Last data update: 24 January 2024 16:39 CET
Plasmid name: pRH003 (LMBP 5414)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5414.gb
(View with Genome Compiler) p5414.txt p5414.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 |
| 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: | - |
| Selection marker: | Ampicillin (amp) 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-4 |
| Further information: | The plasmid was constructed by ligating the Gateway reading frame cassette B (rfB, from Invitrogen) into the EcoRV (Eco32I) opened pBBR1MCS-4 vector. pRH003 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 the destination vector, replacing the ccdB gene. 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 permits counterselection. 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-4 vector corresponds with the EMBL Nucleotide Sequence Database accession number U25060.1. |
| EMBL Accession number: | U25060.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 01/02/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglII, Eco32I, MluI, PvuII and XbaI. |
| 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 + ampicillin (100 μg/ml) + chloramphenicol (34 μg/ml)* |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | *: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin. Medium copy plasmid, use two times more culture than usual for plasmid preparation. |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pRH003 (LMBP 5414) 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.