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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pMcMTNFm2t (LMBP 2655)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p2655.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Mouse tumor necrosis factor cDNA (Tnf, TNF); mutated mature sequence
Promoter: -
Ribosome
binding site:
-
Terminator: Phage fd terminator
Selection marker: Chloramphenicol (cam)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Parental clone: pMaMTNF
Further information: This plasmid was constructed by site-specific mutagenesis, where the nucleotide sequences of codons 2 and 4 of mature mouse TNF (AGA, coding for Arg, and TCT, coding for Ser) were converted to TGA (opal) and TAG (amber), respectively. This mutagenesis creates a unique BclI restriction site.
Restriction enzyme digestions revealed that approx. 60 nucleotides are missing from the 3' untranslated region of mouse TNF, including the 3'-terminal (supposedly restored) EcoRI site.
After mutagenesis, use mutS strains for primary transformation (e.g. sup(-) strain WK6mutS, sup(+) strain BMH71-18mutS); for segregation of possible mutants: sup(-) strains (e.g. WK6).
The nucleotide sequence of the wild type mouse TNF corresponds with the EMBL Nucleotide Sequence Database accession number M11731.1.
EMBL Accession number: M11731.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 25/01/1993
Sequence detail:
Nucleotide sequence at the start of mature TNF:

   presequence  |  mature mTNF
                |  1  2   3   4   5   6   7   8   9   10  11
wild type 5' ACA CTC AGA TCA TCT TCT CAA AAT TCG AGT GAC AAG 3'
             Thr Leu Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys

                  1   2   3   4   5   6   7   8   9   10  11
mutant    5' ACA CTC TGA TCA TAG TCT CAA AAT TCG AGT GAC AAG 3'
             Thr Leu  *  Ser  *  Ser Gln Asn Ser Ser Asp Lys
                     BclI
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 W. Fiers(1) (2). It was constructed by X. Van Ostade(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 SURE
Host reference: Greener, Strategies 3 (1990), 5-6
Cultivation medium: LB-Lennox + chloramphenicol (25 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
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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