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GeneCorner plasmid details

Last data update: 23 October 2020 04:27 CEST

Plasmid name: pAS2-mABIN-1(388-647) (LMBP 5394)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p5394.gb (View with Genome Compiler)
p5394.txt
p5394.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1); C-terminal fragment encoding amino acids 388-647
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 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
Further information: The plasmid was constructed by ligating the following fragments: 1) the 6699 bp SmaI-BsmI fragment of pAS2; 2) the 1699 bp BsmI-EaeI fragment of pAS2; 3) the 965 bp NotI-SmaI PCR fragment, containing the C-terminal fragment of the mABIN-1 coding sequence, encoding amino acids 388 up to 647. As a result, this fragment of the mABIN-1 coding sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-mABIN-1(388-647) fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-mABIN-1(388-647) 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 LT726887.1.
The nucleotide sequence of the pAS2 vector corresponds with the EMBL Nucleotide Sequence Database accession number U30496.1, but adapted as follows: 1) the 'N' nucleotide was removed, restoring the coding sequence of cycloheximide; 2) the sequence GATTTC between GALbd and the HA epitope was replaced by GAATTC, an EcoRI site that has been experimentally confirmed in pAS2; 3) the sequence TTCGAA (Csp45I site) in the ADH1 promoter was replaced by TCGAAG, eliminating the Csp45I site that could not be experimentally confirmed in pAS2.
The nucleotide sequence of the mABIN-1 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number AJ242778.1.
Other name of the plasmid is pAS2-mABIN-1(3).
EMBL Accession number: U30496.1, view at EMBL, GenBank, DDBJ
AJ242778.1, view at EMBL, GenBank, DDBJ
LT726887.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 20/03/2007
Sequence detail:
Primers used to amplify the C-terminal fragment of mABIN-1:

                              388 389 390 391 392
forward: 5' ATAAGAATGCGGCCGCA.GAG.ATG.GAA.GAG.ACC.G 3'
                    NotI      Glu Met Glu Glu Thr

reverse: 5' TCCCCCGGGTCTTGTCAACAGTTCGAGG 3'
               SmaI

Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BglI/EcoRI, Bsp119I/NdeI, Eco32I/PaeI, NheI/SmaI, Psp5II/SspI and SacI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr K. Heyninck(1) (2) and Prof. Dr R. Beyaert(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-mABIN-1(388-647) (LMBP 5394) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. Heyninck and Prof. Dr R. Beyaert .

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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