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GeneCorner plasmid details

Last data update: 26 November 2020 04:30 CET

Plasmid name: Cre excised pRMCE DV1-OSKM (LMBP 8176)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p8176.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: 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)
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
Ribosome
binding site:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Bovine growth hormone polyadenylation signal (BGH polyA)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Mammalian cells
Parental clone: Cre excised pRMCE DV1; pENTR1A-OSKM
Further information: The plasmid was constructed by introducing the mouse OSKM cassette from pENTR1A-OSKM into the Cre excised pRMCE DV1 vector via Gateway LR recombination.
The monisite Gateway expression plasmid is a highly efficient Recombinase-Mediated Cassette Exchange (RMCE)-compatible Gateway vector and allows immediate ROSA26-based OSKM expression in mouse ES cells.
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.
This monosite 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.
Cre-mediated removal of the loxP-flanked stop cassette causes ROSA26-based constitutive expression of the inserted OSKM cDNA.
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. pRMCE DV1 OSKM.
EMBL Accession number: -
Latest sequence update: 01/12/2016
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
Class: Recombinant plasmid
Type: Plasmid
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)
Cultivation temperature: 37°C
Biosafety level: L1
Other culture collection numbers: -

Refer in your Materials and Methods:

Cre excised pRMCE DV1-OSKM (LMBP 8176) 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.

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