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GeneCorner plasmid details

Last data update: 03 December 2020 04:26 CET

Plasmid name: pAS3 (LMBP 4253)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4253.gb (View with Genome Compiler)
p4253.txt
p4253.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: 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 2 micron plasmid (2μ) FLP terminator
Selection marker: Ampicillin (amp)
ADE2; auxotrophic
Replicon: Escherichia coli plasmid pMB1 origin
Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB)
Host range: Escherichia coli
Saccharomyces cerevisiae; ade2(-)
Parental clone: pAS2
Further information: The plasmid was constructed by blunt ligation of the Saccharomyces cerevisiae ADE2 gene under its own promoter into the EcoRV opened pAS2 vector.
pAS3 is designed to generate a fusion of the S. cerevisiae GALbd domain with a sequence encoding a target protein (bait fusion protein).
The GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal, and is followed by the HA epitope sequence, a multicloning site and termination codons in all reading frames.
pAS3 is useful in a yeast three-hybrid approach. The ADE2 auxotrophic selection marker makes the system compatible with the existing yeast strains and libraries that are currently used in Gal4-based two- and three-hybrid systems and which make use of the TRP1, LEU2, HIS3 and URA3 selection markers.
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 LT727334.1.
The nucleotide sequence of the ADE2 cDNA corresponds with the Genbank accession number NC_001147.5.
EMBL Accession number: NC_001147.5, view at EMBL, GenBank, DDBJ
LT727334.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 11/09/2006
Sequence detail:
Nucleotide sequence of the HA epitope and the multi-cloning site:

      GALbd ----->                     ----------- HA epitope ----------->
5' ... ACT.GTA.TCG.CCG.GAA.TTC.ATG.GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.
       Thr Val Ser Pro Glu Phe Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
                       EcoRI                                           NheI


   AGC.TTG.GGT.GGT.CAT.ATG.GCC.ATG.GAG.GCC.CCG.GGG.ATC.CGT.CGA.CCT.GCA.GCC.
   Ser Leu Gly Gly His Met Ala Met Glu Ala Pro Gly Ile Arg Arg Pro Ala Ala
                   NdeI     NcoI         SmaI   BamHI   SalI    PstI
                            StyI
                        BalI
                         SfiI  

                    --------------------------- ADH1 terminator --->
   AAG.CTA.ATT.CCG.GGC.GAA.TTT.CTT.ATG.ATT.TAT.GAT.TTT.TAT.TAT.TAA ... 3'
   Lys Leu Ile Pro Gly Glu Phe Leu Met Ile Tyr Asp Phe Tyr Tyr +++ 
        ++ +                                 + ++

+++: Termination codon.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: AatII/HindIII, AccIII/NdeI, AvaII/Csp45I, BglI/BglII, Bst98I/BstXI, EcoRV/NcoI, MunI, SspI and XhoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr R. Beyaert(1) (2). It was constructed by W. Van Criekinge(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Van Criekinge et al., Anal. Biochem. 263 (1998), 62-66 [PMID: 9750144]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 DH5α
Host reference: Focus 8 (1986), 9
Related host reference: Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
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:

pAS3 (LMBP 4253) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert and was published in Van Criekinge et al., 1998.

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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