol (cam) |
| Replicon: | Broad-host-range Gram-negative Bordetella bronchiseptica S87 plasmid pBBR1 replicon |
| Host range: | Escherichia coli; use a ccdB-resistant strain for propagation Gram-negative bacterial strains |
| Parental clone: | pBBR1MCS; pVV201 |
| Further information: | The plasmid was constructed by inserting a 1997 bp HindIII-EcoRV(Eco32I) fragment from pVV201, containing the Gateway reading frame cassette B (rfB, from Invitrogen) in frame with 3 copies of the HA epitope, into the HindIII-EcoRV(Eco32I) opened pBBR1MCS vector. pRH016 is a Gateway destination vector, containing the ccdB gene of the E. coli F plasmid, flanked by the bacteriophage λ attR recombination sites. A Gateway entry clone, containing the gene of interest flanked by the λ attL recombination sites, recombines with the attR sites of pRH016, replacing the ccdB gene. As a result, the gene of interest is fused to the 3 C-terminal HA epitopes. Standard E. coli strains with the unreacted destination vector or with the by-product plasmid retaining the ccdB gene will fail to grow. Thus, this negative selection provides high-efficiency recovery of only the desired clones. The chloramphenicol resistance gene located between the two attR sites does not permit counterselection as this resistance gene is also present in the plasmid backbone. This plasmid is useful as a medium-copy-number, broad-host-range cloning vector. 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). When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide. The nucleotide sequence of the pBBR1MCS vector corresponds with the EMBL Nucleotide Sequence Database accession number U02374.1. The nucleotide sequence of the wt mob gene corresponds with the EMBL Nucleotide Sequence Database accession numbers U02374.1 and X66730.1. |
| EMBL Accession number: | U02374.1, view at EMBL, GenBank, DDBJ X66730.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 05/01/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AatII/NcoI, BglII/Bsp119I, Bsp1407I/Eco47III, Eco32I/HindIII and SfiI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr X. De Bolle(1). It was constructed by R. Hallez(1). (1) Department of Biology, University of Namur, Namur, Belgium |
| Plasmid reference: | Hallez et al., Appl. Environ. Microbiol. 73 (2007), 1375-1379 [PMID: 17172460] [DOI: 10.1128/AEM.01873-06] |
| Related plasmid reference: | Kovach et al., BioTechniques 16 (1994), 800-802 [PMID: 8068328] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12xB DB3.1 |
| Host reference: | - |
| Related host reference: | Bernard et al., J. Mol. Biol. 226 (1992), 735-745 [PMID: 1324324] |
| Cultivation medium: | LB-Lennox + chloramphenicol (34 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | Take a low-copy-plasmid yield into account while choosing a plasmid DNA isolation procedure. |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pRH016 (LMBP 5427) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr X. De Bolle and was published in Hallez et al., 2007. |
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.