Last data update: 24 January 2024 16:39 CET
Plasmid name: pPSDzeo-FLAG-stuffer-V5-SAG1 (LMBP 9523)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p9523.gb
(View with Genome Compiler) p9523.txt p9523.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Saccharomyces cerevisiae α-mating factor 1 gene (MFα1, GeneID 855914); prepro secretion signal sequence (ppMF) FLAG epitope tag; N-terminal Human galactosidase alpha cDNA (GLA, GALA, GeneID 2717); mature sequence, codon optimised Myc epitope; C-terminal Histidine tag (His-tag); C-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: | Bleomycin (bleo; zeomycin (zeo; Zeocin); phleomycin (phleo)) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli Pichia pastoris; integrative |
| Parental clone: | pPICZ-aGalA-MycHis; pPSDzeoSfiI/PacI |
| Further information: | The plasmid was constructed by cloning a 1.5 kb stuffer DNA fragment from pPICZ-aGalA-MycHis into the SfiI/PacI opened pPSDzeoSfiI/PacI 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 ppMF 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. The stuffer DNA consists of the ppMF, the synthetic human GLA mature sequence, codon optimised for P. pastoris, and the C-terminal Myc epitope and His-tag. 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: | Restriction enzyme pattern analysed at GeneCorner: BamHI, BglI/NotI and NcoI. |
| 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: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] 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 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pPSDzeo-FLAG-stuffer-V5-SAG1 (LMBP 9523) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr N. Callewaert and was published in Morgane Boone, 2017. |
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.