Last data update: 24 January 2024 16:39 CET
Plasmid name: pAS2mproCASP8-3N (LMBP 4300)
New search | Print data sheet |
Price category: | Cat. 1 (cf. price list) |
Status: | GeneCorner non-core plasmid |
Depositor's sequence: | p4300.gb |
Sequence analysis results Genecorner: |
- |
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 |
Ribosome binding site: |
- |
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) Cycloheximide (CYH2) TRP1; auxotrophic |
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(-) |
Parental clone: | pAS2 |
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 |
Sequence detail: | 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. |
Class: | Recombinant plasmid |
Type: | Plasmid |
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 |
Plasmid reference: | - |
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) |
Cultivation temperature: | 37°C |
Biosafety level: | L1 |
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.