Last data update: 26 September 2020 04:22 CEST
Plasmid name: pLVX-EF1alpha-hCYLD-R324A-C601A-IRES-ZsGreen1 (LMBP 9106)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human CYLD lysine 63 deubiquitinase cDNA (CYLD, CYLD1, USPL2, GeneID 1540); mutated sequence
Zoanthus sp. green fluorescent protein cDNA (ZsGreen); human-codon-optimised variant (ZsGreen1)
|Promoter:||Human elongation factor 1α promoter (EF1α)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
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; pCAGGS-E-CYLD-R324A-C601A|
|Further information:||The plasmid was constructed by PCR amplifying the mutant human CYLD coding sequence from pCAGGS-E-CYLD-R324A-C601A and cloning it as a NotI/BapHI fragment into the pLVX-EF1alpha-IRES-ZsGreen1 vector.
The human CYLD coding sequence in this plasmid contains an R324A mutation, making the encoded protein uncleavable by MALT1, and a C601A mutation, making the CYLD protein catalytically inactive. The purpose of this construct is to reconstitute knock-out cells with a mutant variant.
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 CYLD protein 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 CYLD, without time-consuming drug - and clonal selection.
|EMBL Accession number:||-|
|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.