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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMSCV-His-AU-hUb-K48 (LMBP 6802)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: -
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: Escherichia coli; preferably recombination-deficient strains
Mammalian cells
Parental clone: pMSCVpuro; pET3E-His-AU1-hUb-K48R
Further information: The plasmid was constructed by PCR amplifying the mutated human UBB coding sequence from pET3E-His-AU-hUb-K48 and ligating it into the HindIII/ApaI opened pMSCVpuro plasmid.
The human UBBB K48 mutant only encodes the wild type lysine at position 48; the other six lysine codons at positions 6, 11, 27, 29, 33 and 63 are mutated to arginine codons.
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.
The nucleotide sequence of the wild type human UBB coding sequence corresponds with the EMBL Nucleotide Sequence Database accession number BT020104.1, except for some silent mutations.
Other name of the plasmid is pMSCV-His-AU-Ub-K48.
EMBL Accession number: BT020104.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 29/10/2010
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 Dr J. Staal(1) (2) and Prof. R. Beyaert(1) (2).
(1) VIB-UGent Center for Inflammation 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
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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