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GeneCorner plasmid details

Last data update: 20 October 2020 04:14 CEST

Plasmid name: Cre excised pCOIN DV-NLS-lacZ (LMBP 8174)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p8174.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Escherichia coli lac Z gene (lacZ); with modified 5' end
SV40 large T-antigen nuclear localization signal (NLS); N-terminal
Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP)
Photinus pyralis (firefly) luciferase gene (LUC); mutated coding region (LUCm; luc(+))
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)
Ribosome
binding site:
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)
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Mammalian cells
Parental clone: Cre excised pCOIN DV; pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE ; pEntry attR2-IRES-eGFP-luc+-pA-attL3; pENTR1A-NLS-LacZ
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 mouse LacZ coding sequence with a NLS signal from pENTR1A-NLS-LacZ 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 NLS-lacZ will only be achieved upon doxycycline administration and can be monitored by the induced reporter expression (EGFP-LUC).
The G4 ROSALUC ES cells (LMBP 10507CB) are available at BCCM/GeneCorner as well.
Other name of the plasmid is Cre ex. pCOIN DV nls-lacZ.
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
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 pCOIN DV-NLS-lacZ (LMBP 8174) 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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