GREAT AT SMALL THINGS

0

GeneCorner plasmid details

Last data update: 27 November 2020 04:15 CET

Plasmid name: pAS2-hOMI-S306A (LMBP 4578)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4578.gb (View with Genome Compiler)
p4578.txt
p4578.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human serine protease cDNA (OMI, HTRA2); 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: pAS2
Further information: The plasmid was constructed by inserting a 1386 bp NdeI-Sfil PCR fragment, containing the mutated, full-length hOMI coding sequence, into the NdeI-SfiI opened pAS2 vector. As a result, the mutated hOMI coding sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
The full-length hOMI coding sequence includes an N-terminal mitochondrial targeting sequence.
The serine (S) codon at position 306 of the 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-hOMI-S306A fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-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 LT727107.1.
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
LT727107.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 18/09/2007
Sequence detail:
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 ----------->                     ------- 
                                                                       1
       ATG.GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.AGC.TTG.GGT.GGT.CAT.ATG.GCT
       Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly His Met Ala
                                                                   NdeI


       ----------------------------------- hOMIm -----------------------------
                               134                             306
       GCG.CCG.AGG.GCG ... GCC.GCC.GTC.CCT.AGC.CCG ... GGA.AAC.GCC.GGC.GGT ... 
       Ala Pro Arg Ala     Ala Ala Val Pro Ser Pro     Gly Asn Ala Gly Gly
                                A   V   P   S                  ^ ^   ^
                                                               NaeI

       ---------------------->
                       458
       CCT.GAG.GTC.ACA.GAA.TGA.GGCCATGGAGGCCCCGGGGATCCGTCGACCTGCAGCCAAGC ... 3'
       Pro Glu Val Thr Glu +++ SfiI        AvaI BamHI SalI  PstI
                                BglI       SmaI       HindII
                                 NcoI
                                 StyI 

***: 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.
Class: Recombinant plasmid
Type: Plasmid
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
Plasmid reference: -
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)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

Refer in your Materials and Methods:

pAS2-hOMI-S306A (LMBP 4578) 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.

New search