Last data update: 28 October 2020 04:17 CET
Plasmid name: pCAGGS-FLAGAVPShOMI-S306A (LMBP 4843)
|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
FLAG epitope tag; N-terminal
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Further information:||The plasmid was constructed by inserting a KpnI-SalI PCR fragment, containing the mutated hOMI mature sequence, between the KpnI and SalI sites of a pCAGGS-FLAG vector.
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 PdiI site.
The human Omi/HtrA2 serine point mutant is no longer cytotoxic.
pCAGGS-FLAGAVPShOMI-S306A is useful for highly efficient expression of mutated, mature hOMI, fused to the N-terminal FLAG epitope tag, under the control of the AG promoter and the human CMV-IE enhancer in various mammalian cells.
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 LT727047.1.
The nucleotide sequence of the hOMI cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF141305.1.
Other name of the plasmid is pCAGGS-FLAGAVPShOMI-S/A.
|EMBL Accession number:||AF141305.1, view at EMBL, GenBank, DDBJ
LT727047.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||09/02/2004|
Nucleotide sequence of the fusion gene: ----------- ---------- FLAG-tag ----------> 134 5' ... GAATTCCTCGAG.ATG.GAC.TAC.AAG.GAC.GAC.GAT.GAC.AAG.GGT.ACC.ATG.GCC.GTC. EcoRI XhoI Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Thr Met Ala Val *** KpnI A V ---------------------- hOMI mutant ---------------------------> 306 458 CCT.AGC.CCG ... GGA.AAC.GCC.GGC.GGT ... CCT.GAG.GTC.ACA.GAA.TGA.GTCGAC ... 3' Pro Ser Pro Gly Asn Ala Gly Gly Pro Glu Val Thr Glu +++ SalI P S ^ ^ ^ PdiI ***: Start codon. +++: Termination codon. ^ : Mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Alw44I/PdiI, BamHI/PdmI, EcoRI/PstI, HindIII/PvuI, KpnI and NcoI/SalI.|
|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]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061(λ)|
|Host reference:||Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
|Related host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAGGS-FLAGAVPShOMI-S306A (LMBP 4843) 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.