Last data update:
25 April 2019 04:11 CEST
|Name:||pROSA26-COIN-DV-Ins/Ins Add to cart|
|Accession number:||LMBP 8193|
|Sequence file:||Depositor's sequence: p8193.gb View with Genome Compiler|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Mouse gene trap ROSA 26 gene (Gtrosa26, Gt(ROSA)26Sor, beta geo, Gtrgeo26, R26, ROSA26, Thumpd3as1, GeneID 14910); 5' UTR and 3' UTR
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)
Phage T3 promoter
Phage T7 gene 10 promoter (T7g10)
Escherichia coli lac operon promoter
Escherichia coli lac operon promoter; mutant (lacUV5)
|Terminator:||Bovine growth hormone polyadenylation signal (BGH polyA)
Simian virus 40 polyadenylation signal (SV40 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:||The plasmid was constructed by introducing an insulator cassette between the Gateway recombination cassette and the 3' UTR of ROSA26 of pROSA26-CON-DV.
The plasmid is a multisite Gateway destination vector, containing the ccdB gene inserted between the attenuator sites R3 and R4.
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-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-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-attL3 (LMBP 8200)).
The plasmid contains:
- two bacteriophage P1 Cre recombinase target sites (loxP sites)
- a splice acceptor (SA) for linking the rtTA-IRES-puromycin to the endogenous ROSA26 promoter
- 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.
This plasmid is part of a second generation, insulated COIN (GOF/LOF) system, in which the gene of interest is to be targeted to the mouse ROSA26 locus via homologous recombination and can be expressed conditionally and inducibly.
The G4 ROSALUC ES cells are available at BCCM/GeneCorner as well.
Other name of the plasmid is pROSA26 COIN DV Insulated.
|EMBL Accession number:||-|
|Latest sequence update:||01/12/2016|
|Authenticity test:||The restriction enzyme pattern has still to be analysed.|
|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:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|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.