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GeneCorner plasmid details

Last data update: 26 November 2020 04:30 CET

Plasmid name: pAS2htBID (LMBP 4758)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4758.gb (View with Genome Compiler)
p4758.txt
p4758.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human BH3 interacting domain death agonist (hBID); truncated sequence (htBID)
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 constructed by inserting a 416 bp NcoI-SalI fragment, containing the htBID coding sequence, obtained by RT-PCR using HeLa cDNA as template, into the NcoI-SalI opened pAS2 vector. As a result, the htBID coding sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-htBID fusion protein can be used as a bait protein in two-hybrid screening.
pAS2htBID carries the S. cerevisiae tryptophan 1 coding sequence (TRP1) 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727031.1.
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 hBID cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF042083.1.
Other name of the plasmid is pAS2 htBid.
EMBL Accession number: U30496.1, view at EMBL, GenBank, DDBJ
AF042083.1, view at EMBL, GenBank, DDBJ
LT727031.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 31/01/2005
Sequence detail:
Primers used for RT-PCR on HeLa cDNA:

forward primer: 5' CAT.GCC.ATG.GCG.GGC.AAC.CGC.AGC.AGC.CAC.TCC.CGC.TT 3'
                        NcoI

reverse primer: 5' TGCGGTCGAC.TCA.GTC.CAT.CCC.ATT.TCT.GGC.TAA 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
                                                                           

       ---------------------- htBID --------------------->
       61                                          195
       GGC.AAC.CGC.AGC.AGC.CAC ... AGA.AAT.GGG.ATG.GAC.TGA.GTCGACCTGCAGCCAAGC ... 3' 
       Gly Asn Arg Ser Ser His     Arg Asn Gly Met Asp +++ SalI  PstI
                                                           HindII

***: start codon.
+++: termination codon.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: Alw44I/Eco81I, BglI/EcoRI, BglII, NcoI/SalI, NheI/PvuII and SspI/XhoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2). It was constructed by N. Festjens(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:

pAS2htBID (LMBP 4758) 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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