Last data update: 24 January 2024 16:39 CET
Plasmid name: pT73hIFNGEK (LMBP 3283)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | p3283.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Human interferon γ cDNA (IFNG); mutated mature sequence heat-shock chaperone grpE heat-shock chaperone DnaK |
| 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 |
| Terminator: | - |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pT73grpEdnaK; pPLT10hIFNGT |
| Further information: | The plasmid was constructed by cloning the SstI (SacI) - EcoRI fragment from plasmid pPLT10hIFNGT containing the mature human IFNG gene preceded by the phage T7 gene 10 promoter and ribosome binding site, into the SstI and EcoRI (nucleotide position 2725) sites of plasmid pT73grpEdnaK. pT73hIFNGEK is an expression vector for human IFNG and for the heat-shock chaperones GrpE and DnaK under control of the T7 promoter. GrpE and DnaK are useful to study their effect on the folding/solubility of the human IFNG protein expressed in E. coli. The dnaK and the grpE genes are preceded by their own ribosome binding site. 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 3064) 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. Since the nucleotide sequence downstream from the dnaK gene up to the HindIII site is not known, 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: | 31/03/1995 |
| 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: | 37°C |
| Biosafety level: | L1 |
| Cultivation remark: | - |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pT73hIFNGEK (LMBP 3283) 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.