Last data update: 26 September 2020 04:22 CEST
Plasmid name: pLVX-EF1alpha-hMALT1-E4A-IRES-ZsGreen1 (LMBP 9107)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human MALT1 paracaspase cDNA (MALT1, MLT, PCASP1, paracaspase 1); mutated sequence
Zoanthus sp. green fluorescent protein cDNA (ZsGreen); human-codon-optimised variant (ZsGreen1)
|Promoter:||Human elongation factor 1α promoter (EF1α)
Escherichia coli lac operon promoter
Human immunodeficiency virus long terminal repeat (HIV-1 5' LTR)
|Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene|
|Terminator:||Human immunodeficiency virus long terminal repeat (HIV-1 3' LTR); with deleted U3 region (ΔU3/3' LTR)|
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli
|Parental clone:||pLVX-EF1alpha-IRES-ZsGreen1; pCD-F-bio-MK-E4A|
|Further information:||The plasmid was constructed by PCR amplifying the mutant human MALT1 coding sequence from pCD-F-bio-MK-E4A and cloning it as a NotI/BamHI fragment into the pLVX-EF1alpha-IRES-ZsGreen1 vector.
The TRAF6 binding sites of human MALT1 were mutated. As compared to the wt human MALT1 ccoding sequence, the glutamic acid (E) codons at positions 313, 316, 653 and 806 were replaced by alanine (A) codons, creating additional SphI, AgeI and NruI sites and abolishing an Alw44I site.
This bicistronic lentiviral expression vector can be used to generate high-titer lentivirus for transducing virtually any dividing or nondividing mammalian cell type, including primary and stem cells. The vector contains an internal ribosomal entry site (IRES) that allows the tagged MALT1 fragment and the ZsGreen1 fluorescent protein to be simultaneously coexpressed from a single mRNA transcript. Expression of the transcript is driven by the human elongation factor 1 alpha (EF1α) promoter, which continues to be constitutively active even after stable integration of the vector into the host cell genome. Stable expression of the transcript allows the monitoring of a variety of cellular processes (such as differentiation in primary or stem cells) without the transgene silencing associated with CMV promoters. In addition, the vector allows efficient flow cytometric detection of stably or transiently transfected mammalian cells expressing ZsGreen1 and human MALT1, without time-consuming drug - and clonal selection.
The purpose with this construct is to reconstitute MALT1 knock-out cells with a mutant variant.
The nucleotide sequence of the wild type human MALT1 coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number AF130356.2.
|EMBL Accession number:||AF130356.2, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||26/04/2017|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||The plasmid was deposited by DR J. Staal(1)(2) and Prof. Dr R. Beyaert(1)(2).
(1) VIB Center for Inflammation Research, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Restricted distribution:||- BCCM MTA|
|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)|
|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.