Last data update: 04 December 2020 04:17 CET
Plasmid name: pLT10hIFNGT (LMBP 3282)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Human interferon γ cDNA (IFNG); mutated mature sequence|
|Promoter:||Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
|Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)|
|Terminator:||Phage T7 gene 10 terminator (T7g10)|
|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|
|Parental clone:||pLT10T; pTrphIFNGHS2|
|Further information:||The plasmid was constructed by inserting the SphI (blunt)/ BamHI fragment from pTrphIFNGHS2 containing the mature, mutated human interferon γ cDNA (hIFNG) into the ApaI (blunt)/BamHI opened pLT10T vector.
pLT10hIFNGT contains mutated mature human interferon γ cDNA (hIFNG); a unique SalI site and an extra HhaI site are created by silent mutations of amino acid 140 and 130-131 resp., the 3' UTR fragment is appox. 600 bp long.
This is an expression plasmid for mature hIFNG under control of the PL or the T7 promoter.
Restriction analysis suggests the presence of a XbaI site very close to the BamHI site, that is not retrieved in the sequence file.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene, as well as a cI function: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857]. Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
|EMBL Accession number:||-|
|Latest sequence update:||17/09/2003|
Start of the mature human IFN gamma cDNA: |-> mature hIFNG | 1 5 5' CTTTAAGAAGGAGATATACAT.ATG.CAG.GAC.CCA.TAT.GTA.AAA 3' Met Gln Asp Pro Tyr Val Lys Nucleotide sequence at the end of the hIFNG wt DNA: 130 140 143 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 hIFNG mutant: 130 140 143 5' CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CGA.GCA.TCC.CAG.TAA.TGGTTGTC 3' Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln * * * * HhaI SalI *: mutated nucleotide Punctuation indicated reading frame
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by Dr N. Mertens(1) (2) and 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
|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)|
|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.