LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPP1MFmds1 (LMBP 4426)

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


Cloned DNA: Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Trichoderma reesei 1,2-α-D-mannosidase cDNA (mds1); mature sequence
Promoter: Pichia pastoris alcohol oxidase 1 promoter (AOX1)
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; pGEM11mds1
Further information: The vector was constructed by inserting a 1.7 kb NarI-NotI fragment of pGEM11mds1, containing the Trichoderma reesei 1,2-α-D-mannosidase (mds1) mature sequence, into the EcoRI-NotI opened pPIC9 vector. The EcoRI and NarI restriction sites were filled in by Klenow DNA polymerase. As a result, the prepro secretion signal sequence (ppMF) of the Saccharomyces cerevisiae α-mating factor 1 gene (MFα1) was fused to the T. reesei mds1 mature sequence.
At the fusion junction, a (Glu-Ala)-dipeptide, as well as additional (but not essential) Tyr-, Val-, Glu-, Phe-, residues are present. During secretion of the protein, the (Glu-Ala)-dipeptide is removed by a dipeptidase.
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 LT727205.1.
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 names of the plasmid are pPICMFmannosidase and pPIC9MFmannase.
EMBL Accession number: AF212153.1, view at EMBL, GenBank, DDBJ
LT727205.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 03/11/2003
Sequence detail:
Nucleotide sequence at the fusion junction:
  ------ ppMF ------->        |       ||       ||               |-> mannosidase
   Gly Val Ser Leu Glu Lys Arg|Glu Ala||Glu Ala||Tyr Val Glu Phe Ala Thr Lys 
               XhoI           |       ||       ||SnaBI   EcoRI   
               AvaI                      HindIII

| : Kex2 cleavage site.
||: dipeptidase cleavage.
Punctuation indicates reading frame.

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, EcoRI, FspI and NheI.

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 1498, 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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