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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPLc245BHTNFs (LMBP 1408)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p1408.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Ampicillin resistance gene (amp); signal sequence
Human tumor necrosis factor cDNA (TNF); mature sequence
Promoter: Phage λ major leftward promoter (λ PL)
Ribosome
binding site:
Ribosome binding site (RBS) of the phage MS2 polymerase gene
Terminator: -
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli; use strains with a cI function, cIts for PL controlled expression
Parental clone: pPLc245B; pPLcmuHTNF1
Further information: The plasmid was constructed as follows : First, an AvaI linker was ligated to the ApaI site (blunted with T4 polymerase exonuclease) of pPLc245B; Next, pPLc245B was cut with AvaI and HindIII, followed by insertion of the AvaI-HindIII fragment of pPLcmuHTNF1, containing the mature human TNF gene. As a result, the mature human TNF gene was correctly fused to the amp signal sequence.
pPLc245BHTNFs is an expression plasmid, designed for expression of human TNF.
The first seven amino acids of the mature human TNF are identical to the wild type amino acids, but they are encoded by a synthetic linker sequence which differs from the wild type sequence.
Strains containing pPLc245BHTNFs secrete mature human TNF into the periplasmic space upon temperature shift (42°C).
The ampicillin resistance gene does not contain a PstI site.
The nucleotide sequence of the mature human TNF sequence corresponds with the EMBL Nucleotide Sequence Database accession number X01394.1, except for the AvaI linker.
Name mentioned in Leemans et al. (1989) is pPLc245BhTNF.
Other name of the plasmid is pPLc245BTNF.
EMBL Accession number: X01394.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 29/08/1990
Sequence detail:
Nucleotide sequence at the junction between the amp signal sequence and the mature human TNF gene.


wild type TNF     Ala Gln Ala|Val Arg Ser Ser Ser Arg Thr Pro Ser
                  GCC.CAG.GCA|GTC.AGA.TCA.TCT.TCT.CGA.ACC.CCG.AGT
                            *|@                         AvaI



pPLc245BHTNFs     Val Phe Ala|Val Arg Ser Ser Ser Arg Thr Pro Ser
                  GTT.TTT.GCG|GTA.CGT.TCT.TCT.TCT.CGT.ACC.CCG.AGT
                            ^|@
                             |---------------------------
                             |                          AvaI


*: End of the wild type human TNF presequence.
^: End of the ampicillin signal sequence.
@: Start of the mature human TNF gene.
-: AvaI linker 5' GTA.CGT.TCT.TCT.TCT.CGT.ACC       3'
               3' CAT.GCA.AGA.AGA.AGA.GCA.TGG.GGC.T 5'
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 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: Leemans et al., Gene 85 (1989), 99-108 [PMID: 2695405]
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 SG4044[pcI857]
Host reference: Remaut et al., Nucleic Acids Res. 11 (1983), 4677-4688 [PMID: 6308555]
Helper plasmid: pcI857
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml) + kanamycin (50 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
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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