Last data update: 21 September 2022 10:23 CEST
Plasmid name: pRMCE DV1 (LMBP 8870)
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|Price category:||Cat. 2 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
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)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
|Parental clone:||pROSA26-DV1; pROSALUC|
|Further information:||The plasmid was constructed by replacing the 5' UTR and 3' UTR regions of mouse ROSA26 and the neomycin resistance gene in pROSA26-DV1 with the heterospecific FRT sites present in pROSALUC. A PGK-ATG sequence was included to ensure a proper trap-coupled RMCE reaction when targeted into mouse G4 ROSALUC ES cells.
pRMCE DV1 is a highly efficient Recombinase-Mediated Cassette Exchange (RMCE)-compatible, conditional 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 leads to 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.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726806.1, except for the adjustments due to the implementation of the next-generation sequencing results.
|EMBL Accession number:||LT726806.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||23/12/2019|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, EcoRV, PvuII and XmnI.
This plasmid has also been fully sequenced but the NGS sequence data were unfortunately incomplete (because of homology regions: SV40 polyA and part of mPGK1 promoter).
|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. Rep. 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 first publication making use of the MATERIAL.
|Distributed as:||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:||LMBP 08196|
Refer in your Materials and Methods:
|pRMCE DV1 (LMBP 8870) 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.