Last data update: 31 October 2020 04:21 CET
Plasmid name: pAS2-AVPS-hOMI-S306A (LMBP 4579)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human serine protease cDNA (OMI, HTRA2); mutated mature 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 inserting a 990 bp NdeI-Sfil PCR fragment, containing the mutated hOMI mature sequence, into the NdeI-SfiI opened pAS2 vector. As a result, the mutated hOMI mature sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
The hOMI insert does not include a mitochondrial targeting sequence.
The first four amino acids of the mature human Omi/HtrA2 protein are alanine, valine, proline and serine (AVPS).
The serine (S) codon at position 306 of the full-length hOMI coding sequence was replaced by an alanine (A) codon, creating an additional NaeI site.
The human Omi/HtrA2 serine point mutant is no longer cytotoxic.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-AVPS-hOMI-S306A fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-AVPS-hOMI-S306A 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.
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 LT727108..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 hOMI cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF141305.1.
|EMBL Accession number:||AF141305.1, view at EMBL, GenBank, DDBJ
U30496.1, view at EMBL, GenBank, DDBJ
LT727108..1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||18/09/2007|
Nucleotide sequence of the fusion gene: ---------------------- GALbd ---------------------> 1 147 5' ... CTGAAAG.ATG.AAG.CTA.CTG.TCT.TCT ... AGA.CAG.TTG.ACT.GTA.TCG.CCG.GAA.TTC Met Lys Leu Leu Ser Ser Arg Gln Leu Thr Val Ser Pro Glu Phe *** HindII EcoRI ----------- HA epitope -----------> ------- 134 ATG.GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.AGC.TTG.GGT.GGT.CAT.ATG.GCC Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly His Met Ala NdeI A -------------------------- hOMIm ---------------------------------> 306 458 GTC.CCT.AGC.CCG ... GGA.AAC.GCC.GGC.GGT ... CCT.GAG.GTC.ACA.GAA.TGA.GGC Val Pro Ser Pro Gly Asn Ala Gly Gly Pro Glu Val Thr Glu +++ SfiI V P S ^ ^ ^ BglI NaeI NcoI StyI CATGGAGGCCCCGGGGATCCGTCGACCTGCAGCCAAGC ... 3' AvaI BamHI SalI PstI SmaI HindII ***: start codon. +++: termination codon. ^ : mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: BglI/SphI, Csp45I/NheI, EcoRI, EcoRV/XbaI, NdeI/SpeI, SalI and SfiI/StuI.|
|History of deposit:||This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2). It was constructed by G. Van Loo(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Van Loo et al., Cell Death Differ. 9 (2002), 20-26 [PMID: 11803371]
Vande Walle et al., J. Proteome Res. 6 (2007), 1006-1015 [PMID: 17266347]
Suzuki et al., Mol. Cell 8 (2001), 613-621 [PMID: 11583623]
|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-AVPS-hOMI-S306A (LMBP 4579) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr P. Vandenabeele .|
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.