Last data update: 28 October 2020 04:17 CET
Plasmid name: pAS2-hABIN1-P299A (LMBP 5134)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1, NAF1); 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
|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 1928 bp BamHI-XhoI PCR fragment, containing the human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) mutated sequence. As a result, the hABIN-1 mutated 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-1m fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-hABIN1-P299A 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.
Other name of the plasmid is pAS2-hABIN1-FLXP.
|EMBL Accession number:||NM_006058.2, view at GenBank|
|Latest sequence update:||05/01/2006|
Primers used to amplify the hABIN-1 coding sequence: forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAA.GGG.AGA.GGA.CCG ... 3' BamHI NotI *** reverse: 5' ... GCCGCTCGAG.TCA.CTG.AGG.CCC.CTC.ACG ... 3' XhoI +++ 7 9 11 297 299 301 WT (NM_006058): 5' ... TAC.CGG.ATC.TAC.GAC ... GGC.GCA.CCC.GAG.AAG ... 3' Tyr Arg Ile Tyr Asp Gly Ala Pro Glu Lys DpnI AvaI Sau3AI 7 9 11 297 299 301 P299A mutant: 5' ... TAC.CGG.ATT.TAC.GAC ... GGC.GCA.GCC.GAG.AAG ... 3' Tyr Arg Ile Tyr Asp Gly Ala Ala Glu Lys ^ ^ ***: 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-P299A (LMBP 5134) 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.