Last data update:
22 July 2019 04:19 CEST
|Name:||pCOIN DV Add to cart|
|Accession number:||LMBP 8192|
|Sequence file:||LMBP sequence: p8192.gb, p8192.txt View with Genome Compiler|
|Status:||GeneCorner core plasmid|
|Cloned DNA:||B gene of the control of cell death locus of the Escherichia coli F plasmid (ccdB, lethal gene)|
|Promoter:||Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter; mutant (lacUV5)
Escherichia coli lac operon promoter
|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:||pBigT; pDEST R4-R3|
|Further information:||pCOIN DV is a highly efficient Recombinase-Mediated Cassette Exchange (RMCE)-compatible, conditional and inducible Gateway destination vector.
This multisite Gateway destination vector contains the ccdB gene inserted between the attenuator sites attR3 and attR4. Next to this plasmid, three entry clones are needed in the multisite approach to target the gene of interest to the endogeneous ROSA26 locus in mouse G4 ROSALUC ES cells: 1) a 5' entry clone with attL4-attR1 sites and containing the reverse tetracycline transactivator (rtTA), followed by an IRES-puromycin-polyA cassette (e.g. pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1 (LMBP 8212)); 2) a middle entry clone with attL1-attL2 sites and containing the gene of interest and 3) a 3' entry clone with attR2-attL3 sites and containing the IRES-EGFP-Luciferase-polyA cassette (e.g. pEntry attR2-IRES-eGFP-luc+-pA-attL3 (LMBP 8200)).
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 rtTA-IRES-puromycin to the endogenous ROSA26 promoter.
The resulting multisite 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 conditional stop cassette leads to ROSA26-based rtTA expression and puromycin resistance. Inducible expression of the inserted cDNA will only be achieved upon doxycycline administration and can be monitored by the induced reporter expression (EGFP-LUC).
The G4 ROSALUC ES cells are available at BCCM/GeneCorner as well.
The region from the BamHI restriction site (nucleotide position 6376) until the SacI restriction site (nucleotide position 283), spanning the chloramphenicol resistance gene, attR3 site, the BGH terminator and the first 5'HS4 insulator sequence, has been sequenced at BCCM/GeneCorner
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726874.1.
|EMBL Accession number:||LT726874.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||19/01/2016|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BamHI, EcoRI, PvuII, SacI and XhoI.|
|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:||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) + chloramphenicol (34 μg/ml)*|
|Cultivation remark:||*: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin. The plasmid-carrying strain must be cultivated at 28°C, under heavy shaking, to prevent recombination. After each cultivation, the plasmid has to be checked for recombination. Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA.|
|Other culture collection numbers:||-|
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.