Last data update: 26 September 2020 04:22 CEST
Plasmid name: pLacDN6HIFNG (LMBP 1877)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|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 (RBS) of the Escherichia coli ampicillin resistance gene (amp)
Ribosome binding site (RBS) of the Escherichia coli tryptophan operon attenuator peptide (trp)
|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|
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.|
|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]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml) + kanamycin (50 μg/ml)|
|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.