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GeneCorner plasmid details

Last data update: 26 October 2020 04:25 CET

Plasmid name: pCAGGSFLAGmCASP-11m2 (LMBP 3972)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3972.gb (View with Genome Compiler)
p3972.txt
p3972.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse cysteinyl aspartate specific proteinase 11 cDNA (caspase-11, CASP-11, Ich3, Casp4); mutated 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
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-FLAGmA20f3
Further information: The plasmid was constructed by inserting a KpnI/SalI PCR amplicon, encoding mutated mCASP-11, in the KpnI/SalI opened pCAGGS-FLAGmA20f3 vector.
The mCASP-11 insert contains a Cys/Ser mutation at codon position 254, creating a unique NheI site.
pCAGGSFLAGmCASP-11m2 is useful for highly efficient expression of mutated mCASP-11 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 LT727297.1.
The nucleotide sequence of the mCASP-11 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number Y13089.1, except for the 3' end of the coding sequence that corresponds with the nucleotide sequence of the reverse primer.
Other names of the plasmid are pCAGGSFLAGCASP-11M and pCAGGSFLAGCASP-11MCys/Ser.
EMBL Accession number: Y13089.1, view at EMBL, GenBank, DDBJ
LT727297.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 07/09/2007
Sequence detail:
Primers used to amplify mutated mCASP-11:

Forward primer:  5' CCATGGGTACC.ATG.GCT.GAA.AAC.AAA.CAC.CCT.GAC 3'
                         KpnI   ***



Reverse primer:  5' ACGCGTCGAC.TCA.GTT.GCC.AGG.AAA.GAC.GTA.GAA.ATA 3'
                        SalI   +++                   |
                                                     G according to EMBL Y13089
                                                    (C results in Val, G in Leu)
***: start codon.
+++: termination codon.
Punctuation indicates reading frame.



Nucleotide sequence of the mutated mCASP-11 cDNA:

               250         253 254
wild type:  5' ATT.GTG.CAG.GCC.TGC.AGA.GGT.GGG.AAC 3'
               Ile Val Gln Ala Cys Arg Gly Gly Asn
  
               250         253 254                    
mutated:    5' ATT.GTG.CAG.GCT.AGC.AGA.GGT.GGG.AAC 3'
               Ile Val Gln Ala Ser Arg Gly Gly Asn
                           NheI
                             ^ ^
^: mutated nucleotide.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglII, EcoRI, NcoI, NdeI, XbaI and XhoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by 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
Plasmid reference: -
Related plasmid reference: Wang et al., Cell 92 (1998), 501-509 [PMID: 9491891]
Schoen et al., PLoS ONE 8 (2013), e56520 [PMID: 23441200] [DOI: 10.1371/journal.pone.0056520]
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:

pCAGGSFLAGmCASP-11m2 (LMBP 3972) is available at BCCM/GeneCorner. This plasmid was deposited by 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.

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