Last data update: 24 January 2024 16:39 CET
Plasmid name: pRH005 (LMBP 5416)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5416.gb
(View with Genome Compiler) p5416.txt p5416.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) Aequorea victoria green fluorescent protein DNA (GFP); enhanced yellow-green fluorescent variant (EYFP) |
| Promoter: | Escherichia coli lac operon promoter Escherichia coli lac operon promoter; mutant (lacUV5) Transposon Tn5 neomycin resistance gene promoter (neo) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| Terminator: | Phage T4 gene 32 terminator (T4g32) Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1) |
| Selection marker: | Chloramphenicol (cam) Neomycin (neo; 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; use a ccdB-resistant strain for propagation Gram-negative bacterial strains |
| Parental clone: | pMR10cat; pRH008 |
| Further information: | The plasmid was constructed by inserting the 3184 bp XbaI-XhoI fragment from pRH008, containing the Gateway reading frame cassette B (rfB, from Invitrogen) in frame with the C-terminal enhanced yellow-green fluorescent variant of the A. victoria green fluorescent protein DNA (EYFP), into the XbaI-XhoI opened pMR10cat vector. pRH005 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 pRH005, replacing the ccdB gene. As a result, the gene of interest is fused to the C-terminal EYFP coding sequence. 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. The plasmid RK2 par genes encode stabilization proteins that provide stable maintenance of the plasmid. 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. This plasmid is useful as a low 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726890.1. The nucleotide sequence of the pMR10 vector corresponds with the EMBL Nucleotide Sequence Database accession number AJ606312.1. |
| EMBL Accession number: | AJ606312.1, view at EMBL, GenBank, DDBJ LT726890.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 04/01/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglII/SalI, Bsp1407I, Eco32I, EcoRI/NotI, NcoI/PdmI, NdeI/PstI and XbaI/XhoI. |
| 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] |
| 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) + kanamycin (50 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 kanamycin. 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: |
pRH005 (LMBP 5416) 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.