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GeneCorner plasmid details

Last data update: 25 October 2020 04:11 CET

Plasmid name: pAS2.1-MLT5 (LMBP 5539)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p5539.gb (View with Genome Compiler)
p5539.txt
p5539.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human MALT1 paracaspase cDNA (MALT1, MLT, PCASP1, paracaspase 1); N-terminal fragment (amino acids 1-132) with Kozak consensus sequence
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 402 bp EcoRI-SalI fragment, containing the N-terminal fragment of the human MALT lymphoma associated translocation coding sequence, encoding amino acid 1 to 132, into the EcoRI-SalI opened pAS2.1 vector. As a result, this fragment of the hMLT coding sequence, containing a death 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-hMLT5 fusion protein can be used as a bait protein in two-hybrid screening.
pAS2.1-MLT5 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 LT726923.1.
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-MLT5.
EMBL Accession number: U30497.1, view at EMBL, GenBank, DDBJ
AF130356.2, view at EMBL, GenBank, DDBJ
LT726923.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 19/06/2009
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 ----->
                                   1   2   3       130 131 132
       ATG.GCC.ATG.GAG.GCC.GAA.TTC.ATG.GCG.CTG ... ACT.GTA.AAC.GTC.GAC.CTG.CAG.CCA
       Met Ala Met Glu Ala Glu Phe Met Ala Leu     Thr Val Asn 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-MLT5 (LMBP 5539) 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.

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