GREAT AT SMALL THINGS

0

LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLacDN8HIFNG (LMBP 1876)

Add to cart

New search

Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1876.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); fragment
Promoter: Escherichia coli tetracycline resistance gene promoter (tet)
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
Parental clone: pLacDN3HIFNG; pBR322
Further information: The plasmid was constructed by replacing the PstI-SpeI fragment of pLacDN3HIFNG containing the λ PL promoter by the PstI-EcoRV fragment of pBR322 containing the tetracycline promoter. The SpeI site was first blunted with T4 DNA polymerase.
This plasmid carries a hIFNG-lacZ fusion gene.
There is a constitutive low expression of the fusion protein hIFNγ/β-galactosidase under control of the tetracycline promoter.
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.
The chromosomal lacZ gene must be inactivated for detection of β-galactosidase activity of the hIFNG-lacZ fusion protein.
Other name of the plasmid is pDN8G.
EMBL Accession number: -
Latest sequence update: 27/10/1989
Sequence detail:
Nucleotide sequence at the 3' end of the wt hIFNG gene:

   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 3'

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     ^




*: Termination codon of the wt hIFNG gene.
^: 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.

New search