Last data update: 24 January 2024 16:39 CET
Plasmid name: pEMBLyex4 (LMBP 2423)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p2423.gb
(View with Genome Compiler) p2423.txt p2423.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
- |
| Promoter: | Saccharomyces cerevisiae hybrid UDP-glucose 4-epimerase/iso-1-cytochrome C promoter (GAL10/CYC1) |
| Ribosome binding site: |
- |
| Terminator: | Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator |
| 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: | pEMBLyex2 |
| Further information: | pEMBLyex4 was derived from pEMBLyex2 by eliminating (partial digestion followed by filling-in with Klenow DNA polymerase) the XbaI site in the 2μ inverted repeat (IR1). This resulted in a unique XbaI site located in the polylinker of pEMBLyex4. This vector is useful for expression of proteins in yeast by inserting the gene of interest into an expression site of the polylinker, located between the GAL10/CYC1 hybrid promoter and the 2μ FLP terminator. The hybrid promoter sequence consists of the upstream activation sequence of the GAL10 gene (UAS-G), the GAL10 promoter and the 5' untranslated leader sequence of the CYC1 gene. pEMBLyex4 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, EcoRI, AflII or BstEII) or at URA3 (by linearising with ApaI or StuI). pEMBLyex4 also contains the replication origin 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 (leu2-d) is poorly expressed, because there are 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 LT727514.1. The nucleotide sequence of this vector was obtained from Dr Jahm (Cambridge, UK). However, M. Logghe (Dept of Biomedical Molecular Biology, Ghent University, Belgium) found an extra FspI site around position 1550-1560, a region between the f1 sequence and the 2μ fragment where one (or more ?) nucleotide was indicated with a 'N'. |
| EMBL Accession number: | LT727514.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 18/02/1997 |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AflIII, AspI, EcoRI, HindIII, Tth111I and XmnI. The unique Tth111I and AspI sites could not be experimentally confirmed. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr R. Contreras(1) (2). He received it from EMBL(3). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium (3) Heidelberg, Germany |
| Plasmid reference: | - |
| 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: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 28°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pEMBLyex4 (LMBP 2423) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Contreras . |
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.