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LMBP plasmid details

Last data update: 23 September 2020 04:27 CEST

Plasmid name: pLT10hIFNGT (LMBP 3282)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p3282.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Human interferon γ cDNA (IFNG); mutated mature sequence
Promoter: Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
Ribosome
binding site:
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)
Terminator: Phage T7 gene 10 terminator (T7g10)
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: pLT10T; pTrphIFNGHS2
Further information: The plasmid was constructed by inserting the SphI (blunt)/ BamHI fragment from pTrphIFNGHS2 containing the mature, mutated human interferon γ cDNA (hIFNG) into the ApaI (blunt)/BamHI opened pLT10T vector.
pLT10hIFNGT contains mutated mature human interferon γ cDNA (hIFNG); a unique SalI site and an extra HhaI site are created by silent mutations of amino acid 140 and 130-131 resp., the 3' UTR fragment is appox. 600 bp long.
This is an expression plasmid for mature hIFNG under control of the PL or the T7 promoter.
Restriction analysis suggests the presence of a XbaI site very close to the BamHI site, that is not retrieved in the sequence file.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene, as well as a cI function: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857]. Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
EMBL Accession number: -
Latest sequence update: 17/09/2003
Sequence detail:
Start of the mature human IFN gamma cDNA:
                        |-> mature hIFNG 
                        | 1               5
5' CTTTAAGAAGGAGATATACAT.ATG.CAG.GAC.CCA.TAT.GTA.AAA 3'
                         Met Gln Asp Pro Tyr Val Lys

Nucleotide sequence at the end of the hIFNG wt DNA:
       130                                     140         143
5' CGA.AAA.AGG.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.AGA.GCA.TCC.CAG.TAA.TGGTTGTC 3'
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg arg Ala Ser Gln

Nucleotide sequence at the end of the hIFNG mutant:
       130                                     140         143
5' CGA.AAG.CGC.AGT.CAG.ATG.CTG.TTT.CGA.GGT.CGA.CGA.GCA.TCC.CAG.TAA.TGGTTGTC 3'
   Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln
         * * *                                 *
         HhaI                           SalI

*: mutated nucleotide
Punctuation indicated reading frame
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 N. Mertens(1) (2) and 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: -
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061(λ)
Host reference: Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 28°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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