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GeneCorner plasmid details

Last data update: 21 October 2020 04:26 CEST

Plasmid name: pAS2-hFIP2 (LMBP 5133)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p5133.gb (View with Genome Compiler)
p5133.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human optineurin cDNA (OPTN, FIP2, GLC1E, HYPL, HIP7, GeneID 10133)
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 and 3) a 1751 bp BamHI-XhoI PCR fragment, containing the human optineurin (hOPTN, hFIP2) coding sequence. As a result, the hFIP2 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-hFIP2 fusion protein can be used as a bait protein in two-hybrid screening.
pAS2-hFIP2 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 this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727096.1.
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 hFIP2 cDNA was obtained from Genbank (Accession number NM_001008213.1) but contains a silent mutation at codon 34.
EMBL Accession number: NM_001008213.1, view at GenBank
U30496.1, view at EMBL, GenBank, DDBJ
LT727096.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 14/12/2005
Sequence detail:
Primers used to amplify the human optineurin cDNA (hOPTN, hFIP2):

forward primer: 5' ... CGGGATCCTAGCGGCCGCC.ATG.TCC.CAT.CAA.CCT.CTC.AGC.TGC.CTC ... 3'
                         BamHI   NotI      ***

reverse primer: 5' ... GCCGCTCGAG.TTA.AAT.GAT.GCA.ATC.CAT.CAC.GTG.AAT.CTG ... 3'
                           XhoI   +++


                                           32      34      36
wt hFIP2 according to NM_001008213: 5' ... CTG.GAC.ACG.TTT.ACC ... 3'
                                           Leu Asp Thr Phe Thr

                                           32      34      36
hFIP2 silent mutant:                5' ... CTG.GAC.ACA.TTT.ACC ... 3'
                                           Leu Asp Thr Phe Thr
                                                     ^
***: start codon.
+++: termination codon.
^  : mutated nucleotide.
Punctuation indicates reading frame.
Authenticity test: Restriction enzyme pattern analysed at GeneCorner: BamHI/BanI, Csp45I/SnaBI, Eco52I/SspI, EcoRV/PvuI, HindIII and NcoI.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by M. Haegman(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-hFIP2 (LMBP 5133) is available at BCCM/GeneCorner. This plasmid was deposited by M. Haegman 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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