Last data update: 24 September 2020 04:17 CEST
Plasmid name: pSCGAL10-SN (LMBP 2471)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human interferon β gene (IFNB, IFNB1); stuffer fragment|
|Promoter:||Saccharomyces cerevisiae hybrid UDP-glucose 4-epimerase/iso-1-cytochrome C promoter (GAL10/CYC1)|
|Terminator:||Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator
Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1)
|Selection marker:||Ampicillin (amp)
Saccharomyces cerevisiae URA3; auxotrophic
LEU2 defective (leu2-d; auxotrophic)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB)
|Host range:||Escherichia coli
Saccharomyces cerevisiae; ura3(-)
|Parental clone:||pSC-SN2; pEMBLyex4|
|Further information:||The plasmid was constructed by inserting the HindIII/blunted XbaI fragment from pSC-SN2 between the HindIII/SmaI sites of pEMBLyex4 as described in the publication of Goldman et al. (1992).
This E. coli-yeast shuttle plasmid allows efficient cloning and regulated expression of cDNA libraries. Efficient cloning can be carried out by inserting the gene of interest into the unique SfiI and NotI sites of the plasmid, separated from each other by a segment of stuffer DNA, facilitating purification of a SfiI-NotI vector. The stuffer DNA consists of the genomic human interferon β gene (signal and mature sequence). Expression of the cloned cDNA is controlled by the regulatable GAL10/CYC1 hybrid promoter from S. cerevisiae and the CYC1 and 2μ FLP terminators. The hybrid promoter sequence consists of the upstream activation sequence (UAS-G) of the GAL10 gene, the GAL10 promoter and the 5' untranslated leader sequence of the CYC1 gene.
pSCGAL10-SN can be used in a c(+) yeast as a high-copy number extrachromosomal vector using the 2μ plasmid origin, but it can also be directed to integrate into the yeast chromosome at LEU2 (by linearising with BstXI or AflII) or at URA3 (by linearising with ApaI).
pSCGAL10-SN contains three types of replication origin: 1) the origin of replication for E. coli; 2) the origin of replication of plasmid 2μ for extrachromosomal replication in S. cerevisiae; 3) the origin of replication of the single-stranded DNA phage f1, so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage, e.g. M13KO7.
The defective LEU2 gene is poorly expressed, because there is only 30 bp of upstream sequence left in this plasmid; the expression is probably controlled by a promoter within the 2μ sequences.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727521.1.
The nucleotide sequence of the hIFNβ cDNA corresponds with the EMBL Nucleotide Sequence Database accession number V00546.1.
The 3' border region of the hIFNB insert has been sequenced at the Department of Biomedical Molecular Biology (Ghent University, Belgium). The results revealed the presence of the S. cerevisiae CYC1 terminator at the 3' side of the hIFNB insert.
|EMBL Accession number:||V00546.1, view at EMBL, GenBank, DDBJ
LT727521.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||26/03/2001|
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: HindIII/XbaI, PvuI and SmaI.|
|History of deposit:||This plasmid was deposited by J. De Molder(1) and Prof. Dr R. Contreras(1).
(1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Goldman et al., Mol. Gen. Genet. 234 (1992), 481-488 [PMID: 1406594]
|Related plasmid reference:||Chen et al., Exp. Gerontol. 38 (2003), 1051-1063 [PMID: 14580858]
PhD thesis Mladenov Ashikov et al. (2006)
Desmyter et al., Exp. Gerontol. 39 (2004), 707-715 [PMID: 15130665] [DOI: 10.1016/j.exger.2004.01.008]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5α|
|Host reference:||Focus 8 (1986), 9
|Related host reference:||Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051]
Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187]
Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791]
Rodriguez-Quinones et al., Focus 15 (1993), 110-112
Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|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.