Last data update: 24 January 2024 16:39 CET
Plasmid name: pT73hIFNGEKJ (LMBP 3073)
New search | Print data sheet |
Price category: | Cat. 1 (cf. price list) |
Status: | GeneCorner non-core plasmid |
Depositor's sequence: | p3073.gb |
Sequence analysis results Genecorner: |
- |
Cloned DNA: |
Human interferon γ cDNA (IFNG); mutated mature sequence heat-shock chaperone grpE heat-shock chaperone DnaK Heat-shock protein DnaJ |
Promoter: | Phage T7 gene 10 promoter (T7g10) |
Ribosome binding site: |
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10) Ribosome binding site (RBS) of the Escherichia coli heat-shock chaperone GrpE Ribosome binding site (RBS) of the Escherichia coli heat-shock chaperone DnaK DnaJ |
Terminator: | - |
Selection marker: | Ampicillin (amp) |
Replicon: | Escherichia coli plasmid pMB1 origin |
Host range: | Escherichia coli |
Parental clone: | pT73hIFNGEK; pDNAJ-A |
Further information: | The plasmid was constructed by inserting the dnaJ gene as a PstI (blunted with T4 DNA polymerase) - SalI fragment of pDNAJ-A (Ohki et al., J. Biol. Chem. 261 (1986), 1778-1781) into the SalI site (nucleotide position 122) of pT73hIFNGEK. pT73hIFNGEKJ is an expression vector for human IFNG and for the heat-shock chaperones GrpE, DnaK and DnaJ under control of the T7 promoter. GrpE, DnaK and DnaJ are useful to study their effect on the folding/solubility of the human IFNG protein expressed in E. coli. The dnaJ, the dnaK and the grpE genes are preceded by their own ribosome binding site, while the hIFNG gene is preceded by the ribosome binding site of phage T7 gene 10. The cloned hIFNG gene contains a SalI site at position 413 (amino acid 143) and an extra HhaI site at position 390 of the mature sequence. The amino acid sequence of the mature hIFNG peptide is the same as the natural one and starts with Met-Gln. This plasmid also contains an undefined part (approx. 600 bp) of the 3' untranslated region of the hIFNG gene. This 3' UTR region ends at the BamHI site and contains a BglI site (presumably at position 5328) and an XbaI site (very close to the BamHI site). For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3). Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid. The nucleotide sequence of the dnaJ gene corresponds with the EMBL Nucleotide Sequence Database (Release 33; Accession number M12544). Since the nucleotide sequence downstream from the dnaJ gene up to the HindIII site is unknown, and the included part of the 3' UTR region of the hIFNG gene is undefined, there is uncertainty as to the cutting frequency of the restriction enzymes indicated. |
EMBL Accession number: | - |
Latest sequence update: | 07/06/1995 |
Sequence detail: | Nucleotide sequence at the 3' end of the wt hIFNG gene. Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln * CGA.AAA.AGG.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.AGA.GCA.TCC.CAG.TAA Nucleotide sequence at the 3' end of the mutated hIFNG gene. Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln * CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CGA.GCA.TCC.CAG.TAA ^ ^ ^ ^ HhaI SalI *: Termination codon ^: Mutated nucleotide Punctuation indicates reading frame. |
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 J. Robbens(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 |
Plasmid reference: | PhD thesis Johan Robbens (1994) |
Restricted distribution: | - BCCM MTA |
Distributed as: | H/P active culture or plasmid DNA |
Host for distribution: | Escherichia coli K12 MC1061 |
Host reference: | Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
Related host reference: | Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111] |
Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
Cultivation temperature: | 28°C |
Biosafety level: | L1 |
Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pT73hIFNGEKJ (LMBP 3073) is available at BCCM/GeneCorner. This plasmid was deposited by J. Robbens and Prof. Dr E. Remaut and was published in Johan Robbens, 1994. |
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.