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LMBP plasmid details

Last data update: 26 September 2020 04:22 CEST

Plasmid name: pLacDN7FHIFNG (LMBP 2020)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p2020.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
Ampicillin resistance gene (amp); signal sequence
Promoter: Escherichia coli β-lactamase promoter (amp)
Phage λ major leftward promoter (λ PL)
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: Phage fd terminator
Selection marker: Ampicillin (amp; contains an amber codon)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli; use strains with a cI function and an amber suppressor function, preferably supF.
Parental clone: pLacDN6FHIFNG; pTM3HIFNG
Further information: The plasmid was constructed by replacing the PstI-SalI fragment of pLacDN6FHIFNG by the PstI-SalI fragment of pTM3HIFNG. This is the only plasmid of the type pLacDN with an amber mutation in ampicillin hIFNγ is a mutant, containing a unique SalI site and an extra HhaI site at position 390 of the coding region.
The natural EcoRI site near the end of lacZ was removed by mutation.
Expression from either one or both promoters results in the production of two polypeptides: a small 31 amino acids 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 from the ampicillin promoter, enough for rapid colour development on X-Gal indicator plates at 28°C. High expression levels of fusion protein can be obtained by shifting to 42°C.
pLacDN7FHIFNG can be used to obtain ssDNA for site-directed mutagenesis on hIFNγ.
This plasmid can be used in conjunction with pLacDN6FHIFNG to obtain selection for the opposite strand.
The chromosomal lacZ gene must be inactivated for detection of β-galactosidase activity of the hIFNG-lacZ fusion protein.
Other names of the plasmid are pDN6faG and pLacDN6faHIFNG.
EMBL Accession number: -
Latest sequence update: 07/03/1990
Sequence detail:
Nucleotide sequence around the start of the fusion protein in pLacDN6FHIFNG and pLacDN7FHIFNG:

-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.
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 H12R8a
Host reference: Garen et al., J. Mol. Biol. 14 (1965), 167-178 [PMID: 5327650]
Related host reference: Remaut et al., J. Mol. Biol. 71 (1972), 243-261 [PMID: 4564480]
Weigert et al., J. Mol. Biol. 14 (1965), 522-527 [PMID: 4286570]
Cultivation medium: LB-Lennox
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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