Last data update: 24 January 2024 16:39 CET
Plasmid name: pAS2.1htBID (LMBP 4759)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p4759.gb
(View with Genome Compiler) p4759.txt p4759.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human BH3 interacting domain death agonist (BID); truncated sequence (tBID) 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 |
| Further information: | The plasmid was constructed by inserting a 416 bp NcoI-SalI fragment, containing the human tBID coding sequence, obtained by RT-PCR using HeLa cDNA as template, into the NcoI-SalI opened pAS2.1 vector. As a result, the human tBID 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 pAS2htBID, pAS2.1htBID 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-htBID fusion protein can be used as a bait protein in two-hybrid screening. pAS2.1htBID carries the S. cerevisiae tryptophan 1 coding sequence (TRP1) 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727032.1. 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 human BID cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF042083 .1. Other name of the plasmid is pAS2.1 htBid. |
| EMBL Accession number: | U30497.1, view at EMBL, GenBank, DDBJ AF042083.1, view at EMBL, GenBank, DDBJ LT727032.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 31/01/2005 |
| Sequence detail: | Primers used for RT-PCR on HeLa cDNA:
forward primer: 5' CAT.GCC.ATG.GCG.GGC.AAC.CGC.AGC.AGC.CAC.TCC.CGC.TT 3'
NcoI
reverse primer: 5' TGCGGTCGAC.TCA.GTC.CAT.CCC.ATT.TCT.GGC.TAA 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
---------------------- htBID ------
61
TAC.CCA.GCT.TTG.ACT.CAT.ATG.GCC.ATG.GCG.GGC.AAC.CGC.AGC.AGC.CAC ... AGA.AAT
Tyr Pro Ala Leu Thr His Met Ala Met Ala Gly Asn Arg Ser Ser His Arg Asn
NdeI NcoI
-------------->
195
GGG.ATG.GAC.TGA.GTCGACCTGCAGCCAAGC ... 3'
Gly Met Asp +++ SalI PstI
HindII
***: start codon.
+++: termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: Alw44I/Eco81I, BglI/EcoRI, BglII, NcoI/SalI, NheI/PvuII and SspI/XhoI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr P. Vandenabeele(1) (2). It was constructed by N. Festjens(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: |
pAS2.1htBID (LMBP 4759) 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.