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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLacDN1HIFNG (LMBP 1574)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1574.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: pPLc321HIFNGv; pPLcZCYA0
Further information: The plasmid encodes a fusion protein: human interferon γ/β-galactosidase (HIFNG-lacZ). The fusion was made at the level of the unique SalI site, introduced at position 415 of the mature sequence. The chromosomal lacZ gene must be inactivated for detection of β-galactosidase activity of the hIFNG-lacZ fusion protein.
This plasmid easily gives rise to deletion derivatives after cutting and religation.
The natural EcoRI site near the end of lacZ was removed by mutation.
The used human IFNγ gene is a mutated variant, containing a unique SalI site at position 415 and an extra HhaI site at position 390. The amino acid sequence is unaffected by the mutations.
Other name of the plasmid is pDN1G.
EMBL Accession number: -
Latest sequence update: 30/01/1989
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: PhD thesis Erwin Sablon (1990)
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061[pcI857]
Host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Helper plasmid: pcI857
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml) + kanamycin (50 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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