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GeneCorner plasmid details

Last data update: 03 December 2020 04:26 CET

Plasmid name: pAS2mBIDG94E (LMBP 4764)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p4764.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse BH3 interacting domain death agonist (BID); 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; pAS2mBID
Further information: The plasmid was constructed by inserting a 596 bp NcoI-SalI fragment, containing the mutated sequence of mBID, into the NcoI-SalI opened pAS2 vector. As a result, the mutated sequence of mBID is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
The mutated sequence of mBID was obtained by fusion PCR using pAS2mBID as template. As compared to the wt mBID cDNA, the glycine (G) codon at position 94 was replaced by a glutamic acid (E) codon.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-mBIDG94E fusion protein can be used as a bait protein in two-hybrid screening.
pAS2mBIDG94E 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 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.
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 6050 is substituted by an A- residue, resulting in a Glu/Lys replacement for amino acid 14.
Other name of the plasmid is pAS2 mBid G94E.
EMBL Accession number: U30496.1, view at EMBL, GenBank, DDBJ
NM_007544.2, view at GenBank
Latest sequence update: 08/02/2005
Sequence detail:
Primers used to amplify mBIDm:

forward primer: 5' CATGCCATGGCG.ATG.GAC.TCT.GAG.GTC.AGC.AAC.GG 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.GAA.TTC.ATG
               Met Lys Leu Leu Ser Ser     Arg Gln Leu Thr Val Ser Pro Glu Phe Met
               ***                               HindII                EcoRI


           ----------- HA epitope ----------->                      
       GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.AGC.TTG.GGT.GGT.CAT.ATG.GCC.ATG.GCG
       Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly His Met Ala Met Ala
                                           NheI                NdeI     NcoI
                                                                        StyI

       ---------------------------------- mBIDm --------------------------------->
       1                                   94                              195
       ATG.GAC.TCT.GAG.GTC.AGC ... CAA.ATA.GAG.GAT.GAG ... AGA.AAC.GAG.ATG.GAC.TGA
       Met Asp Ser Glu Val Ser     Gln Ile Glu Asp Glu     Arg Asn Glu Met Asp +++
       ***                                  ^^


       GTCGACCTGCAGCCAAGC ... 3' 
       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:

pAS2mBIDG94E (LMBP 4764) 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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