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LMBP plasmid details

Last data update: 23 September 2020 04:27 CEST

Plasmid name: pLacDN9HIFNG (LMBP 1880)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1880.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); 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: pLacDN5HIFNG; pLacDN6HIFNG
Further information: The plasmid was constructed by replacing the PstI-SalI fragment of pLacDN5HIFNG by the PstI-SalI fragment of pLacDN6HIFNG.
This plasmid carries a hIFNG-lacZ fusion gene.
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.
There is a constitutive weak expression of the fusion protein under control of the ampicillin promoter, enough for rapid colour development on X-Gal indicator plates at 28°C. High (PL controlled) expression levels of the fusion protein can be obtained by shifting to 42°C. The chromosomal lacZ gene must be inactivated for detection of β-galactosidase activity of the hIFNG-lacZ fusion protein.
The incomplete lacY gene (first 69 codons) is still functional, but activity is lower than that of the wild type lacY gene. As such, β-galactosidase activity measurement by X-Gal hydrolysis is slower.
The hIFNγ gene is a mutant, containing a unique SalI site at position 413 (amino acid 143) and an extra HhaI site at position 390 of the mature sequence.
The natural EcoRI site near the 3' end of the lacZ gene was removed by mutation.
Other name of the plasmid is pDN9G.
EMBL Accession number: -
Latest sequence update: 06/03/1990
Sequence detail:
Nucleotide sequence around the start of the hIFNG-lacZ fusion gene:

-23         -20                                     -10
ATG.AGT.ATT.CAA.CAT.TTC.CGT.GTC.GCC.CTT.ATT.CCC.TTT.TTT.GCG.GCA.TTT
*

                     -1  1               5           8
TGC.CTT.CCT.GTT.TTT.GCT.CAC.CGC.CTA.GTA.CGC.AAG.TTC.ACG.TAA AAAGGGTAT
                    +                                   @

CGATTCC ATG.CAG.GAC
        ^

*: Start codon of the first peptide (23 amino acids of the amp signal sequence and 8 other amino acids).
+: End of the signal sequence of the amp gene.
@: Termination codon of the first peptide.
^: Start codon of the hIFNG-lacZ fusion protein.


Nucleotide sequence at the 3' end of the hIFNG variant:

   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Pro Ser Arg Ile Pro 
5' CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CCC.TCT.AGA.ATT.CCC.
         HhaI                           SalI       XbaI EcoRI  AvaI
                                                               SmaI
                                                               XmaI


Gly Asp Pro Val Val 3'
GGG.GAT.CCC.GTC.GTT
  BamHI     ^

^: Start of the lacZ gene (at the 9th amino acid)


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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