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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLacDN6HIFNG (LMBP 1877)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1877.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 A gene (lacA); fragment
Ampicillin resistance gene (amp); signal sequence
Promoter: Phage λ major leftward promoter (λ PL)
Escherichia coli β-lactamase promoter (amp)
Ribosome
binding site:
Ribosome binding site (RBS) of the Escherichia coli ampicillin resistance gene (amp)
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: pPLc2820B; pLacDN3HIFNG
Further information: hIFNγ is a mutated variant, containing a unique SalI site at position 413 and an extra HhaI site at position 390 of the coding region (sequence represented at page 235).
Expression from either one or both promoters results in the production of two polypeptides: a small 31 amino acid peptide (of which 23 amino acids are encoded by the signal sequence of the ampicillin resistance gene) and the hIFNγ/β-galactosidase fusion protein (bicistronic cistron).
There is a constitutive weak expression of the fusion protein from the ampicillin promoter, enough for rapid coloration on X-Gal indicator plates at 28°C. High expression levels of fusion protein can be obtained by shifting to 42°C.
The natural EcoRI site near the end of lacZ was removed by mutation.
Other name of the plasmid is pDN6G.
EMBL Accession number: -
Latest sequence update: 09/02/1989
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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