Last data update: 24 January 2024 16:39 CET
Plasmid name: pOPINE-ME-BRIL-hDRD2-EGFP-His (LMBP 10448)
New search | Print data sheet |
Price category: | Cat. 1 (cf. price list) |
Status: | GeneCorner non-core plasmid |
Depositor's sequence: | p10448.gb |
Sequence analysis results Genecorner: |
- |
Cloned DNA: |
Apis mellifera melittin cDNA (Melt, GeneID 406130); signal sequence Escherichia coli cytochrome b562 cDNA (GeneID 13703733); codon-optimized fragment (BRIL), N-terminal Human dopamine receptor D2 cDNA (DRD2, GeneID 1813) Human rhinovirus 3C protease cleavage site (PreScission site, PRS); C-terminal Aequorea victoria green fluorescent protein DNA (GFP); enhanced red-shifted variant (EGFP), C-terminal Histidine tag (His-tag); C-terminal |
Promoter: | Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only Chicken β-actin promoter (ACTB) Phage T7 gene 10 promoter (T7g10) with lac operator (T7lac) Autographa californica nuclear polyhedrosis virus (AcNPV) p10 promoter (p10) |
Ribosome binding site: |
Internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) polyprotein gene |
Terminator: | Rabbit β-globin polyadenylation signal (β-globin polyA) Phage T7 gene 10 terminator (T7g10) |
Selection marker: | Ampicillin (amp) Neomycin (neo; G418; kanamycin (kan)) |
Replicon: | Escherichia coli plasmid pMB1 origin |
Host range: | Escherichia coli Mammalian cells Insect cells; e.g. Sf9 cells |
Parental clone: | pOPINE |
Further information: | The plasmid was constructed by cloning the BRIL-DRD2 coding sequence, with the Apis mellifera mellitin signal sequence and C-terminal EGFP reporter gene, into the pOPINE vector. The dopamine receptor D2 belongs to the G protein-coupled receptors. This vector contains the hybrid CMV-enhancer/Chicken β-actin promoter that has been reported to give higher expression levels compared to vectors using CMV-derived promoter and enhancer. In addition this vector contains a Kozak consensus sequence for efficient initiation of translation in eukaryotic hosts. The presence of the p10 baculoviral promoter and the flanking lef2/ORF603 and ORF1629 baculoviral recombination sites allow the construction of recombinant baculoviruses and, finally, a T7 polymerase promoter with lacO operator offers high level inducible expression in E.coli harbouring the λ (DE3) prophage. The BRIL soluble domain promotes expression by stabilizing the receptor and increasing the solubility. The C-terminal EGFP reporter gene and the C-terminal His-tag can be removed from the encoded protein through the HRV-3C protease cleavage site or PreScission site. The nucleotide sequence of the pOPINE vector corresponds with the EMBL Nucleotide Sequence Database accession number EF372397.1. Other name of the plasmid is pOPINE-ME-BRIL-D2-EGFP-His. |
EMBL Accession number: | EF372397.1, view at EMBL, GenBank, DDBJ |
Latest sequence update: | 16/08/2017 |
Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
Class: | Recombinant plasmid |
Type: | Plasmid |
History of deposit: | The plasmid was deposited by Prof. Dr S. Savvides(1)(2). It was constructed by Dr. I. Ramou(1)(2). (1) Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium (2) VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium |
Plasmid reference: | - |
Related plasmid reference: | Berrow et al., Nucleic Acids Res. 35 (2007), e45 [PMID: 17317681] [DOI: 10.1093/nar/gkm047] |
Restricted distribution: | - BCCM MTA |
Distributed as: | H/P active culture or plasmid DNA |
Host for distribution: | Escherichia coli K12 DH5α |
Host reference: | Focus 8 (1986), 9 |
Related host reference: | Woodcock et al., Nucleic Acids Res. 17 (1989), 3469-3478 [PMID: 2657660] Rodriguez-Quinones et al., Focus 15 (1993), 110-112 Hanahan, J. Mol. Biol. 166 (1983), 557-580 [PMID: 6345791] [DOI: 10.1016/s0022-2836(83)80284-8] Hanahan, in 'DNA Cloning: A Practical Approach Volume I', IRL Press, Oxford (1985), 109-135 [ISSN/ISBN: 0947946187] Grant et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 4645-4649 [PMID: 2162051] |
Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
Cultivation temperature: | 37°C |
Biosafety level: | L1 |
Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pOPINE-ME-BRIL-hDRD2-EGFP-His (LMBP 10448) is available at BCCM/GeneCorner. The plasmid was deposited by Prof. Dr S. Savvides. |
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.