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LMBP plasmid details

Last data update: 29 October 2020 04:15 CET

Plasmid name: pTM3HIFNG (LMBP 1896)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1896.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human interferon γ cDNA (IFNG); mutated mature sequence
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
Parental clone: pMc58; pLacDN6HIFNG
Further information: The plasmid was constructed by replacing the XbaI/XmnI fragment of pTM1HIFNG the XbaI/XmnI (nucleotide position 7493) fragment of pLacDN6HIFNG.
pTM3HIFNG is a Target Mutagenesis plasmid.
The human interferon γ gene (IFNG) is a truncated and mutated variant, derived from the ancestral pPLc321HIFNGHS vector; it contains an extra HhaI site by silent mutations of codons 130 and 131, and a unique SalI site at codons 138-140. The SalI site was used for ligation to pMc58, resulting in the loss of 4 carboxy terminal codons of human IFNG. After induction at 42°C, this plasmid gives rise to a larger human IFNG peptide, due to the read-through (at the SalI-junction). The plasmid should not be used for expression as the human IFNG protein has additional nonsense information at its carboxy terminal end. Expression from either one or both promoters results in the production of two polypeptides: a small 31 amino acids peptide (the signal sequence of AMP + 8 nonsense amino acids) and the human IFNG variant.
pTM3HIFNG can be used to obtain ssDNA for site-directed mutagenesis on human IFNG.
This plasmid can be used in conjunction with pTM4HIFNG 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.
Other name of the plasmid is pTM3G.
EMBL Accession number: AM903379.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 02/03/1990
Sequence detail:
Start of the mature human IFN gamma cDNA:
                         |-> mature human IFNG 
                         | 1               5
5' AAAAAGGGTATCGATTCC.ATG.CAG.GAC.CCA.TAT.GTA.AAA 3'
            ClaI      Met Gln Asp Pro Tyr Val Lys

Nucleotide sequence at the end of the human IFNG wt DNA:
       130                                 139 140         143
5' CGA.AAA.AGG.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.AGA.GCA.TCC.CAG.TAA.TGGTTGTC 3'
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln

Nucleotide sequence at the end of the human IFNG variant:
                                 human IFNG ->|-> pMc58
       130 131                             139|
5' CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CCC.TCT.AGA.GGT.CGA... 3'
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Pro Ser Arg Gly Arg
         * * *                          SalI       XbaI
         HhaI

*: mutated nucleotide
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 Prof. Dr E. Remaut(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) 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 CA275[pcI857]
Host reference: -
Helper plasmid: pcI857
Cultivation medium: LB-Lennox + 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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