Last data update: 19 October 2020 04:30 CEST
Plasmid name: pCAGGS-EmCASP-1m3 (LMBP 3913)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Mouse cysteinyl aspartate specific proteinase 1 cDNA (caspase-1, CASP-1, ICE, Casp1); p45-C285S-2DE mutant
|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 mouse caspase-1 coding sequence contains the following point mutations (p45-C285S-2DE): (i) two Asp (D) codons of the prodomain were converted to Glu (E) to destroy potential cleavage sites; (ii) the Cys codon in the active site of the conserved QACRG sequence was converted to Ser (QASRG); and (iii) additional BalI (MscI), KpnI and NheI restriction sites were created.
The Cys conversion is described as C285S, according to the numbering of the codons in the human caspase-1 coding sequence, although the mutated mouse serine is located on position 284.
pCAGGS-EmCASP-1m3 is useful for highly efficient expression of mutated mouse caspase-1 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 LT727285.1.
The nucleotide sequence of the wt mCASP-1 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number BC008152.1.
Other name of the plasmid is pCAGGSEICECYSSERASP.
|EMBL Accession number:||BC008152.1, view at EMBL, GenBank, DDBJ
LT727285.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||24/10/2001|
E-tag --------------------------------------------------- 5' ... GAATTCCACC ATG.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAC.CCG.CTG.GAA.CCG.CGT.GCG.GCC. Met Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala EcoRI BspMII NotI |--> mouse CASP-1 | 2 3 4 100 103 110 GCT.GAC.AAG...TTT.GTT.GCT.ACA.GAA.GAA.TCT.AAA.GGT.GGC.CAT.CCT.TCA.TCC.TCA.GAA Ala Asp Lys Phe Val Ala Thr Glu Glu Ser Lys Gly Gly His Pro Ser Ser Ser Glu --* * * E BalI (MscI) prodomain --> | --> p30 120 122| ACA.AAG.GAA.GAA.CAG.AAC.AAA.GAA.GAG.GGT.ACC.TTT.CCA.GGA.CTG.ACT. ... 3' Thr Lys Glu Glu Gln Asn Lys Glu Glu Gly Thr Phe Pro Gly Leu Thr --* * * E KpnI Sequence of the inactivated (mutated) proteolytic activity site: 280 284 ...GAC.AAG.CCC.AAG.GTG.ATC.ATT.ATT.CAA.GCT.AGC.CGT.GGA.GAG.AAA.CAA... Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Ser Arg Gly Glu Lys Gln * * *-- Q A S R G NheI *: mutated nucleotide. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: KpnI, NcoI and NheI.|
|History of deposit:||This plasmid was deposited by P. Schotte(1) (2) and 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
|Related plasmid reference:||Lamkanfi et al., J. Biol. Chem. 279 (2004), 24785-24793 [PMID: 15039421]
Van Criekinge et al., J. Biol. Chem. 271 (1996), 27245-27248 [PMID: 8910297]
|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:
|pCAGGS-EmCASP-1m3 (LMBP 3913) is available at BCCM/GeneCorner. This plasmid was deposited by P. Schotte and 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.