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GeneCorner plasmid details

Last data update: 30 October 2020 04:11 CET

Plasmid name: pAS2hA20S565A (LMBP 3772)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3772.gb (View with Genome Compiler)
p3772.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human TNF alpha induced protein 3 cDNA (TNFAIP3, A20, OTUD7C, GeneID 7128); 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: pAS2A20
Further information: The plasmid was constructed by site-specific mutagenesis of the human zinc finger protein A20 coding sequence (hA20) by the use of primers. As a result the serine codon at position 565 was replaced by an alanine codon, creating an additional PvuI site.
The S. cerevisiae GALbd domain is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The mutated hA20 (hA20S565A) does not interact with 14-3-3 proteins.
The GALbd-HA-hA20S565A fusion protein can be used as a bait protein in two-hybrid screening.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727382.1.
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 hA20 coding sequence corresponds with the Genbank accession number M59465.1.
Other name of the plasmid is pAS2HuA20S565A.
EMBL Accession number: M59465.1, view at EMBL, GenBank, DDBJ
LT727382.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 05/02/2004
Sequence detail:
Mutator oligonucleotide used:       5' GTCACCAGCGATCGAAGGCAGATCCCTCG 3'
                                               PvuI

The nucleotide sequence of the fusion gene:

               ------------------ GALbd ----------------->
5' ... CTGAAAG.ATG.AAG.CTA.CTG.TCT ... CAG.TTG.ACT.GTA.TCG.CCG.GAA.TTC.ATG.GCT.
               Met Lys Leu Leu Ser     Gln Leu Thr Val Ser Pro Glu Phe Met Ala
               ***                                             EcoRI

       ----------- HA epitope ----------->                     ---------------
       TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.AGC.TTG.GGT.GGT.CAT.ATG.GCT.GAA.CAA
       Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly His Met Ala Glu Gln
                                       NheI                NdeI

       ---------------- hA20m ------------------------------->
                               565    
       GTC ... CAG.CGA.TCG.AAG.GCA.GAT ... CAG.ATG.TAT.GGC.TAA.CCGGAAA ... 3'
       Val     Gln Arg Ser Lys Ala Asp     Gln Met Tyr Gly +++
                   PvuI
                     ^   ^     ^

***: start codon.
+++: termination codon.
^  : mutated nucleotide.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglII, EcoNI and NaeI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr R. Beyaert(1)(2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: De Valck et al., Biochem. Biophys. Res. Commun. 238 (1997), 590-594 [PMID: 9299557]
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
Other culture collection numbers: -

Refer in your Materials and Methods:

pAS2hA20S565A (LMBP 3772) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert and was published in De Valck et al., 1997.

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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