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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPP1mds1 (LMBP 4484)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4484.gb (View with Genome Compiler)
p4484.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Trichoderma reesei 1,2-α-D-mannosidase cDNA (mds1)
Promoter: Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Ribosome
binding site:
-
Terminator: Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Selection marker: Ampicillin (amp)
Pichia pastoris HIS4; auxotrophic
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Pichia pastoris; his4(-), integrative
Parental clone: pPIC9; pGEM4mds1
Further information: The vector was constructed as follows: 1) The BglII fragment from pAJ401mds1, containing the intact Trichoderma reesei 1,2-α-D-mannosidase coding sequence (mds1), was inserted into the BamHI opened pGEM4 vector. 2) This intermediate plasmid pGEM4mds1 was treated with EcoRI. This EcoRI fragment was isolated and partially digested with PvuII (PvuI in Maras et al. (2000) is a typing error). 3) The resulting fragment, still containing the intact mds1 coding sequence, was then inserted into the BamHI(Klenow)-EcoRI opened pPIC9 vector.
This plasmid is useful in P. pastoris strains for secreted expression of the mds1 gene under control of the strong, highly-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter.
The presence of the P. pastoris HIS4 gene and two P. pastoris AOX1 regions (the AOX1 promoter and the AOX1 3' flanking region) allows integration of the linearized vector into the Pichia genome via homologous recombination at the his4 locus or AOX1 locus respectively.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727222.1.
The nucleotide sequence of the T. reesei mds1 cDNA corresponds with the Genbank accession number AF212153.1. Be aware that the resulting amino acid sequence differs from the one described by Maras et al. (2000). Furthermore, additional amino acid changes have been noticed by a BCCM/GeneCorner customer (02/09/02). Some of the latter changes could be confirmed by the latest sequence analysis results obtained with this GeneCorner plasmid by Dr Maras (11/03/03), others couldn't. Referring to the customer's sequence analysis, mds1 codon 116 encodes for isoleucine (I) instead of serine (S); consequently, the BamHI site at this position is absent. This confirms the results of our authenticity test.
Other name of the plasmid is pPIC9mannosidase.
EMBL Accession number: AF212153.1, view at EMBL, GenBank, DDBJ
LT727222.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 29/08/2006
Sequence detail:
Overview of the amino acid differences of the mds1 coding sequence:

AA position     EMBL     Maras2000     Customer     Maras2003

116             S        S             I            S
292             G        G             A            A
298             G        G             A            A
301             G        G             A            A
332             G        G             A            G
361             G        G             A            G
364             T        N             N            N
438             L        F             F            F
496             T        N             N            N
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BamHI/BglII, BglI/EcoRV, EcoRI/XhoI, NcoI, PvuII and StuI/XmnI.

As compared to the compiled nucleotide sequence, the restriction site analysis pattern revealed that BamHI only cuts once instead of twice. The BamHI site at position 1297, corresponding to amino acids 115 and 116 of mds1, could not be experimentally confirmed. As the compiled nucleotide sequence has not been adapted accordingly, BamHI is not indicated as a unique site.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Prof. Dr R. Contreras(1)
(1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
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: -

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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