Last data update: 22 October 2020 04:25 CEST
Plasmid name: Cre excised pRMCE DV1 (LMBP 8195)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|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:||Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter; mutant (lacUV5)
Escherichia coli lac operon promoter
|Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene|
|Terminator:||Bovine growth hormone polyadenylation signal (BGH polyA)|
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
|Parental clone:||pRMCE DV1|
|Further information:||The plasmid was obtained via Cre-mediated removal of the loxP-flanked conditional stop cassette of pRMCE DV1.
The plasmid Cre excised pRMCE DV1 is a highly efficient Recombinase-Mediated Cassette Exchange (RMCE)-compatible Gateway destination vector.
This monosite Gateway destination vector contains the ccdB gene inserted between the attenuator sites attR1 and attR2.
The plasmid contains:
- two heterospecific Saccharomyces cerevisiae 2μ FLP Recombinase Target sites (one wt and one mutant FRT site)
- two bacteriophage P1 Cre recombinase target sites (loxP sites)
- two adjacent copies of the chicken β-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator core sequence. Insulators are DNA sequences which possess the ability to protect expressing genes from inappropriate signals emanating from their surrounding environment by acting as barriers that prevent the advance of nearby condensed chromatin that may otherwise silence expression.
- a splice acceptor (SA) for linking the cDNA of interest to the endogenous ROSA26 promoter.
Next to this plasmid, an entry clone, containing the gene of interest flanked by the attL1-attL2 sites, is needed in the monosite approach to target the gene of interest to the endogeneous ROSA26 locus in mouse G4 ROSALUC ES cells.
The resulting monosite Gateway expression vector, by co-transfection of a FlpE plasmid, can subsequently be targeted to the ROSA26 locus of G4 ROSALUC ES cells using a trap-coupled RMCE approach, hereby restoring neomycin resistance.
Cre-mediated removal of the loxP-flanked stop cassette causes ROSA26-based constitutive expression of the inserted cDNA.
The G4 ROSALUC ES cells (LMBP 10507CB) are available at BCCM/GeneCorner as well.
This Gateway destination vector is compatible with the human orfeome library which is also available at BCCM/GeneCorner.
Other name of the plasmid is Cre ex. pRMCE DV1.
|EMBL Accession number:||-|
|Latest sequence update:||08/07/2013|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, EcoRV, PvuII and XmnI.
As compared to the compiled nucleotide sequence file, the expected 1344 bp XmnI fragment appears to be approximately 100 bp longer.
|History of deposit:||This plasmid was deposited by Dr L. Haenebalcke(1) (2) and Prof. Dr J. Haigh(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Haenebalcke et al., Stem Cell Rev 9 (2013), 774-785 [PMID: 23877658] [DOI: 10.1007/s12015-013-9458-z]
|Related plasmid reference:||PhD thesis Lieven Haenebalcke
|Restricted distribution:||- VIB/BCCM MTA
- The depositor will be informed of the customer's identity and of the enduser's research described in the VIB/BCCM MTA upon release of a sample outside the depositor's department.
- When appropriate in accordance with academic customs, RECIPIENT agrees to include the depositor(s) as co-author(s) in the initial publication describing the MATERIAL.
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12xB DB3.1|
|Related host reference:||Bernard et al., J. Mol. Biol. 226 (1992), 735-745 [PMID: 1324324]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|Cre excised pRMCE DV1 (LMBP 8195) is available at BCCM/GeneCorner. This plasmid was deposited by Dr L. Haenebalcke and Prof. Dr J. Haigh and was published in Haenebalcke et al., 2013.|
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.