Last data update: 28 November 2020 04:24 CET
Plasmid name: pMacBlaHiDsbA (LMBP 3060)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Ampicillin resistance gene (amp)
Human immunoglobulin γ3 cDNA (hIG3); heavy chain hinge region
Escherichia coli gene facilitating disulfide bond formation (dsbA)
Escherichia coli lac repressor gene (lacI)
|Promoter:||Escherichia coli hybrid tryptophan/lacUV5 promoter (trc)
Escherichia coli lac repressor promoter; mutant (lacI(q))
|Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)|
|Terminator:||Phage fd terminator|
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli|
|Parental clone:||pMacBlaHirPDI; pSE380DsbA|
|Further information:||The plasmid was constructed by substituting the SphI-XbaI fragment from pMacBlaHirPDI containing the lacI(q) gene and the rat PDI gene with the corresponding fragment from pSE380DsbA containing the lacI(q) gene and the dsbA gene.
The BlaHi gene, consisting of the ampicillin resistance gene fused in phase to the start of the first hinge region H1 of the human immunoglobulin γ3 heavy chain, is cloned downstream from the constitutive kanamycin phosphoribosyltransferase promoter. The translation of the fusion gene stops 8 amino acids after the end of the hinge region. The fusion product still provides resistance to ampicillin.
The mature dsbA gene, preceded by its own signal sequence, is expressed under control of the IPTG-inducible trc promoter, followed by the lacZ ribosome binding site.
The phasmid also contains the lacI(q) gene, leading to overproduction of the lac repressor (LacI), to repress the trc promoter in order to expand the host range of the vector to non-lacI(q) strains (e.g. MC1061).
The f1 part of the phasmid contains the f1 origin, but also the sequence around the gene II promoter.
pMacBlaHiDsbA was used for the coproduction of DsbA and the cysteine-rich fusion protein BlaHi in E. coli in order to study the effect of DsbA on the formation of inter-chain disulfide bonds in the human γ3 hinge region.
The nucleotide sequence of the dsbA coding region (starting at the ATG start codon) up to the SspI site was taken from the publication of Bardwell et al. Since this sequence has not been experimentally verified, we cannot exclude that some nucleotides in the present gene are different.
Since most of the nucleotide sequence of the 3' flanking region of the dsbA gene is not known, there is uncertainty as to the cutting frequency of the restriction enzymes indicated.
|EMBL Accession number:||-|
|Latest sequence update:||23/01/1997|
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|History of deposit:||This plasmid was deposited by Dr K. De Sutter(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||De Sutter et al., Mol. Microbiol. 6 (1992), 2201-2208 [PMID: 1406260]
|Related plasmid reference:||Bardwell et al., Cell 67 (1991), 581-589 [PMID: 1934062]
|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) + chloramphenicol (25 μg/ml)*|
|Cultivation remark:||*: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin.|
|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.