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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMSCV-His-AU-Ub-K63 (LMBP 6594)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p6594.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human ubiquitin B cDNA (UBB, Ub, HEL-S-50, GeneID 7314); mutated sequence
Histidine tag (His-tag); N-terminal
AU1-tag; N-terminal
Promoter: Mouse stem cell PCMV virus long terminal repeat (5' MSCV-PCMV LTR)
Mouse phosphoglycerate kinase 1 promoter (PGK1)
Escherichia coli lac operon promoter
Ribosome
binding site:
-
Terminator: Mouse stem cell PCMV virus long terminal repeat (3' MSCV-PCMV LTR)
Selection marker: Ampicillin (amp)
Puromycin (puro)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Mammalian cells
Parental clone: pMSCVpuro; pET-His-AU-Ub-K63
Further information: The plasmid was constructed by PCR amplifying the mutated human UBB cds from pET-His-AU-Ub-K63 and ligating it into the HindIII/ApaI opened pMSCVpuro plasmid.
This plasmid is generated for high-affinity isolation of ubiquitinated proteins of a specific linkage. For even greater specificity, this plasmid can be combined with the corresponding bio-tagged ubiquitin for 2D (first His-purification, then biotin precipitation) isolation of ubiquitinated proteins under denaturing conditions, for example for proteomic studies. Denaturing (8M Urea, 2% SDS) conditions also ensures that no deubiquitinase activity is present during purification.
EMBL Accession number: -
Latest sequence update: 04/02/2010
Sequence detail:
Primers used to amplify the His-tagged human UBB:

Forward: 5' CATAAGCTTAAA.ATG.GTA.CAT.CAC.CAT.CAC.CAT.CAC
               HindIII   ***
Reverse: 5' TAAGGGCCC.CTA.CCC.ACC.TCT.GAG.AC
               ApaI   +++

***: start codon.
+++: 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: -
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: -

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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