Last data update: 24 January 2024 16:39 CET
Plasmid name: pAS2.1mBIDdeltaC (LMBP 4769)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p4769.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Mouse BH3 interacting domain death agonist (BID); with C-terminal deletion (BIDΔC) Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd) |
| Promoter: | Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1) Escherichia coli lac operon promoter |
| Ribosome binding site: |
- |
| Terminator: | Saccharomyces cerevisiae alcohol dehydrogenase terminator (ADH1) Saccharomyces cerevisiae iso-1-cytochrome C terminator (CYC1) Saccharomyces cerevisiae 2 micron plasmid (2μ) FLP terminator |
| Selection marker: | Ampicillin (amp) Cycloheximide (CYH2) TRP1; 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; trp1(-) |
| Parental clone: | pAS2.1; pAS2mBID |
| Further information: | The plasmid was constructed as follows: the mBID coding sequence with C-terminal deletion of helix 6, 7 and 8 (mBIDΔC, encoding amino acids 1-144) was amplified by PCR on pAS2mBID and inserted as a 443 bp NcoI-SalI fragment into the NcoI-SalI opened pAS2.1 vector. As a result, the mBIDΔC coding sequence is fused in phase to the N-terminal S. cerevisiae GALbd domain. pAS2.1 was derived from pAS2 by removing the Influenza HA epitope, encoding the haemagglutinin tagging peptide. Therefore, unlike pAS2mBIDdeltaC, pAS2.1mBIDdeltaC transformants lack aspecific β-galactosidase activity. The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal. The GALbd-mBIDΔC fusion protein can be used as a bait protein in two-hybrid screening. pAS2.1mBIDdeltaC carries the S. cerevisiae tryptophan 1 coding sequence and the CYH2 sequence encoding ribosomal protein L29 for dominant cycloheximide sensitivity which facilitates the elimination of false positives. 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 the pAS2.1 vector corresponds with the EMBL Nucleotide Sequence Database accession number U30497.1, corresponding to the Clontech website (version 05/01/1996). The nucleotide sequence of the mBID cDNA corresponds with the Genbank accession number NM_007544.2 and adapted to the sequence analysis results of the Department of Biomedical Molecular Biology (Ghent University, Belgium). The following difference is observable: the nucleotide G at position 6023 is substituted by an A- residue, resulting in a Glu/Lys replacement for amino acid 14. Other name of the plasmid is pAS2.1 mBid C-term del. |
| EMBL Accession number: | U30497.1, view at EMBL, GenBank, DDBJ NM_007544.2, view at GenBank |
| Latest sequence update: | 29/03/2005 |
| Sequence detail: | Primers used to amplify mBIDdeltaC:
forward primer: 5' CATGCCATGGCG.ATG.GAC.TCT.GAG.GTC.AGC.AAC.GG 3'
NcoI ***
reverse primer: 5' ACGCGTCGAC.TCA.GTT.CTC.CAT.GTC.TCT 3'
SalI +++
Nucleotide sequence of the fusion gene:
---------------------- GALbd --------------------->
1 147
5' ... CTGAAAG.ATG.AAG.CTA.CTG.TCT.TCT ... AGA.CAG.TTG.ACT.GTA.TCG.CCG.GTA.TTG.CAA
Met Lys Leu Leu Ser Ser Arg Gln Leu Thr Val Ser Pro Val Leu Gln
*** HindII
------------------- mBIDdeltaC ----
1
TAC.CCA.GCT.TTG.ACT.CAT.ATG.GCC.ATG.GCG.ATG.GAC.TCT.GAG.GTC.AGC ... CCC.AGA
Tyr Pro Ala Leu Thr His Met Ala Met Ala Met Asp Ser Glu Val Ser Pro Arg
NdeI NcoI ***
StyI
-------------->
144
GAC.ATG.GAG.AAC.TGA.GTCGACCTGCAGCCAAGC ... 3'
Asp Met Glu Asn +++ SalI PstI
HindII
***: start codon.
+++: termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| 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: |
pAS2.1mBIDdeltaC (LMBP 4769) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr P. Vandenabeele . |
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.