Last data update: 24 November 2020 04:18 CET
Plasmid name: pPCAzeo-FLAG-stuffer-V5 (LMBP 10237)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Saccharomyces cerevisiae α-mating factor 1 gene (MFα1, GeneID 855914); prepro secretion signal sequence (ppMF)
FLAG epitope tag; N-terminal
V5 epitope; C-terminal
|Promoter:||Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Saccharomyces cerevisiae translation elongation factor 1α promoter (TEF1α)
Synthetic prokaryotic EM7 promoter
|Terminator:||Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1)
|Selection marker:||Zeocin (zeo)|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli
Pichia pastoris; integrative
|Further information:||The plasmid was constructed by cloning a 1.5 kb stuffer fragment between the SfiI and PacI sites of pPSDalpha-FLAG-stuffer-V5-SAG1, replacing the stuffer fragment and removing the SAG1 coding sequence.
The plasmid is intended for secretable expression of proteins in P. pastoris.
The protein of interest is cloned behind a preproMF secretion signal and the N-terminal FLAG tag, and in front of the V5 tag, using SfiI-PacI restriction enzymes to replace the stuffer DNA.
Protein expression is regulated by the methanol-induced AOX1 promoter.
Integration into the AOX1 promoter can be obtained via linearisation with MssI or PmeI.
Other name of the plasmid is pPCAzeoSfiIPacI-FLAGV5-AOX1-stuffer.
|EMBL Accession number:||-|
|Latest sequence update:||10/08/2017|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by Prof. Dr N. Callewaert(1) (2).
(1) VIB-UGent Center for Medical Biotechnology, VIB, Ghent, Belgium
(2) Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium
|Plasmid reference:||PhD thesis Morgane Boone (2017)
|Restricted distribution:||- BCCM MTA
- The depositor will be informed of the customer's identity upon release of a sample outside the depositor's department or outside the departments in which BCCM/GeneCorner is embedded, namely UGent-DBMB and VIB-IRC.
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 TOP10|
|Host reference:||Invitrogen Instruction Manual
|Related host reference:||Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
|Cultivation medium:||LB-Lennox + zeocin (25 μg/ml)|
|Other culture collection numbers:||-|
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.