GeneCorner plasmid details

Last data update: 18 January 2021 04:24 CET

Plasmid name: pEntry attR2-IRES-eGFP-luc+-pA-attL3 (LMBP 8200)

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Price category: Cat. 3 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: (View with Genome Compiler)
analysis results

NGS: d10-gc-dec2018.fasta

Cloned DNA: Photinus pyralis (firefly) luciferase gene (LUC); mutated coding region (LUCm; luc(+))
Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP)
Promoter: Phage T7 gene 10 promoter (T7g10)
binding site:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Rabbit β-globin polyadenylation signal (β-globin polyA)
Escherichia coli rrnB operon T1 terminator
Escherichia coli rrnB operon T2 terminator
Selection marker: Neomycin (neo; kanamycin (kan))
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Parental clone: pEntry R2L3
Further information: The plasmid was constructed by cloning the IRES-EGFP-luc+-pA cassette as an XhoI-XhoI fragment into the XhoI-SalI restricted pEntry R2L3 vector.
This Gateway entry vector is to be used with the pROSA26-DV2 or pROSA26-DV3 multisite plasmids and with the plasmids of the recombinase-mediated cassette exchange (RMCE), conditional and inducible multisite system.
As compared to pEntry attR2-IRES-eGFP-attL3 (LMBP 6454, formerly LMBP 6353), the depositor believes that pEntry attR2-IRES-eGFP-luc+-pA-attL3 is a better reporter with optimal pA+ sequence allowing expression of both EGFP and luciferase.
The region between the luc+ coding sequence and the neomycin resistance gene and the region downstream from the T1 terminator were analysed at the Department for Molecular Biomedical Research (VIB, Ghent, Belgium).
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726875.1.
The nucleotide sequence of the EGFP DNA corresponds with the EMBL Nucleotide Sequence Database accession number U55761.1.
The nucleotide sequence of the 5' untranslated region of the EMCV polyprotein gene, containing the IRES sequence, corresponds with the EMBL Nucleotide Sequence Database accession number M81861.1, except for an additional A at position 3227 of the plasmid sequence.
The depositor suggests that the products of the LR reactions using this vector should be sequenced at their junctions as is outlined in Nyabi et al. (2009) to ensure correct plasmid recombination before final electroporation of the targeting constructs into ES cells.
Other names of the plasmid are pEntry R2 IRES eGFP luciferase pA L3 and pEntry R2 IRES-EGFP-Luciferase-pA L3.
EMBL Accession number: U55761.1, view at EMBL, GenBank, DDBJ
M81861.1, view at EMBL, GenBank, DDBJ
LT726875.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 11/01/2013
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglI, HaeII, HincII and ScaI.

This plasmid has also been fully sequenced but the NGS sequence data still need to be implemented.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by 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: Nyabi et al., Nucleic Acids Res. 37 (2009), e55 [PMID: 19279185] [DOI: 10.1093/nar/gkp112]
Sherf et al., Promega Notes Magazine 49 (1994), 14-21
PhD thesis Lieven Haenebalcke
Restricted distribution: - VIB/BCCM MTA
- 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: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: LMBP 06440

Refer in your Materials and Methods:

pEntry attR2-IRES-eGFP-luc+-pA-attL3 (LMBP 8200) is available at BCCM/GeneCorner. This plasmid was deposited by 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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