Last data update: 24 November 2020 04:18 CET
Plasmid name: pPLT10mIFNBmT (LMBP 3659)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Mouse interferon β cDNA (IFNB); mutated mature sequence|
|Promoter:||Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
|Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)|
|Terminator:||Phage fd terminator
Phage T7 gene 10 terminator (T7g10)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli; use strains with a cI function, cIts for PL controlled expression|
|Parental clone:||pLT10T3; pMacT10mIFNBm|
|Further information:||The plasmid was constructed by inserting the NdeI/HindIII fragment of pMacT10mIFNBm, containing the mature, mouse interferon β (IFNB) gene, between the NdeI and HindIII sites of the pLT10T3 vector.
pPLT10mIFNBmT is an expression phasmid for mutated mouse IFNB in Escherichia coli under control of the strong and efficiently regulatable λ PL or T7 gene 10 promoter.
The mutations in the mature mouse IFNB gene should lead to the formation of intra-chain disulfide bonds in the expressed protein, resulting in a more stable product.
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.
Other name of the plasmid is pPLT10mIFNBm.
|EMBL Accession number:||-|
|Latest sequence update:||04/05/1999|
Nucleotide sequence around the Shine-Dalgarno (SD) position of the T7 gene 10 ribosome binding site: | 5' TAATACGACTCACTATA|GGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTT ---------------->| XbaI T7 promoter 1 2 3 4 5 6 TAAGAAGGAGATATACAT ATG.ATC.ACC.TAT.AAG.CAG.CTC … 3' ------ NdeI |--> mature IFNB SD Nucleotide sequence around the codon at amino acid position 17 of the mature mIFNB gene: 14 15 16 17 18 19 20 Ile Arg Lys Cys Gln Glu Leu wt mIFNB: 5' ... .ATT.CGG.AAA.TGT.CAG.GAG.CTC. ... 3' SacI 14 15 16 17 18 19 20 Ile Arg Lys Ser Gln Glu Leu mutated mIFNB: 5' ... .ATT.CGG.AAA.TCC.CAG.GAG.CTC. ... 3' ** SacI BsaJI Nucleotide sequence around the codon at amino acid position 29 of the mature mIFNB gene: 26 27 28 29 30 31 32 Gly Lys Ile Asn Leu Thr Tyr wt mIFNB: 5' ... .GGA.AAG.ATC.AAC.CTC.ACC.TAC. ... 3' 26 27 28 29 30 31 32 Gly Lys Ile Cys Leu Thr Tyr mutated mIFNB: 5' ... .GGA.AAG.ATC.TGC.CTC.ACC.TAC. ... 3' BglII ** Nucleotide sequence around the codons at amino acid positions 136 and 137 of the mature mIFNB gene: 133 134 135 136 137 138 139 Tyr Asn Ser Tyr Ala Trp Met wt mIFNB: 5' ... .TAC.AAC.AGC.TAC.GCC.TGG.ATG. ... 3' BstNI Tyr Asn Ser Cys Ala Trp Met mutated mIFNB: 5' ... .TAC.AAC.AGC.TGC.GCA.TGG.ATG. ... 3' PvuII * * FspI SD: Shine-Dalgarno. *: Mutated nucleotide Punctuation indicates reading frame.
|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
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061[pICA2]|
|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.