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GeneCorner plasmid details

Last data update: 04 July 2022 09:25 CEST

Plasmid name: pET-28a-MBP-TEV-Canf5 (LMBP 11977)

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Price category: Cat. 3 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p11977.gb (View with Genome Compiler)
p11977.txt
p11977.pdf
Sequence
analysis results
Genecorner:

NGS: f04-gc-dec2019.fasta

Cloned DNA: Canis lupus familiaris kallikrein-related peptidase 2 cDNA (KLK2, Can f5, Can f 5, GeneID 403967); mature sequence
Escherichia coli maltose ABC transporter periplasmic binding protein cDNA (malE, GeneID 948538); mature sequence, N-terminal
Thrombin recognition site; N-terminal
Tobacco etch virus (TEV) protease recognition site; N-terminal
Histidine tag (His-tag); N-terminal
Escherichia coli DNA-binding transcriptional repressor LacI cDNA (lacI, GeneID 945007)
Histidine tag (His-tag); C-terminal
Promoter: Phage T7 gene 10 promoter (T7g10) with lac operator (T7lac)
Escherichia coli lac repressor promoter (lacI)
Ribosome
binding site:
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)
Terminator: Phage T7 gene 10 terminator (T7g10)
Selection marker: Neomycin (neo; kanamycin (kan))
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Parental clone: pET28a-MBP-TEV
Further information: The plasmid was constructed by cloning the dog KLK2 coding sequence, optimised for E. coli, into the BamHI/XhoI opened pET28a-MBP-TEV vector.
The plasmid was designed to express the malE-KLK2 fusion protein in E. coli BL21(DE3). The plasmid encodes for the mature form of the E. coli maltose binding protein, which does not contain a signal sequence, hence the protein will remain in the cytosol.
The N-terminal His-tag can be removed via a thrombin recognition site, while the maltose binding protein can be separated from the dog KLK2 allergen via the TEV protease recognition site. The His-tag at the C-terminal end of the allergen is not functional in this plasmid.
The amino acid sequence of the dog Can f 5 allergen corresponds with GenBank accession number NP_001003284.1 except for a K106E substitution.
EMBL Accession number: NP_001003284.1, view at EMBL, GenBank, DDBJ
Latest sequence update: 06/11/2019
Authenticity test: This plasmid has been fully sequenced.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: The plasmid was deposited by Dr G. Zvirblis(1). It was constructed by K. Juskaitė(1) and Dr G. Zvirblis(1).
(1) Institute of Biotechnology, Vilnius University, Vilnius, Lithuania
Plasmid reference: -
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 DH5αT1R
Host reference: -
Related host reference: Killmann et al., J. Bacteriol. 178 (1996), 6913-6920 [PMID: 8955314] [DOI: 10.1128/jb.178.23.6913-6920.1996]
Cultivation medium: LB-Lennox + kanamycin (50 μg/ml)
Cultivation temperature: 37°C
Biosafety level: L1
Other culture collection numbers: -

Refer in your Materials and Methods:

pET-28a-MBP-TEV-Canf5 (LMBP 11977) is available at BCCM/GeneCorner. The plasmid was deposited by Dr G. Zvirblis.

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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