GeneCorner plasmid details

Last data update: 18 January 2021 04:24 CET

Plasmid name: pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1 (LMBP 8212)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: (View with Genome Compiler)
analysis results


Cloned DNA: Reverse tetracycline-responsive transcriptional activator (rtTA)
Promoter: Human cytomegalovirus immediate early promoter (CMV-IE); minimal promoter with tet operator (CMVT)
binding site:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Bovine growth hormone polyadenylation signal (BGH polyA)
Escherichia coli rrnB operon T1 terminator
Escherichia coli rrnB operon T2 terminator
Selection marker: Neomycin (neo; G418; kanamycin (kan))
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Mammalian cells
Parental clone: pENTRY-L4-R1
Further information: The plasmid is a multisite Gateway entry vector, designed as a 5' entry clone (with attL4-attR1 sites) for the multisite approach using the pCOIN DV vector.
The plasmid contains:
- a reverse tetracyclin transactivator (rtTA)
- an IRES-puromycin-pA drug-selectable cassette to select for rtTA expressing cells after Cre excision of the pCOIN DV vector.
- a tetracycline responsive element (TRE), consisting of the minimal human CMV promoter, combined with the tet operator.
- two adjacent copies of the chickenβ-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.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726877.1.
The nucleotide sequence of the rtTA corresponds with the EMBL Nucleotide Sequence Database accession number U89930.1.
EMBL Accession number: U89930.1, view at EMBL, GenBank, DDBJ
LT726877.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 08/07/2013
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: ApaI, HindIII, PvuI, PvuII and SacII.

As compared to the compiled nucleotide sequence, the restriction enzyme pattern revealed that the plasmid is about 150 to 200 bp shorter. This fragment is probably located between the Tn903 neomycin resistance and the pMB1 ori.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr L. Haenebalcke(1) (2) and Prof. Dr J. Haigh(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Haenebalcke et al., Stem Cell Rev 9 (2013), 774-785 [PMID: 23877658] [DOI: 10.1007/s12015-013-9458-z]
Related plasmid reference: PhD thesis Lieven Haenebalcke
Restricted distribution: - VIB/BCCM MTA
- The depositor will be informed of the customer's identity and of the enduser's research described in the VIB/BCCM MTA upon release of a sample outside the depositor's department.
- When appropriate in accordance with academic customs, RECIPIENT agrees to include the depositor(s) as co-author(s) in the initial publication describing the MATERIAL.
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 + kanamycin (50 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
Cultivation remark: This culture grows very slow: take 24 hours into account for saturated growth!
Other culture collection numbers: -

Refer in your Materials and Methods:

pEntry L4 rtTA-IRES-Puro-pA-Ins/Ins-TRE R1 (LMBP 8212) is available at BCCM/GeneCorner. This plasmid was deposited by Dr L. Haenebalcke and Prof. Dr J. Haigh and was published in Haenebalcke et al., 2013.

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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