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GeneCorner plasmid details

Last data update: 03 December 2020 04:26 CET

Plasmid name: pAS2mCASP-11m (LMBP 3922)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p3922.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse cysteinyl aspartate specific proteinase 11 cDNA (caspase-11, CASP-11, Ich3, Casp4); 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 as follows: the plasmid pAS2 was opened with BalI(MscI)/SalI and the mutated mouse cysteinyl aspartate specific proteinase 11 cDNA (caspase-11, mCASP-11) was inserted by the use of primers.
mCASP-11 contains a C254S mutation. The cysteine codon at position 254 was replaced by a serine codon, creating an additional NheI site.
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.
Other name of the plasmid is pAS2PROCASP-11M.
EMBL Accession number: U30496.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 06/02/2004
Sequence detail:
Nucleotide sequence around the HA epitope and the mutated mouse CASP-11 coding sequence:


  GALbd -> 
       147                     ----------- HA epitope ----------->
5' ... TCG.CCG.GAA.TTC.ATG.GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.AGC.TTG.GGT.GGT.
       Ser Pro Glu Phe Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly
               EcoRI                                           NheI


                       ------------------------ mCASP-11m ------------------------>
                       1                            254                         373
   CAT.ATG.GCC.ACC.ACC.ATG.GCT.GAA.AAC.AAA  ... GCT.AGC.AGA ... TTC.TTT.CCT.GGC.AAC.
   His Met Ala Thr Thr Met Ala Glu Asn Lys      Ala Ser Arg     Phe Phe Pro Gly Asn
        BalI        NcoI                        NheI
                    StyI                          ^ ^
                           

   GTC.GAC.CTG.CAG.CCA.AGC.TAA.TTCCGGGCG ... 3'
   Val Asp Leu Gln Pro Ser +++
   SalI    PstI
   HindII


^  : Mutated nucleotide.
+++: Termination codon.
Punctuation indicates reading frame.
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
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]
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:

pAS2mCASP-11m (LMBP 3922) 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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