Last data update: 21 October 2021 10:13 CEST
Plasmid name: pROSA26-RMCE-COIN-DV (LMBP 8188)
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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)|
Mouse gene trap ROSA 26 gene (Gtrosa26, Gt(ROSA)26Sor, beta geo, Gtrgeo26, R26, ROSA26, Thumpd3as1, GeneID 14910); 5' UTR and 3' UTR
|Promoter:||Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)|
|Terminator:||Simian virus 40 polyadenylation signal (SV40 polyA)
Bovine growth hormone polyadenylation signal (BGH polyA)
Mouse phosphoglycerate kinase 1 polyadenylation signal (PGK1 polyA)
Herpes simplex virus (HSV) thymidine kinase polyadenylation signal (TK polyA)
|Selection marker:||Ampicillin (amp)
Neomycin (neo; G418)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli; use a ccdB-resistant strain for propagation
|Further information:||This multisite Gateway Destination vector was constructed by cloning an FRT-loxP-pPGK1-neo-pA-loxP cassette between the 5' UTR of ROSA26 and the attR4 site, and a pA-FRT-neo cassette between the attR3 site and the 3' UTR of ROSA26 of pROSA26-DV3.
The plasmid is a multisite Gateway destination vector, containing the ccdB gene inserted between the recombination sites attR4 and attR3. 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 ES cells: 1) a 5' entry clone with attL4-R1 sites and containing the rtTA transactivator, an IRES-Puro-pA selection cassette and a CMVTRE promoter (e.g. pEntry-attL4-rtTA-IRES-Puro-pA-TRE-attR1 (LMBP 8214)) ; 2) a middle entry clone with attL1-L2 sites and containing the gene of interest and 3) a 3' entry clone with attR2-L3 sites and containing the IRES-EGFP reporter (e.g. pEntry-attR2-IRES-eGFP-luc+-pA-Ins/Ins-attL3 (LMBP 8199)). In this approach, an exogeneous promoter is used to drive transgene expression, resulting in higher expression levels as compared to the monosite approach using the endogeneous ROSA26 promoter (see pROSA26-DV1 (LMBP 6451)).
The G4 ROSALUC ES cells (LMBP 10507CB) are available at BCCM/GeneCorner as well.
Other name of the plasmid is pROSA26 RMCE COIN DV.
|EMBL Accession number:||-|
|Latest sequence update:||28/07/2021|
|Authenticity test:||Restriction enzyme pattern analysed at BCCM/GeneCorner: BamHI, HindIII, KpnI and NotI.
Additionally, the polyA region between Tn5 neomycine resistance gene and the ccdB gene was sequenced at BCCM/GeneCorner.
|History of deposit:||This plasmid was deposited by Prof. Dr J. Haigh(1).
(1) Australian Centre for Blood Diseases, Central Clinical School, Monash University, Melbourne, Australia
|Plasmid reference:||PhD thesis Lieven Haenebalcke
|Restricted distribution:||- VIB/BCCM MTA
- The depositor will be informed of the customer's identity upon release of a sample outside the depositor's department or outside the departments in which BCCM/GeneCorner is embedded, namely UGent-DBMB and VIB-IRC.
|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 instead of 37°C, under heavy shaking, to prevent recombination. After each cultivation, the plasmid has to be checked for recombination. Because of the size of the plasmid, use electro-competent bacterial strains for propagation.|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pROSA26-RMCE-COIN-DV (LMBP 8188) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr J. Haigh and was published in Lieven Haenebalcke, .|
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.