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GeneCorner plasmid details

Last data update: 23 October 2020 04:27 CEST

Plasmid name: pAS2.1mtBIDG94E (LMBP 4763)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p4763.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse BH3 interacting domain death agonist (BID); mutated truncated sequence (tBIDm)
Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd)
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: pAS2.1; pAS2mBID
Further information: The plasmid was constructed by inserting a 419 bp NcoI-SalI fragment, containing the mutated truncated sequence of mBID, into the NcoI-SalI opened pAS2.1 vector. As a result, the mtBIDm coding sequence is fused in phase to the N-terminal S. cerevisiae GALbd domain.
The mutated sequence of mtBID was obtained by fusion PCR using pAS2mBID as template. As compared to the wt mtBID cDNA, the glycine (G) codon at position 94 of mBID was replaced by a glutamic acid (E) codon.
pAS2.1 was derived from pAS2 by removing the Influenza HA epitope, encoding the haemagglutinin tagging peptide. Therefore, unlike pAS2mtBIDG94E, pAS2.1mtBIDG94E 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-mtBIDG94E fusion protein can be used as a bait protein in two-hybrid screening.
pAS2.1mtBIDG94E 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 mtBID cDNA corresponds with the Genbank accession number NM_007544.2.
Other name of the plasmid is pAS2.1 mtBid G94E.
EMBL Accession number: U30497.1, view at EMBL, GenBank, DDBJ
NM_007544.2, view at GenBank
Latest sequence update: 09/02/2005
Sequence detail:
Primers used to amplify mtBIDm:

forward primer: 5' CAT.GCC.ATG.GCG.GGC.AGC.CAG.GCC.AGC.CGC.TCC.TTC 3'
                        NcoI

forward primer for fusion PCR:  5' TC.GCC.CAA.ATA.GAG.GAT.GAG.ATG.GA 3'
                                                   ^^

reverse primer for fusion PCR:  5' TC.CAT.CTC.ATC.CTC.TAT.TTG.GGC.GA 3'
                                                  ^^              

reverse primer: 5' TGCGGTCGAC.TCA.GTC.CAT.CTC.GTT.TCT.AAC.CAA.GTT.C 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                


                                               ---------------------------- mtBIDm
                                               60
       TAC.CCA.GCT.TTG.ACT.CAT.ATG.GCC.ATG.GCG.GGC.AGC.CAG.GCC.AGC.CGC ... CAA.ATA
       Tyr Pro Ala Leu Thr His Met Ala Met Ala Gly Ser Gln Ala Ser Arg     Gln Ile 
                           NdeI     NcoI


       mtBIDm ------------------------------->
       94                              195
       GAG.GAT.GAG ... AGA.AAC.GAG.ATG.GAC.TGA.GTCGACCTGCAGCCAAGC ... 3' 
       Glu Asp Glu     Arg Asn Glu Met Asp +++ SalI  PstI
        ^^                                     HindII


***: start codon.
^  : mutated nucleotide.
+++: termination codon.
Punctuation indicates reading frame.
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
Cultivation remark: -
Other culture collection numbers: -

Refer in your Materials and Methods:

pAS2.1mtBIDG94E (LMBP 4763) 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.

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