Last data update: 28 November 2020 04:24 CET
Plasmid name: pMSCV-His-AU-hUb-7R (LMBP 6800)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human ubiquitin B cDNA (UBB, Ub, HEL-S-50, GeneID 7314); mutated sequence
Histidine tag (His-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
|Terminator:||Mouse stem cell PCMV virus long terminal repeat (3' MSCV-PCMV LTR)|
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli; preferably recombination-deficient strains
|Parental clone:||pMSCVpuro; pET3E-His-AU1-hUb-7R|
|Further information:||The plasmid was constructed by PCR amplifying the mutated human UBB coding sequence from pET3E-His-AU1-hUb-7R and ligating it into the HindIII/ApaI opened pMSCVpuro plasmid.
In the human UBB 7R cDNA all seven lysine codons at positions 6, 11, 27, 29, 33, 48 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-7R.
|EMBL Accession number:||BT020104.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||29/10/2010|
Primers used to amplify the human UBB coding sequence: Forward: 5' CATAAGCTTAAA.ATG.GTA.CAT.CAC.CAT.CAC.CAT.CAC HindIII Reverse: 5' TAAGGGCCC.CTA.CCC.ACC.TCT.GAG.AC ApaI
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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
|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)|
|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.