GREAT AT SMALL THINGS

0

GeneCorner plasmid details

Last data update: 23 October 2020 04:27 CEST

Plasmid name: pCAGGS-EmCASP-8-C362A (LMBP 4942)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4942.gb (View with Genome Compiler)
p4942.txt
p4942.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse cysteinyl aspartate specific proteinase 8 cDNA (caspase-8, CASP-8, Casp8, MCH5, MACH, FLICE); C362A mutant
E-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
Ribosome
binding site:
-
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
Parental clone: pCAGGS-HAmCASP-8-C362A; pCAGGS-EmCASP-1
Further information: The plasmid was constructed by inserting the NotI/BamHI fragment of pCAGGS-HAmCASP-8-C362A between the NotI and BglII sites of pCAGGS-EmCASP-1.
This plasmid carries a mouse caspase-8 cDNA mutant, with the cysteine codon in the catalytic site of the conserved QACQG sequence converted to an alanine codon (QAAQG), resulting in an additional MstI site. In Vanden Berghe et al. (2004), this C362A mutation is numbered C347A by mistake.
pCAGGS-EmCASP-8-C362A is useful for highly efficient expression of mutated mouse caspase-8 under the control of the AG promoter and the human CMV-IE enhancer in various mammalian cells. The expressed protein is catalytically inactive.
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 LT727060.1.
The nucleotide sequence of the wild type mouse CASP-8 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AJ007749.1.
Other names of the plasmid are pCAGGS-E-proC8-C/A and pCAGGS-mCASP-8C/A.
EMBL Accession number: AJ007749.1, view at EMBL, GenBank, DDBJ
LT727060.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 27/10/2008
Sequence detail:
Nucleotide sequence at the 5' end, the mutated region and the 3' end of the mouse CASP-8 cDNA:

                      ---------------------- E-tag --------------------->
5' ... GAATTCCACC.ATG.GGT.GCG.CCG.GTG.CCG.TAT.CCG.GAC.CCG.CTG.GAA.CCG.CGT.GCG.GCC.
       EcoRI      *** Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala 
               NcoI                                                       NotI


           -------- mouse CASP-8   
            1               5                      360     362         365  
       GCC.ATG.GAT.TTC.CAG.AGT ... ATC.TTT.TTC.ATT.CAG.GCT.GCG.CAA.GGA.AGT.AAC... 
       Ala Met Asp Phe Gln Ser     Ile Phe Phe Ile Gln Ala Ala Gln Gly Ser Asn
           ***                                             ^^^  
        NcoI                                        Q   A   A   Q   G  
                                                         MstI
                                                         FspI
                        mCASP-8 ->|
       475                 480    |
   ... AAG.CTC.TTC.TTC.CCT.CCC.TAA.GGATCT ... 3'
       Lys Leu Phe Phe Pro Pro +++

***: start codon.                    
+++: termination codon.
^  : mutated nucleotide.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglII, FspI and NotI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr T. Vanden Berghe(1) (2) and Prof. Dr P. Vandenabeele(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: Vanden Berghe et al., J. Biol. Chem. 279 (2004), 7925-7933 [PMID: 14668343]
Related plasmid reference: Maelfait et al., J. Exp. Med. 205 (2008), 1967-1973 [PMID: 18725521] [DOI: 10.1084/jem.20071632]
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:

pCAGGS-EmCASP-8-C362A (LMBP 4942) is available at BCCM/GeneCorner. This plasmid was deposited by Dr T. Vanden Berghe and Prof. Dr P. Vandenabeele and was published in Vanden Berghe et al., 2004.

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