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GeneCorner plasmid details

Last data update: 03 December 2020 04:26 CET

Plasmid name: pAS2-hABIN1-N (LMBP 5135)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p5135.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human A20-binding inhibitor of NF-κB activation 1 cDNA (ABIN-1, TNIP1, NAF1); mutated N-terminal fragment
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 three following fragments: 1) the 5686 bp SalI-SacI fragment from pAS2. 2) the 2721 bp SacI-BamHI fragment from pAS2. 3) a 1145 bp BamHI-XhoI PCR fragment, containing the N-terminal fragment of the human A20-binding inhibitor of NF-κB activation 1 (hABIN-1) coding sequence. As a result, the hABIN-1-N coding sequence is fused in phase to the N-terminal HA epitope and the S. cerevisiae GALbd domain.
As compared to the wt hABIN-1 cDNA, the proline (P) codon at position 299 is replaced by an alanine (A) codon resulting in the loss of an AvaI site and the isoleucine (I) codon at position 9 contains a silent mutation (atc -> att) resulting in the loss of a DpnI and a Sau3AI site.
The S. cerevisiae GALbd is composed of the first 147 codons from GAL4, containing the nuclear localization signal.
The GALbd-HA-hABIN-1-N fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-hABIN1-N 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.
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 hABIN-1 cDNA corresponds with the Genbank accession number NM_006058.2.
EMBL Accession number: NM_006058.2, view at GenBank
Latest sequence update: 05/01/2006
Sequence detail:
Primers used to amplify the mutated N-terminal fragment of the hABIN-1 coding sequence:

                                     1               5
forward: 5' ... CGGGATCCTAGCGGCCGCC.ATG.GAA.GGG.AGA.GGA.CCG ... 3'
                  BamHI   NotI      ***

                               375                 370
reverse: 5' ... GCCGCTCGAG.TCA.TTC.AAT.CTT.GGA.CTT.GGC ... 3'
                    XhoI   +++


                            9              299    
WT (NM_006058): 5' ... CGG.ATC.TAC ... GCA.CCC.GAG ... 3'
                       Arg Ile Tyr     Ala Pro Glu
                         DpnI              AvaI
                        Sau3AI

                            9              299    
mutant:         5' ... CGG.ATT.TAC ... GCA.GCC.GAG ... 3'
                       Arg Ile Tyr     Ala Ala Glu
                             ^             ^
                                       
***: start codon.
+++: termination codon.
^  : mutated nucleotide.
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 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-hABIN1-N (LMBP 5135) is available at BCCM/GeneCorner. This plasmid was deposited by 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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