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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPCAzeo-FLAG-stuffer-V5 (LMBP 10237)

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

-

Cloned DNA: Saccharomyces cerevisiae α-mating factor 1 gene (MFα1, GeneID 855914); prepro secretion signal sequence (ppMF)
Stuffer fragment
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
Ribosome
binding site:
-
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
Parental clone: pPSDalpha-FLAG-stuffer-V5-SAG1
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.
Class: Recombinant plasmid
Type: Plasmid
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)
Cultivation temperature: 37°C
Biosafety level: L1
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.

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