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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPIC9-TSE (LMBP 4166)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4166.gb (View with Genome Compiler)
p4166.txt
p4166.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Trypanosoma cruzi trans-sialidase gene (TS); mature sequence
E-tag; C-terminal
Phage fd gene 3 (g3); fragment
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: pCAGGS-TSE; pPIC9-TS
Further information: The plasmid was constructed by exchanging the MluI/NotI (filled in with Klenow DNA polymerase) fragment from pPIC9-TS, containing the C-terminal part of the mature T. cruzi TS gene, by the MluI/ClaI (filled in with Klenow DNA polymerase) fragment from pCAGGS-TSE, containing the C-terminal part of the mature T. cruzi TS gene provided with a C-terminal E-tag.
This plasmid is useful in P. pastoris strains for secreted expression of trans-sialidase ligated to an E-tag under control of the strong, highly-inducible P. pastoris AOX1 promoter.
At the fusion junction between the S. cerevisiae ppMF and the mature T. cruzi TS gene, a (Glu-Ala)-dipeptide and an additional (but not essential) Tyr-residue 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 plasmid contains a modified P. pastoris HIS4 gene: the internal BamHI site is removed.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727317.1.
The nucleotide sequence of the T. cruzi TS cds corresponds with the EMBL Nucleotide Sequence Database accession number AJ276679.1.
The nucleotide sequence of the pPIC9 vector corresponds with the online Invitrogen sequence.
Other name of the plasmid is pPIC9-TSjE.
EMBL Accession number: AJ276679.1, view at EMBL, GenBank, DDBJ
LT727317.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 17/12/2012
Sequence detail:
Nucleotide sequence around the start of the trans-sialidase gene:
                                                             
      -- ppMF --->                |       ||       ||    -- mature TS -->
5' ... GGG.GTA.TCT.CTC.GAG.AAA.AGA|GAG.GCT||GAA.GCT||TAC.GTG.CTG.GCA.CCC ... 3'
       Gly Val Ser Leu Glu Lys Arg|Glu Ala||Glu Ala||Tyr Val Leu Ala Pro 
                   XhoI           |       ||       ||-------
                                             HindIII SnaBI/PmlI
| : Kex2 cleavage site.                              fusion
||: Dipeptidase cleavage.
Punctuation indicates reading frame.


Nucleotide sequence at the end of the trans-sialidase gene:

      -- mature TS -->             -- E-tag -->
5' ... AGC.AGC.GAC.ACG.AAG.GAA.TTG.GGT.GCG.CCG ... 3'
       Ser Ser Asp Thr Lys Glu Leu Gly Ala Pro

Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglI, Eco47III and NcoI.
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: Laroy et al., Protein Expr. Purif. 20 (2000), 389-393 [PMID: 11087678]
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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