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LMBP plasmid details

Last data update: 23 September 2020 04:27 CEST

Plasmid name: pLenti6-strep-hMLKL(211-471)-Q356A-FLAG (LMBP 8810)

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

-

Cloned DNA: Human mixed lineage kinase domain like pseudokinase cDNA (MLKL, GeneID 197259); mutated fragment
Strep-tag II; N-terminal
FLAG epitope tag; C-terminal
Promoter: Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Escherichia coli lac operon promoter
Simian virus 40 early promoter (SV40 early)
Phage T3 promoter
Phage T7 gene 10 promoter (T7g10)
Ribosome
binding site:
-
Terminator: 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)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: pLenti6-Flag puro; pENTR3C-strep-hMLKL(211-471)-Q356A
Further information: This Lentiviral Gateway Expression vector was constructed by cloning the strep-tagged N-terminal fragment of the human MLKL coding sequence, consisting of codons 211 up to and including 471 and containing a Q356A mutation, into the pLenti6-Flag puro vector via Gateway recombination with pENTR3C-strep-hMLKL(211-471)-Q356A.
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 of the high risk for recombination, plasmid integrity should be checked via PvuII restriction digest after each subcultivation.
The nucleotide sequence of the wild type human MLKL coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number AK091708.1.
The nucleotide sequence of the pLenti6-Flag puro vector corresponds with the EMBL Nucleotide Sequence Database accession number LT009452.1.
Other name of the plasmid is pLenti6(puro)-FLAG-dest-strep-hMLKL(211-471)-Q356A-stop.
EMBL Accession number: AK091708.1, view at EMBL, GenBank, DDBJ
LT009452.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 05/10/2016
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 Dr Y. Dondelinger(1) (2) and Prof. Dr M. Bertrand(1) (2).
(1) Inflammation Research Center, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Dondelinger et al., Cell Rep 7 (2014), 971-981 [PMID: 24813885] [DOI: 10.1016/j.celrep.2014.04.026]
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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