Last data update: 22 January 2021 04:11 CET
Plasmid name: Cre excised pCOIN DV-OSKM (LMBP 8172)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
Thosea asigna virus 2A peptide (T2A)|
Foot-and-mouth disease virus 2A peptide (F2A)
Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP)
Photinus pyralis (firefly) luciferase gene (LUC); mutated coding region (LUCm; luc(+))
Mouse Kruppel-like factor 4 (gut) cDNA (Klf4, EZF, Gklf, Zie)
Mouse myelocytomatosis oncogene cDNA (Myc, bHLHe39, c-myc, Myc2, Niard, Nird)
Mouse SRY (sex determining region Y)-box 2 cDNA (Sox2, Icc, Sox-2, ysb)
Mouse POU domain, class 5, transcription factor 1 cDNA (Pou5f1, Oct-3, Oct-3/4, Oct3/4, Oct4, Oct-4, Otf3, Otf-3, Otf3g, Otf3-rs7, Otf4, Otf-4)
|Promoter:||Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter
Human cytomegalovirus immediate early promoter (CMV-IE); minimal promoter with tet responsive element (CMVTRE)
|Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene|
|Terminator:||Bovine growth hormone polyadenylation signal (BGH polyA)
Rabbit β-globin polyadenylation signal (β-globin polyA)
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli
|Parental clone:||Cre excised pCOIN DV; pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE ; pEntry attR2-IRES-eGFP-luc+-pA-attL3; pENTR1A-OSKM|
|Further information:||The plasmid was constructed by introducing a reverse tetracycline transactivator (rtTA), followed by an IRES-puromycin-polyA cassette from pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1, the OSKM cassette from pENTR1A-OSKM and an IRES-EGFP-Luciferase-polyA cassette from pEntry attR2-IRES-eGFP-luc+-pA-attL3 into the Cre excised pCOIN DV vector via Gateway LR recombination.
The plasmid contains:
- two heterospecific Saccharomyces cerevisiae 2μ FLP Recombinase Target sites (one wt and one mutant FRT site)
- one remaining bacteriophage P1 Cre recombinase target site (loxP site)
- 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.
This multisite Gateway expression vector, by co-transfection of a FlpE plasmid, can be targeted to the ROSA26 locus of G4 ROSALUC ES cells using a trap-coupled RMCE approach, hereby restoring neomycin resistance. The absence of the loxP-flanked stop casette compared to pCOIN DV-derived expression vectors causes ROSA26-based rtTA expression and puromycin resistance. Inducible expression of OSKM will only be achieved upon doxycycline administration and can be monitored by the induced reporter expression (EGFP-LUC).
The coding sequences in the OSKM cassette are connected via the self-cleaving T2A and F2A peptides, fulfilling the same role as an IRES, but with a much shorter sequence.
The G4 ROSALUC ES cells (LMBP 10507CB) are available at BCCM/GeneCorner as well.
Other name of the plasmid is Cre ex. pCOIN DV OSKM.
|EMBL Accession number:||-|
|Latest sequence update:||01/12/2016|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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
|Related plasmid reference:||Yusa et al., Nat. Methods 6 (2009), 363-369 [PMID: 19337237]
|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:||-|
Refer in your Materials and Methods:
|Cre excised pCOIN DV-OSKM (LMBP 8172) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr J. Haigh and was published in Lieven Haenebalcke, .|