Last data update: 24 January 2024 16:39 CET
Plasmid name: pRH008 (LMBP 5419)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5419.gb
(View with Genome Compiler) p5419.txt p5419.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) Phage T3 promoter Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
- |
| Terminator: | Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1) |
| Selection marker: | Ampicillin (amp) Chloramphenicol (cam) |
| 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: | pKSoriT; pFA6-YFP-NatMX6 |
| Further information: | The plasmid was constructed as follows: 1) the SmaI fragment from pFA6-YFP-NatMX6, containing the enhanced yellow-green fluorescent variant of the A. victoria green fluorescent protein DNA (EYFP), was inserted into the EcoRV opened pKSoriT vector, resulting in pKSoriT-yfp; 2) the Gateway reading frame cassette B (rfB, from Invitrogen) was cloned into the SmaI linearized pKSoriT-yfp vector. As a result, the rfB cassette was fused in phase to the C-terminal EYFP coding sequence. pRH008 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 pRH008, 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 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 LT726893.1. |
| EMBL Accession number: | LT726893.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 04/01/2007 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: Alw44I/NcoI, BamHI, BamHI/Bsu15I, BglII/SalI, Bsp1407I/PdiI, PdmI, PdmI/PstI, 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)* |
| 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. |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pRH008 (LMBP 5419) 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.