Last data update: 15 January 2021 04:14 CET
Plasmid name: pLPPHIs (LMBP 2587)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Escherichia coli outer membrane protein II gene (ompA); signal sequence
Human immunoglobulin γ3 cDNA (hIG3); heavy chain hinge region
Escherichia coli lac repressor gene (lacI)
|Promoter:||Escherichia coli lipoprotein promoter (lpp)
Escherichia coli lac operon promoter; mutant (lacUV5)
Escherichia coli lac repressor promoter; mutant (lacI(q))
|Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ)|
|Terminator:||Escherichia coli lipoprotein terminator (lpp)|
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin|
|Host range:||Escherichia coli|
|Parental clone:||p714; pMcHIs|
|Further information:||The plasmid was constructed by substituting the XbaI-BamHI fragment of p714, containing the sompA-hB2M fusion gene, by the XbaI-BamHI fragment of pMcHIs, containing the human immunoglobulin γ3 hinge region fused in phase to the ompA signal sequence.
The expression of the sompA-Hi fusion gene is controlled by the E. coli lpp promoter.
The translation of the fusion protein stops two amino acids after the fourth hinge region (H4), caused by the presence of a termination codon in the CH2 domain.
The plasmid also contains the lacI(q) repressor cassette.
The nucleotide sequence of the inserted human γ3 fragment was obtained from Genbank 'Release 55; Locus: HUMIGHAF'. The unknown nucleotides in the Genbank-obtained sequence have been sequenced (Dr R. Schoonjans, Department of Biomedical Molecular Biology, Ghent University, Belgium). These nucleotides correspond with the nucleotides of the genomic E6Hy3 fragment of pATE6Hy3.
The nucleotide sequence at the 5' and 3' end of the E. coli lacI insert of the plasmid pLPPsOmpArPDI has been sequenced at Roche Research USA and was kindly provided by Dr. Yves Guisez from Roche Research Ghent. The unknown nucleotides were replaced by the corresponding nucleotides of the E. coli K12 genome sequence obtained from Genbank (Accession number NC_000913.1).
Based on these data, the nucleotide sequence of pLPPHIs has been adapted.
|EMBL Accession number:||NC_000913.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||26/11/2003|
Nucleotide sequence at the fusion between the ompA signal sequence and the hIG3 hinge region: sompA H1-4 CH2 fragment -------> ------------------> ----------------------> 5' ... CAG.GCC.GAG.CTC ... TGC.CCA.GGG.CCC.TAA ... AAGTGGT ... 3' Gln Ala Glu Leu Cys Pro Gly Pro +++ SacI ^^ ^ ^ ApaI ^ : Mutated nucleotide. +++: Termination codon. 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 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
|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)|
|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.