Last data update: 01 December 2020 04:11 CET
Plasmid name: pCAGGS-FLAG-hUb-K63 (LMBP 5903)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human ubiquitin B cDNA (UBB, Ub, HEL-S-50, GeneID 7314); K63 mutated mature sequence
FLAG epitope tag; N-terminal
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||pET3E-His-AU1-hUb-K63; pCAGGS-FLAGmA20|
|Further information:||The plasmid was constructed as follows: 1) the human ubiquitin B mutated mature sequence was PCR amplified on the template pET3E-His-AU1-hUb-K63; 2) the KpnI and partially SalI digested amplicon was then inserted into the KpnI and SalI opened pCAGGS-FLAGmA20 plasmid.
The human ubiquitin B 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.
pCAGGS-FLAG-hUb-K63 is useful for highly efficient expression of human Ub-K63 under the control of the AG promoter and the human CMV-IE enhancer in various mammalian cells.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726812.1.
The nucleotide sequence of the human Ub insert, which was the result of the sequence analysis at BCCM/GeneCorner, differs from the nucleotide sequence obtained from the EMBL Nucleotide Sequence Database accession number BT020104.1 in some silent mutations next to the 6 K->R mutations.
|EMBL Accession number:||BT020104.1, view at EMBL, GenBank, DDBJ
LT726812.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||03/02/2009|
Primers used to amplify hUb-K63: sense: 5' gatgggtaccgcg.ATG.CAG.ATC.TTC.GTG.A 3' KpnI *** antisense: 5' gtagcgtcgac.CTA.CCC.ACC.TCT.GAG.ACG.C 3' SalI +++ ^ ^ : 'G' according to the depositor's information, but 'C' according to our sequence analysis results. ***: start codon. +++: termination codon. Punctuation indicates reading frame. Schematic representation of the hUb-K63 mutant: 6 11 27 29 33 48 63 wild type: 5' --K--K--K--K--K--K--K-- 3' K63 mutant: 5' --R--R--R--R--R--R--K-- 3'
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: Acc65I/SalI, Alw44I/ApaI, AvaII/HindIII, NcoI/PvuI and PvuII.
The ApaI site at position 3778 could not be experimentally confirmed.
|History of deposit:||This plasmid was deposited by Prof. Dr R. Beyaert(1) (2). It was constructed by B. Coornaert(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:
|pCAGGS-FLAG-hUb-K63 (LMBP 5903) 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.