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GeneCorner plasmid details

Last data update: 29 November 2020 04:28 CET

Plasmid name: pAS2-mRIP1-KD-ID (LMBP 6735)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p6735.gb (View with Genome Compiler)
p6735.txt
p6735.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse receptor TNFRSF-interacting serine-threonine kinase 1 cDNA (Ripk1, Rip1, Rinp, GeneID 19766); kinase domain and intermediate domain (Ripk1-KD-ID)
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-mRIP1; pGEM-RIP
Further information: The plasmid was constructed by PCR amplifying the kinase domain and the intermediate domain of the mRIP1 coding sequence from pGEM-RIP and ligating it as an NheI/BamHI fragment into the BamHI-NheI opened pAS2-mRIP1 vector, replacing the mRIP1 cDNA. As a result, the mRIP1-KD-ID coding sequence was fused in phase to the N-terminal GAL4 DNA-binding domain and HA epitope.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-mRIP1-KD-ID fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-mRIP1-KD-ID-2 carries the S. cerevisiae tryptophan 1 coding sequence (TRP1) and the CYH2 sequence encoding ribosomal protein L29 for dominant cycloheximide sensitivity which facilitates the elimination of false positives.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726842.1.
The nucleotide sequence of the pAS2 vector corresponds with the EMBL Nucleotide Sequence Database accession number U30496.1, except for the following adaptations: 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.
The nucleotide sequence of the mouse RIP1 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number BC058162.1.
Other name of the plasmid is pAS2-mRIP1-KD-ID-2.
EMBL Accession number: BC058162.1, view at EMBL, GenBank, DDBJ
U30496.1, view at EMBL, GenBank, DDBJ
LT726842.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 25/02/2009
Sequence detail:
Primers used for isolating the kinase domain and intermediate domain of mouse RIP1 cDNA:

Forward: 5' GGTACGCTAGC.ATG.CAA.CCA.GAC.ATG.TCC 3'
                 NheI   ***

Reverse:  5' CGCGGATCC.CGA.CTC.TTC.TTT.GCA.AGT 3'
                BamHI


Nucleotide sequence at the 3' end of the mRIP1-KD-ID cDNA:

GAG.TCG.GGA.TCC.GTC.GAC.CTG.CAG.CCA.AGC.TAA.TTCCGGGCGAATTTCTTATGATTT
mRIP1-> BamHI                           +++       -> ADH1 terminator

***: start codon.
+++: termination codon.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BamHI/NheI, BglII, EcoRI and XmnI.
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: -
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:

pAS2-mRIP1-KD-ID (LMBP 6735) 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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