Last data update: 26 November 2020 04:30 CET
Plasmid name: pAS2-hABIN1-C (LMBP 5136)
|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); C-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 803 bp BamHI-XhoI PCR fragment, containing the C-terminal fragment of the human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) coding sequence. As a result, the hABIN-1-C 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-hABIN-1-C fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-hABIN1-C 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 C-terminal fragment of the hABIN-1 coding sequence: 376 380 forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAG.GAG.ACC.GAC.AAG.G ... 3' BamHI NotI *** 636 635 reverse: 5' ... GCCGCTCGAG.TCA.CTG.AGG.CCC.CTC.ACG ... 3' XhoI +++ ***: start codon. +++: termination codon. 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-C (LMBP 5136) 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.