Last data update: 31 October 2020 04:21 CET
Plasmid name: pCD-F-bio-Ub-K6 (LMBP 6598)
|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
FLAG epitope tag; N-terminal
Biotinylation sequence (bio-tag); N-terminal
|Promoter:||Human cytomegalovirus immediate early promoter (CMV-IE) and enhancer
Phage T7 gene 10 promoter (T7g10)
Simian virus 40 early promoter (SV40 early)
Escherichia coli lac operon promoter
|Terminator:||Bovine growth hormone polyadenylation signal (BGH polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)
Neomycin (neo; G418)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||pCD-F-bio-MK-N149; pET-His-AU-Ub-K6|
|Further information:||The plasmid was constructed by PCR amplifying the human UBB cds from pET-His-AU-Ub-K6 and ligating it as a BamHI/ApaI fragment into Ubiquitin was PCR amplified and ligated intopCD-F-bio-MK-N149, replacing the human MALT1 cds.
This plasmid is generated for high-affinity isolation of ubiquitinated proteins of a specific linkage. Biotinylation requires co-transfection of a mammalian expression vector with BirA. For even greater specificity, this plasmid can be combined with the corresponding His-tagged ubiquitin for 2D (first His-purification, then biotin precipitation) isolation of ubiquitinated proteins under denaturing conditions, for example for proteomic studies.
|EMBL Accession number:||-|
|Latest sequence update:||04/02/2010|
Primers used to amplify Ubiquitin: Forward: 5' TGAGGATCCT.ATG.CAG.ATC.TTC.GTG BamHI *** 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.|
|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
|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:||-|
Refer in your Materials and Methods:
|pCD-F-bio-Ub-K6 (LMBP 6598) 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.