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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLacDN4HIFNG (LMBP 1578)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1578.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human interferon γ cDNA (IFNG)
Escherichia coli lac Z gene (lacZ); starting at the 9th codon
Escherichia coli lac Y gene (lacY)
Escherichia coli lac A gene (lacA)
Promoter: Phage λ major leftward promoter (λ PL)
Ribosome
binding site:
Ribosome binding site (RBS) of the Escherichia coli tryptophan operon attenuator peptide (trp)
Terminator: -
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli; use strains with a cI function, cIts for PL controlled expression
Parental clone: pLacDN1HIFNG
Further information: pLacDN4HIFNG is derived from pLacDN1HIFNG by removing the SalI site at nucleotide position 6176 (filled in with T4 DNA polymerase), resulting in a unique SalI site between human interferon γ (hIFNG) and lacZ gene. The chromosomal lacZ gene must be inactivated for detection of β-galactosidase activity of the hIFNG-lacZ fusion protein.
The natural EcoRI site near the end of lacZ was removed by mutation.
hIFNγ is a mutated variant, containing a unique SalI site and an extra HhaI site at position 390 of the coding region.
Other name of the plasmid is pDN4G.
EMBL Accession number: -
Latest sequence update: 31/01/1989
Sequence detail:
Nucleotide sequence at the end of the hIFNG wt gene:
           131                             139 140
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln *
5' CGA.AAA.AGG.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.AGA.GCA.TCC.CAG.TAA 

   TGGTTG 3'

Nucleotide sequence at the end of the hIFNG variant:
           131                             139 140 
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Pro Ser Arg Gly Cys Ser 
5' CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CCC.TCT.AGA.GGC.TGC.AGC.
         HhaI                           SalI       XbaI      PstI

   Leu *
   CTC.TAG.A 3'
    XbaI

*: Termination codon of hIFNG gene.

Nucleotide sequence around the SalI site of hIFNG at the junction with lacZ:
           139                                 AA9 of lacZ
   Arg Gly Arg Pro Ser Arg Ile Pro Gly Asp Pro Val Val
5' CGA.GGT.CGA.CCC.TCT.AGA.ATT.CCC.GGG.GAT.CCC.GTC.GTT 3'
        SalI       XbaI EcoRI  SmaI  BamHI     
                               XmaI

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 E. Sablon(1) and Prof. Dr E. Remaut(1).
(1) 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(λ)
Host reference: Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
Other culture collection numbers: -

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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