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LMBP plasmid details

Last data update: 20 October 2018 04:23 CEST  


Name: pDG2i-mBeclin1-N-Frag-131-Flag-puro   Add to cart
Accession number: LMBP 8601
Sequence file: Depositor's sequence: p8601.gb     View with Genome Compiler
Status: LMBP non-core plasmid
Cloned DNA: Mouse beclin 1, autophagy related cDNA (Becn1, Atg6); fragment
Reverse tetracycline-responsive transcriptional activator (rtTA); variant 3 (rtTA3)
FLAG epitope tag; C-terminal
Promoter: Human cytomegalovirus immediate early promoter (CMV-IE); minimal promoter with tet responsive element (CMVTRE)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Simian virus 40 early promoter (SV40 early)
Escherichia coli lac operon promoter
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Ribosome
binding site:
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene
Terminator: Simian virus 40 polyadenylation signal (SV40 polyA)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 3' LTR); modified HIV-1 3' LTR with deleted U3 region (ΔU3/3' LTR)
Selection marker: Ampicillin (amp)
Puromycin (puro)
Replicon: Simian virus 40 bidirectional origin (SV40)
Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: pDG2i-Flag puro; pENTR3C-mBeclin1-N-Frag-131-stop
Further information: The plasmid was constructed via Gateway LR recombination between pDG2i-Flag puro and pENTR3C-mBeclin1-N-Frag-131-stop and contains the N-terminal fragment of the mouse Becn1 coding sequence, consisting of the first 131 codons.
This is a lentiviral Gateway expression vector, with the tet-inducible minimal human CMV-IE promotor and rtTA3 on the same plasmid.
The U3 region of the HIV-1 3' LTR is deleted (ΔU3 or dU3) and facilitates self-inactivation of the HIV-1 5' LTR after transduction to enhance the biosafety of the vector. The element also contains a polyadenylation signal for transcription termination and polyadenylation of mRNA in transduced cells.
The vector contains the psi packaging signal, allowing viral packaging, and the rev response element (RRE) from HIV-1, permitting Rev-dependent nuclear export of unspliced viral mRNA.
Because the risk for recombination after each subcultivation is high, this plasmid is only available under the format of isolated plasmid DNA. Plasmid integrity should be checked via PvuII restriction digest after each subcultivation.
The nucleotide sequence of the mouse Becn1 coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number BC005770.1.
The nucleotide sequence of the parental pDG2i-Flag puro vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009444.1.
Other name of the plasmid is pDG2-FLAG-rtTA3puro-mBeclin1-N-term_131-stop.
EMBL Accession number: BC005770.1, view at EMBL, GenBank, DDBJ
LT009444.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 24/08/2016
Authenticity test: The restriction enzyme pattern has still to be analysed.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: The plasmid was deposited by Prof. Dr P. Vandenabeele(1)(2). It was constructed by I. Bruggeman(1)(2).
(1) Inflammation Research Center, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: De Groote et al., BioTechniques 60 (2016), 252-259 [PMID: 27177818] [DOI: 10.2144/000114417]
Restricted distribution: - BCCM MTA
Distributed as: 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: 28°C
Biosafety level: L1 in E. coli; L2 in mammalian cells
Other culture collection numbers: -

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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