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LMBP plasmid details

Last data update: 25 September 2020 04:24 CEST

Plasmid name: pLacDN6FHIFNG (LMBP 2011)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p2011.gb (View with Genome Compiler)
p2011.txt
p2011.pdf
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human interferon γ cDNA (IFNG); mutated mature sequence
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: 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: Phage fd terminator
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli; use strains with a cI function, cIts for PL controlled expression, the detection host must also be lacZ(-).
Parental clone: pLacDN6HIFNG; pTM2HIFNG
Further information: The plasmid was constructed as follows: the XmnI (Asp700: nucleotide position 5561) - PstI fragment of pLacDN6HIFNG was replaced by the XbaI (filled in with T4 DNA polymerase) - PstI (nucleotide position 2488) fragment of pTM2HIFNG.
The hIFNG cDNA 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 λ PL and amp 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 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.
pLacDN6FHIFNG can be used to obtain ssDNA for site-directed mutagenesis on hIFNG.
This phasmid can be used in conjunction with pLacDN7FHIFNG to obtain selection for the opposite strand.
The nucleotide sequence of the wt human IFNG DNA corresponds with the EMBL Sequence Database accession number AM903379.1.
The nucleotide sequence of the wt E. coli lactose operon cDNA was obtained from the EMBL Sequence Database accession number J01636.1.
Other name of the plasmid is pDN6fG.
EMBL Accession number: AM903379.1, view at EMBL, GenBank, DDBJ
J01636.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 07/03/1990
Sequence detail:
Nucleotide sequence around the start of the hIFNG-lacZ fusion gene in pLacDN6FHIFNG:

-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: Restriction enzyme pattern analysed at GeneCorner: AvaI, BanII, BglI/SacI, Bsu36I, EcoRI and NaeI/SacI.
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(λ)
Host reference: Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
Cultivation remark: -
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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