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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPSDalpha-FLAG-stuffer-V5-SAG1 (LMBP 9523)

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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
Saccharomyces cerevisiae alpha-agglutinin cDNA (SAG1, AG(ALPHA)1, GeneID 853460); C-terminal fragment
Promoter: Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Synthetic prokaryotic EM7 promoter
Saccharomyces cerevisiae translation elongation factor 1α promoter (TEF1α)
Ribosome
binding site:
-
Terminator: Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1)
Selection marker: Zeomycin (zeo)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Pichia pastoris; integrative
Parental clone: pPSDalphaSfiI/SfiI
Further information: The plasmid was constructed by cloning a 1.5 kb stuffer DNA fragment into the SfiI/PacI opened pPSDalphaSfiI/SfiI vector.
The plasmid is intended for surface display of proteins in P. pastoris using the S. cerevisiae SAG1 protein.
Protein expression is controlled by the methanol-induced AOX1 promoter.
The protein of interest is cloned behind a preproMF secretion signal and the N-terminal FLAG tag, and in front of the V5 tag and the SAG1 protein, using SfiI-PacI restriction enzymes to replace the stuffer DNA.
Integration into the AOX1 promoter can be obtained via linearisation with MssI or PmeI.
Other name of the plasmid is pPSDzeoSfiIPacI_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)
Related plasmid reference: Ryckaert et al., J. Biotechnol. 145 (2010), 93-98 [PMID: 19861136] [DOI: 10.1016/j.jbiotec.2009.10.010]
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 DH5α
Host reference: Focus 8 (1986), 9
Related host reference: Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
Cultivation medium: LB-Lennox + zeocin (25 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
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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