Last data update: 30 October 2020 04:11 CET
Plasmid name: pCD-F-bio-hUb-K63 (LMBP 6796)
|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; pET3E-His-AU1-hUb-K63|
|Further information:||The plasmid was constructed by PCR amplifying the human UBB coding sequence from pET3E-His-AU1-hUb-K63 and ligating it as a BamHI/ApaI fragment into pCD-F-bio-MK-N149, replacing the human MALT1 coding sequence.
The human UBB K63 cDNA only encodes the wild type lysine at position 63; the other six lysine codons at positions 6, 11, 27, 29, 33 and 48 are mutated to arginine codons.
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.
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 pCD-F-bio-Ub-K63.
|EMBL Accession number:||BT020104.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||29/10/2010|
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 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:||-|
Refer in your Materials and Methods:
|pCD-F-bio-hUb-K63 (LMBP 6796) is available at BCCM/GeneCorner. This plasmid was deposited by Dr J. Staal and Prof. 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.