Last data update: 24 November 2020 04:18 CET
Plasmid name: pPLTACHTNFs (LMBP 2078)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Escherichia coli alkaline phosphatase A gene (phoA); signal sequence
Human tumor necrosis factor cDNA (TNF); mature sequence
|Promoter:||Escherichia coli hybrid tryptophan/lacUV5 promoter (tac)
Phage λ major leftward promoter (λ PL)
|Ribosome binding site (RBS) of the Escherichia coli alkaline phosphatase A gene (phoA)|
|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|
|Further information:||This plasmid was constructed as follows :first a KpnI site was created at the end of the signal sequence of phosphatase A (phoA) in pMC-TPBS1by site-specific mutagenesis. Then, after T4 exonuclease treatment of this KpnI site, a blunt end AvaI linker was added by ligation. The EcoRI-AvaI fragment of this plasmid (containing the tac promoter, the RBS and the start codons of mature human TNF) was inserted into the EcoRI and AvaI sites of pPLc245BHTNF
pPLTACHTNF expresses mature human TNF, and secretes it into the periplasmic space.
As a result, the mature human TNF gene was correctly fused to the amp signal sequence.
Expresssion in λ wt lysogen : constitutive synthesis through leaky tac promoter at any physiological temperature.
The presence of the lacI(q) repressor gene is necessary to control the activity of the tac promoter.
Expression in cIts : λ-PL induction at 42°C.
The first seven amino acids of the mature human TNF are identical to the wild type amino acids, but they are encoded by a synthetic linker sequence which differs from the wild type nucleotide sequence.
Other name of the plasmid is pPLTAHTNF.
|EMBL Accession number:||-|
|Latest sequence update:||29/08/1990|
AvaI linker 5' GTA.CGT.TCT.TCT.TCT.CGT.ACC 3' 3' CAT.GCA.AGA.AGA.AGA.GCA.TGG.GGC.T 5'
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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 Ronny Leemans
|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.