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GeneCorner plasmid details

Last data update: 21 October 2020 04:26 CEST

Plasmid name: pAG25-AOX1-MFmds1E393A (LMBP 5073)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p5073.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Trichoderma reesei 1,2-α-D-mannosidase cDNA (mds1); mutated sequence
Promoter: Eremothecium gossypii translation elongation factor 1α promoter (TEF1α)
Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Ribosome
binding site:
-
Terminator: Eremothecium gossypii translation elongation factor 1α terminator (TEF1α)
Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Selection marker: Ampicillin (amp)
Nourseothricin (ntc)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Pichia pastoris; integrative
Parental clone: pAG25; pPP1MFmds1(9-10)
Further information: The plasmid was created by opening the pAG25 vector with the enzymes BglII and PvuII. The expression cassette for the mutated Trichoderma reesei 1,2-α-D-mannosidase cDNA (mds1) was isolated via a BglII/EcoRV digest on plasmid pPP1MFmds1(M9-10) and ligated sticky-blunt into the opened pAG25 plasmid. Plasmid pPP1MFmds1(M9-10) was derived from pPP1MFmds1 via a site-directed mutagenesis, resulting in the replacement of Glu393 (codon GAG) into Ala (codon GCG). So, an additional NaeI (NgoMIV) site was also created.
The codon replacement results in an inactive variant of the T. reesei alpha-1,2-mannosidase.
The nucleotide sequence of the T. reesei mds1 cDNA was obtained from Genbank (Accession number AF212153.1), with codon 116 encoding for isoleucine (I) instead of serine (S); consequently, the BamHI site at this position is absent. Furthermore, taking the numbering of Genbank into account, Glu413 (codon GAG) is replaced by Ala (codon GCG), resulting in the mutation designation E413A instead of E393A.
Other name of the plasmid is pAG25-AOX1-MFmds1(M9-10).
EMBL Accession number: AF212153.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 20/09/2005
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) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Related plasmid reference: Maras et al., J. Biotechnol. 77 (2000), 255-263 [PMID: 10682284]
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)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

Refer in your Materials and Methods:

pAG25-AOX1-MFmds1E393A (LMBP 5073) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr N. Callewaert .

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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