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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPLT10igapT (LMBP 3243)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p3243.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Neisseria gonorrhoeae NCTC8375 gene encoding IgA1 protease (iga); fragment encoding the protease domain
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; pPLT10igaT; pMc514igam2
Further information: The plasmid was constructed via a three-point-ligation of the following fragments: 1) the PstI-BglII fragment of pPLT10igaT, encoding the N-terminal part of the IgA1 protease gene downstream from the PL and the T7 promoters, as well as part of the ampicillin resistance gene; 2) the BglII-ScaI fragment of pMc514igam2, encoding the C-terminal part of the IgA1 protease gene; 3) The SmaI-PstI vector fragment of pLT10T, encoding the origin of replication and the remaining part of the ampicillin resistance gene.
Due to ligation of the ScaI site (located in the iga gene at the mutated end of the protease domain) to the SmaI site (a restriction site of the expression vector pLT10T), translation stops 20 amino acids after the end of the iga protease domain: see SD.
This is an expression plasmid for the mature IgA1 protease (containing 20 additional amino acids at the C-terminus) under control of the PL or the T7 promoter.
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.
To reconstruct the nucleotide sequence of this plasmid, the sequence of the IgA1 protease gene from Neisseria gonorrhoeae MS11 corresponds with the EMBL Nucleotide Sequence Database (Release 36; Accession number X04835). However, the cloned IgA1 protease gene was derived from Neisseria gonorrhoeae NCTC8375 and differs in several nucleotides. As such, there is uncertainty as to the cutting frequency of the restriction enzymes indicated.
Other name of the plasmid is pLT10tIgAprot.
EMBL Accession number: -
Latest sequence update: 26/07/1995
Sequence detail:
Nucleotide sequence at the C-terminus of the protease domain of the IgA1 protease gene.


   Ala Ser|Gly Phe Glu Ile Asp Lys Leu Gly Ser Gly Glu Leu Pro
5' GCC.AGT|GGG.TTC.GAA.ATC.GAT.AAG.CTT.GGA.TCC.GGA.GAG.CTC.CCA.
         ^|---> vector part
       ---|---
      ScaI/SmaI fusion


   Thr Arg Trp Met Ser Asn Asn *
   ACG.CGT.TGG.ATG.AGC.AAT.AAC.TAG 3'


^: End of the protease domain of the IgA1 protease gene.
*: Termination codon.
Punctuation indicates 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 I. Poelaert(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: PhD thesis Irmgard Poelaert (1995)
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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