Last data update: 23 July 2021 09:43 CEST
Plasmid name: pAS2-hABIN1-N (LMBP 5135)
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|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
Human A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1, NAF1); mutated N-terminal fragment|
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
|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)
|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(-)
|Further information:||The plasmid was constructed by ligating the three following fragments: 1) the 5686 bp SalI-SacI fragment from pAS2. 2) the 2721 bp SacI-BamHI fragment from pAS2. 3) a 1145 bp BamHI-XhoI PCR fragment, containing the N-terminal fragment of the human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) coding sequence. As a result, the hABIN-1-N coding sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
As compared to the wt hABIN-1 cDNA, the proline (P) codon at position 299 is replaced by an alanine (A) codon resulting in the loss of an AvaI site and the isoleucine (I) codon at position 9 contains a silent mutation (atc -> att) resulting in the loss of a DpnI and a Sau3AI site.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-hABIN-1-N fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-hABIN1-N 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.
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 hABIN-1 cDNA corresponds with the Genbank accession number NM_006058.2.
|EMBL Accession number:||NM_006058.2, view at GenBank|
|Latest sequence update:||05/01/2006|
Primers used to amplify the mutated N-terminal fragment of the hABIN-1 coding sequence: 1 5 forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAA.GGG.AGA.GGA.CCG ... 3' BamHI NotI *** 375 370 reverse: 5' ... GCCGCTCGAG.TCA.TTC.AAT.CTT.GGA.CTT.GGC ... 3' XhoI +++ 9 299 WT (NM_006058): 5' ... CGG.ATC.TAC ... GCA.CCC.GAG ... 3' Arg Ile Tyr Ala Pro Glu DpnI AvaI Sau3AI 9 299 mutant: 5' ... CGG.ATT.TAC ... GCA.GCC.GAG ... 3' Arg Ile Tyr Ala Ala Glu ^ ^ ***: start codon. +++: termination codon. ^ : mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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
|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)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pAS2-hABIN1-N (LMBP 5135) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert .|
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.