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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMacBlaHirPDI (LMBP 3143)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p3143.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Ampicillin resistance gene (amp)
Human immunoglobulin γ3 cDNA (hIG3); heavy chain hinge region
Rat protein disulfide isomerase cDNA (rPDI)
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:
Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)
Terminator: Phage fd terminator
Selection marker: Ampicillin (amp)
Chloramphenicol (cam)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Parental clone: pMac254BLAHI; pSE380rPDI
Further information: The plasmid was constructed as follows: 1) pSE380rPDI was cleaved with AccI , filled in with Klenow DNA polymerase and further digested with BglII; 2) Then, the AccI-BglII fragment containing the lacI(q) gene and the rat PDI gene was inserted into the SmaI-BamHI vector fragment of pMac254BLAHI.
This plasmid is similar to pMacBlaHiDsbA, except for the cloned 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 phosphoribosyl transferase 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 rPDI gene, preceded by its own signal sequence, is expressed under control of the strong, 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.
pMacBlaHirPDI was used for the coproduction of rat protein disulfide isomerase and the cysteine-rich protein BlaHi in E. coli in order to study the effect of rat PDI on the formation of inter-chain disulfide bonds in the human γ3 hinge region.
The nucleotide sequence of the rPDI cDNA corresponds with the EMBL Nucleotide Sequence Database (Release 36; Accession number X02918).
EMBL Accession number: X02918, view at EMBL, GenBank, DDBJ
Latest sequence update: 23/01/1997
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 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: -
Related plasmid reference: De Sutter et al., Mol. Microbiol. 6 (1992), 2201-2208 [PMID: 1406260]
De Sutter et al., Gene 141 (1994), 163-170 [PMID: 8163184]
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 temperature: 37°C
Biosafety level: L1
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.

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