Last data update: 30 October 2020 04:11 CET
Plasmid name: pAS2mproCASP8-3N (LMBP 4300)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Mouse cysteinyl aspartate specific proteinase 8 cDNA (caspase-8, CASP-8, Casp8, MCH5, MACH, FLICE); mutated sequence
Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd)
Influenza HA epitope encoding the haemagglutinin tagging peptide; N-terminal
|Promoter:||Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1)
Escherichia coli lac operon promoter
|Terminator:||Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1)
Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1)
Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB)
|Host range:||Escherichia coli
Saccharomyces cerevisiae; trp1(-)
|Further information:||The plasmid was obtained by PCR mutagenesis on pAS2mproCASP8-WT, replacing 3 aspartic acid residues between the p10, P20 and prodomain subunits of CASP8 by asparagin amino acids. These mutations prevent the caspase from activating itself.
pAS2 is a yeast two-hybrid vector, expressing the cloned mproCASP8 cds as a fusion protein with GAL4DBD.
The nucleotide sequence of the pAS2 vector corresponds with the EMBL Nucleotide Sequence Database accession number U30496.1, but adapted as follows: 1) the 'N' nucleotide was removed, restoring the coding sequence of cycloheximide; 2) the sequence GATTTC between GALbd and the HA epitope was replaced by GAATTC, an EcoRI site that has been experimentally confirmed in pAS2; 3) the sequence TTCGAA (Csp45I site) in the ADH1 promoter was replaced by TCGAAG, eliminating the Csp45I site that could not be experimentally confirmed in pAS2.
|EMBL Accession number:||U30496.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||21/04/2009|
Mutagenesis primers used: Forward 1: 5' G.CCA.AGA.GAA.CAA.AAC.AGT.GAG.TCA.CG *** Reverse 1: 5' GA.AGT.CCG.TGA.CTC.ACT.GTT.TTG.TTC.TCT.TGG.C *** Forward 2: 5' C.CAG.AAA.GGA.GTG.CCT.AAT.GAG.GCA.GGC.TTC.G *** Reverse 2: 5' G.TTG.CTC.GAA.GCC.TGC.CTC.ATT.AGG.CAC.TCC.TTT.CTG.G *** Forward 3: 5' C.ACT.TTA.GAA.GTG.AAT.TCA.TCA.TCT.CAC *** Reverse 3: 5' GTT.CTT.GTG.AGA.TGA.TGA.ATT.CAC.TTC.TAA.AGT.G *** ***: Mutated codons
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2).
(1) Department for Molecular Biomedical Research, VIB, 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 MC1061|
|Host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Related host reference:||Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pAS2mproCASP8-3N (LMBP 4300) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr P. Vandenabeele .|
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.