Last data update: 24 January 2024 16:39 CET
Plasmid name: pAS2.1-MLT3b (LMBP 5542)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p5542.gb
(View with Genome Compiler) p5542.txt p5542.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human MALT1 paracaspase cDNA (MALT1, MLT, PCASP1, paracaspase 1); C-terminal fragment (amino acids 304-824) Saccharomyces cerevisiae GAL4 DNA-binding domain (GALbd) Escherichia coli IS10R transposase gene |
| Promoter: | Saccharomyces cerevisiae alcohol dehydrogenase promoter (ADH1) Escherichia coli lac operon promoter |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| 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 1569 bp EcoRI-SalI fragment, containing the C-terminal fragment of the human MALT lymphoma associated translocation coding sequence, encoding amino acid 304 to 824, into the EcoRI-SalI opened pAS2.1 vector. As a result, this fragment of the hMLT coding sequence, containing a caspase-like domain, is fused in phase to the N-terminal S. cerevisiae GAL4 DNA-binding domain. The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal. The GALbd-hMLT3b fusion protein can be used as a bait protein in two-hybrid screening. pAS2.1-MLT3b 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT726926.1. 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 wt hMLT cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AF130356.2. The region between the ADH1 promoter and the lac promoter was sequenced by LMBP. Other name of the plasmid is pAS2-MLT3b. |
| EMBL Accession number: | U30497.1, view at EMBL, GenBank, DDBJ AF130356.2, view at EMBL, GenBank, DDBJ LT726926.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 11/06/2009 |
| Sequence detail: | The nucleotide sequence of the fusion gene:
---------- GALbd --------->
1 2 3 145 146 147
5' ... CTGAAAG.ATG.AAG.CTA ... ACT.GTA.TCG.CCG.GTA.TTG.CAA.TAC.CCA.GCT.TTG.ACT.CAT
Met Lys Leu Thr Val Ser Pro Val Leu Gln Tyr Pro Ala Leu Thr His
*** NdeI
------ hMLT fragment ----->
304 305 306 822 823 824
ATG.GCC.ATG.GAG.GCC.GAA.TTC.GTA.GAA.ATC ... TCT.GAA.AAA.GTC.GAC.CTG.CAG.CCA
Met Ala Met Glu Ala Glu Phe Val Glu Ile Ser Glu Lys Val Asp Leu Gln Pro
NcoI EcoRI SalI PstI
AGC.TAA.TTCCGG ... 3'
Ser +++
***: start codon.
+++: termination codon.
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BglII, EcoRI/SalI, NcoI/PvuI, PdmI, PvuII and SspI/XhoI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr M. Baens(1). (1) Department of Human Genetics, KULeuven, 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: |
pAS2.1-MLT3b (LMBP 5542) is available at BCCM/GeneCorner. This plasmid was deposited by Dr M. Baens. |
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.