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GeneCorner plasmid details

Last data update: 01 December 2020 04:11 CET

Plasmid name: pCAGGS-prepro-TSE (LMBP 4172)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p4172.gb (View with Genome Compiler)
p4172.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
Promoter: Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
Ribosome
binding site:
-
Terminator: Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
Host range: Escherichia coli
Mammalian cells; SV40 permissive cells
Parental clone: pCAGGS; pPIC9-TSE
Further information: This plasmid was constructed by inserting a 3520 bp AseI fragment (filled in with Klenow DNA polymerase) from pPIC9-TSE, containing the prepro secretion signal sequence (ppMF) of the Saccharomyces cerevisiae α-mating factor 1 gene (MFα1) fused to the mature Trypanosoma cruzi trans-sialidase gene (TS) with C-terminal E-tag, in the MscI site of pCAGGS.
pCAGGS-prepro-TSE is useful for highly efficient expression of the fusion protein under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.
The AG promoter sequence consists of the chicken β-actin promoter, the first exon and part of the first intron (that seems to have a strong enhancer-like activity) linked to a rabbit β-globin fragment, consisting of a 3' part of the second intron (inclusive a branch point which is required for normal splicing reactions) and a 5' part of the third exon.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727319.1.
Other name of the plasmid is pCAGGS-MFTSjE.
EMBL Accession number: LT727319.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 28/08/2000
Sequence detail:
Nucleotide sequence around the start of the trans-sialidase gene:

                                                      trans-sialidase-->
      - ppMF --->                                     ---------------
      Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala Tyr Val Leu Ala Pro 
5' …  GGG.GTA.TCT.CTC.GAG.AAA.AGA.GAG.GCT.GAA.GCT.TAC.GTG.CTG.GCA.CCC … 3'
                  XhoI                     HindIII-------
                                                  SnaBI/PmlI fusion

Punctuation indicates reading frame.


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

5'… AGC.AGC.GAC.ACG.AAG.GAA.TTG|GGT.GCG.CCG.GTG … 3'
             trans-sialidase-->|-->E-tag

Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglII, HincII, PstI/StuI and SphI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr W. Laroy(1) (2) and Prof. Dr R. Contreras(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: -

Refer in your Materials and Methods:

pCAGGS-prepro-TSE (LMBP 4172) is available at BCCM/GeneCorner. This plasmid was deposited by Dr W. Laroy and Prof. Dr R. Contreras and was published in Laroy et al., 2000.

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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