Last data update: 21 October 2020 04:26 CEST
Plasmid name: pAG25-AOX1-MFmds1E393A (LMBP 5073)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|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)
|Terminator:||Eremothecium gossypii translation elongation factor 1α terminator (TEF1α)
Pichia pastoris alcohol oxidase 1 terminator (AOX1)
|Selection marker:||Ampicillin (amp)
|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.|
|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
|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)|
|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.