Gateway vector pdonr207

Manufactured by Thermo Fisher Scientific

The Gateway vector pDONR207 is a plasmid designed for use in the Gateway cloning system. It facilitates the transfer of DNA sequences between different vector systems through site-specific recombination. The vector contains attP recombination sites that allow for the efficient and directional insertion of DNA fragments into the vector.

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Lab products found in correlation

Phusion polymerase, by Thermo Fisher Scientific (1 mentions) Lr clonase, by Thermo Fisher Scientific (1 mentions) Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions) Sp 2 software package, by Leica (1 mentions)

Spelling variants of this product

PDONR207 (106 citations) PDONR207 vector (25 citations) GATEWAY entry vector pDONR207 (2 citations)

5 protocols using gateway vector pdonr207

Transgenic Fly Lines for p38c Study

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UAS-p38c construct: A full-length cDNA of p38c was amplified from total cDNA of Oregon^R flies and cloned into the pDONR207 Gateway vector (Invitrogen) and subsequently sub-cloned in the pTW (Drosophila Genomics Resource Center plasmid) transgenesis vector and used to generate transgenic flies. A fly line carrying the transgene on the third chromosome was established and used as UAS-p38c.
P[p38c] rescue construct: To generate a rescue transgene of p38c, we amplified by PCR a fragment comprising the coding region of p38c with the 3′UTR and 300 bp of the upstream sequence (corresponding to the sequence between p38c and p38a,). The amplicon was cloned into pCasper4 plasmid using the restriction sites Not1 and Xho1, and used for generating transgenic flies according to standard procedures. Fly line carrying the transgene on the second chromosome, and was introgressed into the p38c^7B1 mutant.

Chakrabarti S., Poidevin M, & Lemaitre B. (2014). The Drosophila MAPK p38c Regulates Oxidative Stress and Lipid Homeostasis in the Intestine. PLoS Genetics, 10(9), e1004659.

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Cloning and Characterization of IRE1 Promoters

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DNA extraction was performed from 1 month old plants with 200 μl CTAB extraction buffer (2% cetyl-trimethyl ammonium bromide, 100 mM tris [pH 8.0], 20 mM EDTA pH [8.0], 1.4 M NaCl, 0.5% β-Mercaptoethanol, 2% polyvinyl pyrrolidone). The promoter region of IRE1a (~ 1.267 kb) and IRE1b (~ 1.477 kb) from different accessions were amplified from genomic DNA by PCR using Phusion Polymerase (Thermo Scientific) with attB-flanked primers (Table S1). The PCR products were cloned into pDONR207 Gateway vector via BP reactions (Invitrogen). After confirming the entry clones through PCR and Sanger sequencing (primers listed in Table S1), destination clones were constructed by LR reactions with binary Gateway vector pAM-PAT-GW-GUS and confirmed through PCR and Sanger sequencing. The plant expression vector pAM-PAT-35S-GW-GUS was a gift from Drs. Nico Dissmeyer and Imre Somssich (Addgene plasmid # 80,678; https://n2t.net/addgene:80678; RRID:Addgene_80678). The resulting pAM-PAT-promoter-GUS constructs were transformed into Agrobacterium tumefaciens (strain GV3101) for transient expression assays.

Afrin T., Seok M., Terry B.C, & Pajerowska-Mukhtar K.M. (2020). Probing natural variation of IRE1 expression and endoplasmic reticulum stress responses in Arabidopsis accessions. Scientific Reports, 10, 19154.

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Bimolecular Fluorescence Complementation Assay

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For the generation of BiFC constructs, the full length open reading frame (ORF), excluding the stop codon, encoding Trx y2 and FBPase were amplified with iProof™ High-Fidelity DNA Polymerase (Bio-Rad) using oligonucleotides specified in Table S2, which added attB recombination sites at the 5′ and 3′ ends, respectively. The PCR products were cloned in the Gateway vector pDONR207 (Invitrogen) and sequenced. The cloned Trx y2 and FBPase fragments were then transferred to the BiFC vectors [44 (link)] pSPYNE-35S_GW and pSPYCE-35S_GW, respectively, using the LR clonase (Invitrogen). The resulting plasmids, pSPYNE:Trx y2 and pSPYCE:FBPase, were then transformed into the A. tumefaciens strain GV301. For BiFC assays, Agrobacterium strains carrying individual Trx y2, FBPase, Trx x [45 (link)], Trx f1 [25 (link)], glutamine synthetase 2 (GS2) [28 (link)] or 2-Cys Prxs [45 (link)] constructs were mixed at a 1:1 ratio and infiltrated into leaves of 4-week-old Nicotiana benthamiana plants. Leaf sections were analyzed 4 days later by confocal microscopy performed with a Leica SP/2 inverted microscope. Image analysis was performed with the Leica SP/2 software package and the ImageJ bundle provided by the Wright Cell Imaging facility.

Jurado-Flores A., Delgado-Requerey V., Gálvez-Ramírez A., Puerto-Galán L., Pérez-Ruiz J.M, & Cejudo F.J. (2020). Exploring the Functional Relationship between y-Type Thioredoxins and 2-Cys Peroxiredoxins in Arabidopsis Chloroplasts. Antioxidants, 9(11), 1072.

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Characterization of Arabidopsis lincRNA11195

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Seeds of C. rubella and A. thaliana T-DNA insertion mutants including 11195-1 (CS843057), 11195-2 (CS834193) and ddm1-10 (SALK_093009) were obtained from Arabidopsis Biological Resource Center (ABRC). ABA insensitive mutant used in this study is pyr1/pyl1/pyl4 (Park et al., 2009 (link)). For generating transgenic lincRNA11195 plants with or without the LTR, DNA fragments containing 1.5 kb upstream of lincRNA11195 and the fulllength or lacking LTR region lincRNA sequence plus a 200-bp downstream sequence with attB sites were amplified from Col- 0 genomic DNA, and were then cloned into Gateway vector pDONR207 (Invitrogen). Each insert was subsequently introduced into the Gateway pGWB1 vector by LR reaction (Invitrogen). All plasmids were transformed into Agrobacterium tumefaciens strain GV3101, and then transformed into A. thaliana plants of the mutant backgrounds via the floral dip method. Stress treatment was carried out as described previously (Zeller et al., 2009 (link)). Preparation of cDNA and real-time quantitative PCR were performed according to the previous description (Wang et al., 2014 (link)). RT-PCR and strand-specific RTPCR were carried out as described previously (Wierzbicki et al., 2008 (link)). All experiments were carried out with at least three biological replicates. Details of the primers used in this study are listed in Table S8.

Wang D., Qu Z., Yang L., Zhang Q., Liu Z.H., Do T., Adelson D.L., Wang Z.Y., Searle I, & Zhu J.K. (2017). Transposable elements (TEs) contribute to stress-related long intergenic noncoding RNAs in plants. The Plant journal : for cell and molecular biology, 90(1), 133-146.

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Virus-Induced Gene Silencing in Tomato Fruit

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Vector construction, infiltration, and fruit harvesting procedures were performed as previously described (Orzaez et al., 2006 (link), 2009 (link)). Briefly, an approximately 300-bp fragment of the candidate gene was amplified from tomato M82 fruit cDNA using gateway compatible primers and recombined into the GATEWAY vector pDONR207 (Invitrogen, http://www.invitrogen.com/) by the BP reaction following the manufacturer’s protocol to generate an entry clone. An error-free entry vector was confirmed by sequencing and then recombined with the pTRV2-Ros/Del/GW destination vector using an LR reaction to produce the expression clones pTRV2-Ros/Del/GW-Respective Gene ID. Agrobacterium tumefaciens strain GV3101:pMP90 was then transformed with sequenced expression vectors by electroporation. In order to infiltrate fruit for VIGS, purple MicroTom tomato was used and agroinfiltration was performed as previously described (Alseekh et al., 2015 (link)).

Zhu F., Jadhav S.S., Tohge T., Salem M.A., Lee J.M., Giovannoni J.J., Cheng Y., Alseekh S, & Fernie A.R. (2022). A comparative transcriptomics and eQTL approach identifies SlWD40 as a tomato fruit ripening regulator. Plant Physiology, 190(1), 250-266.

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