GREAT AT SMALL THINGS

0

GeneCorner plasmid details

Last data update: 25 October 2020 04:11 CET

Plasmid name: pCD-F-bio-hUb-K33 (LMBP 6693)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p6693.gb
Sequence
analysis results
Genecorner:

-

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
Ribosome
binding site:
-
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: pET3E-His-AU1-hUb-K33; pCD-F-bio-MK-N149
Further information: The plasmid was constructed by PCR amplifying the human UBB coding sequence from pET3E-His-AU1-hUb-K33 and ligating it as a BamHI/ApaI fragment into pCD-F-bio-MK-N149, replacing the human MALT1 coding sequence.
The human ubiquitin B K33 cDNA only encodes the wild type lysine at position 33; the other six lysine codons at positions 6, 11, 27, 29, 48 and 63 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-K33.
EMBL Accession number: BT020104.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 10/06/2010
Sequence detail:
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.
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: -

Refer in your Materials and Methods:

pCD-F-bio-hUb-K33 (LMBP 6693) 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.

New search