Last data update: 24 January 2024 16:39 CET
Plasmid name: pRH012 (LMBP 5423)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5423.gb
(View with Genome Compiler) p5423.txt p5423.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 red-shifted variant (EGFP) |
| 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: |
- |
| Terminator: | - |
| Selection marker: | Ampicillin (amp) Chloramphenicol (cam) Neomycin (neo; kanamycin (kan)) |
| Replicon: | Escherichia coli plasmid pMB1 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; integrative |
| Parental clone: | pRH009; pUC4K |
| Further information: | The plasmid was constructed by inserting the 1252 bp SalI fragment from pUC4K (Pharmacia), containing the coding sequence of the kanamycin resistance gene, into the XhoI opened pRH009 vector. pRH012 carries the Gateway reading frame cassette B (rfB, from Invitrogen) fused in phase to the C-terminal EGFP coding sequence. pRH012 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 pRH012, replacing the ccdB gene. As a result, the gene of interest is fused to the C-terminal EGFP 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 permits counterselection. 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 LT726897.1. The nucleotide sequence of the EGFP DNA corresponds with the EMBL Nucleotide Sequence Database accession number U55761.1. |
| EMBL Accession number: | U55761.1, view at EMBL, GenBank, DDBJ LT726897.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 04/01/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: Alw44I/NcoI, BamHI/Bsu15I, BglII/SalI, Bsp1407I/PdiI, PstI and 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] |
| Related plasmid reference: | Tibor et al., Infect. Immun. 70 (2002), 5540-5546 [PMID: 12228280] |
| 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) + 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 ampicillin and kanamycin. |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pRH012 (LMBP 5423) 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.