Last data update: 24 January 2024 16:39 CET
Plasmid name: pAS3 (LMBP 4253)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4253.gb
(View with Genome Compiler) p4253.txt p4253.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd) Influenza HA epitope encoding the haemagglutinin tagging peptide; N-terminal |
| Promoter: | Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1) Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1) Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator |
| Selection marker: | Ampicillin (amp) ADE2; auxotrophic |
| Replicon: | Escherichia coli plasmid pMB1 origin Saccharomyces cerevisiae 2 micron plasmid origin (2μ); incl. region conferring stability (STB) |
| Host range: | Escherichia coli Saccharomyces cerevisiae; ade2(-) |
| Parental clone: | pAS2 |
| Further information: | The plasmid was constructed by blunt ligation of the Saccharomyces cerevisiae ADE2 gene under its own promoter into the EcoRV opened pAS2 vector. pAS3 is designed to generate a fusion of the S. cerevisiae GALbd domain with a sequence encoding a target protein (bait fusion protein). The GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal, and is followed by the HA epitope sequence, a multicloning site and termination codons in all reading frames. pAS3 is useful in a yeast three-hybrid approach. The ADE2 auxotrophic selection marker makes the system compatible with the existing yeast strains and libraries that are currently used in Gal4-based two- and three-hybrid systems and which make use of the TRP1, LEU2, HIS3 and URA3 selection markers. 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 LT727334.1. The nucleotide sequence of the ADE2 cDNA corresponds with the Genbank accession number NC_001147.5. |
| EMBL Accession number: | NC_001147.5, view at EMBL, GenBank, DDBJ LT727334.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 11/09/2006 |
| Sequence detail: | Nucleotide sequence of the HA epitope and the multi-cloning site:
GALbd -----> ----------- HA epitope ----------->
5' ... ACT.GTA.TCG.CCG.GAA.TTC.ATG.GCT.TAC.CCA.TAC.GAT.GTT.CCA.GAT.TAC.GCT.
Thr Val Ser Pro Glu Phe Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
EcoRI NheI
AGC.TTG.GGT.GGT.CAT.ATG.GCC.ATG.GAG.GCC.CCG.GGG.ATC.CGT.CGA.CCT.GCA.GCC.
Ser Leu Gly Gly His Met Ala Met Glu Ala Pro Gly Ile Arg Arg Pro Ala Ala
NdeI NcoI SmaI BamHI SalI PstI
StyI
BalI
SfiI
--------------------------- ADH1 terminator --->
AAG.CTA.ATT.CCG.GGC.GAA.TTT.CTT.ATG.ATT.TAT.GAT.TTT.TAT.TAT.TAA ... 3'
Lys Leu Ile Pro Gly Glu Phe Leu Met Ile Tyr Asp Phe Tyr Tyr +++
++ + + ++
+++: Termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: AatII/HindIII, AccIII/NdeI, AvaII/Csp45I, BglI/BglII, Bst98I/BstXI, EcoRV/NcoI, MunI, SspI and XhoI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr R. Beyaert(1) (2). It was constructed by W. Van Criekinge(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Van Criekinge et al., Anal. Biochem. 263 (1998), 62-66 [PMID: 9750144] |
| 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: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pAS3 (LMBP 4253) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr R. Beyaert and was published in Van Criekinge et al., 1998. |
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.