Last data update: 05 December 2020 04:15 CET
Plasmid name: pAS2.1mBIDdeltaC (LMBP 4769)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
Mouse BH3 interacting domain death agonist (BID); with C-terminal deletion (BIDΔC)|
Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd)
|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(-)
|Parental clone:||pAS2.1; pAS2mBID|
|Further information:||The plasmid was constructed as follows: the mBID coding sequence with C-terminal deletion of helix 6, 7 and 8 (mBIDΔC, encoding amino acids 1-144) was amplified by PCR on pAS2mBID and inserted as a 443 bp NcoI-SalI fragment into the NcoI-SalI opened pAS2.1 vector. As a result, the mBIDΔC coding sequence is fused in phase to the N-terminal S. cerevisiae GALbd domain.
pAS2.1 was derived from pAS2 by removing the Influenza HA epitope, encoding the haemagglutinin tagging peptide. Therefore, unlike pAS2mBIDdeltaC, pAS2.1mBIDdeltaC transformants lack aspecific β-galactosidase activity.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-mBIDΔC fusion protein can be used as a bait protein in two-hybrid screening.
pAS2.1mBIDdeltaC carries the S. cerevisiae tryptophan 1 coding sequence and the CYH2 sequence encoding ribosomal protein L29 for dominant cycloheximide sensitivity which facilitates the elimination of false positives.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of the pAS2.1 vector corresponds with the EMBL Nucleotide Sequence Database accession number U30497.1, corresponding to the Clontech website (version 05/01/1996).
The nucleotide sequence of the mBID cDNA corresponds with the Genbank accession number NM_007544.2 and adapted to the sequence analysis results of the Department of Biomedical Molecular Biology (Ghent University, Belgium). The following difference is observable: the nucleotide G at position 6023 is substituted by an A- residue, resulting in a Glu/Lys replacement for amino acid 14.
Other name of the plasmid is pAS2.1 mBid C-term del.
|EMBL Accession number:||U30497.1, view at EMBL, GenBank, DDBJ
NM_007544.2, view at GenBank
|Latest sequence update:||29/03/2005|
Primers used to amplify mBIDdeltaC: forward primer: 5' CATGCCATGGCG.ATG.GAC.TCT.GAG.GTC.AGC.AAC.GG 3' NcoI *** reverse primer: 5' ACGCGTCGAC.TCA.GTT.CTC.CAT.GTC.TCT 3' SalI +++ Nucleotide sequence of the fusion gene: ---------------------- GALbd ---------------------> 1 147 5' ... CTGAAAG.ATG.AAG.CTA.CTG.TCT.TCT ... AGA.CAG.TTG.ACT.GTA.TCG.CCG.GTA.TTG.CAA Met Lys Leu Leu Ser Ser Arg Gln Leu Thr Val Ser Pro Val Leu Gln *** HindII ------------------- mBIDdeltaC ---- 1 TAC.CCA.GCT.TTG.ACT.CAT.ATG.GCC.ATG.GCG.ATG.GAC.TCT.GAG.GTC.AGC ... CCC.AGA Tyr Pro Ala Leu Thr His Met Ala Met Ala Met Asp Ser Glu Val Ser Pro Arg NdeI NcoI *** StyI --------------> 144 GAC.ATG.GAG.AAC.TGA.GTCGACCTGCAGCCAAGC ... 3' Asp Met Glu Asn +++ SalI PstI HindII ***: start codon. +++: termination codon. Punctuation indicates reading frame.
|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:
|pAS2.1mBIDdeltaC (LMBP 4769) 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.