Last data update: 24 January 2024 16:39 CET
Plasmid name: pMSCV-His-AU-hUb-K48 (LMBP 6802)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p6802.gb |
| 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: | - |
Refer in your Materials and Methods: |
pMSCV-His-AU-hUb-K48 (LMBP 6802) 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.