pdonr221 vector  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pdonr221 vector
    Schematics of the DNA template construction and the CASP3-substrate-screening assay. Protein kinase ( PK ) genes were obtained from the human MGC and mouse FANTOM libraries, and from a library of PK genes that we had cloned. The PK genes were PCR amplified with the Flag and the biotin ligation site (bls) tags added to the upstream and downstream ends, respectively. The modified genes were each inserted into a Gateway <t>pDONR221</t> vector (pDONR-Flag-PK-bls) and DNA templates (SP6-E02-Flag- PK -bls) were constructed by split-primer PCR and then expressed in the wheat cell-free protein synthesis system that included biotin ligase and -biotin to give Flag-PK-bls∼biotin constructs. The Flag and biotin tags were bound to protein A-conjugated acceptor beads via an anti-Flag antibody and streptavidin-conjugated donor beads, respectively. An intact complex luminesced strongly, whereas after CASP3 cleavage and dissociation of the protein fragments, the luminescence was abolished or reduced
    Pdonr221 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pdonr221 vector - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system"

    Article Title: Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.65

    Schematics of the DNA template construction and the CASP3-substrate-screening assay. Protein kinase ( PK ) genes were obtained from the human MGC and mouse FANTOM libraries, and from a library of PK genes that we had cloned. The PK genes were PCR amplified with the Flag and the biotin ligation site (bls) tags added to the upstream and downstream ends, respectively. The modified genes were each inserted into a Gateway pDONR221 vector (pDONR-Flag-PK-bls) and DNA templates (SP6-E02-Flag- PK -bls) were constructed by split-primer PCR and then expressed in the wheat cell-free protein synthesis system that included biotin ligase and -biotin to give Flag-PK-bls∼biotin constructs. The Flag and biotin tags were bound to protein A-conjugated acceptor beads via an anti-Flag antibody and streptavidin-conjugated donor beads, respectively. An intact complex luminesced strongly, whereas after CASP3 cleavage and dissociation of the protein fragments, the luminescence was abolished or reduced
    Figure Legend Snippet: Schematics of the DNA template construction and the CASP3-substrate-screening assay. Protein kinase ( PK ) genes were obtained from the human MGC and mouse FANTOM libraries, and from a library of PK genes that we had cloned. The PK genes were PCR amplified with the Flag and the biotin ligation site (bls) tags added to the upstream and downstream ends, respectively. The modified genes were each inserted into a Gateway pDONR221 vector (pDONR-Flag-PK-bls) and DNA templates (SP6-E02-Flag- PK -bls) were constructed by split-primer PCR and then expressed in the wheat cell-free protein synthesis system that included biotin ligase and -biotin to give Flag-PK-bls∼biotin constructs. The Flag and biotin tags were bound to protein A-conjugated acceptor beads via an anti-Flag antibody and streptavidin-conjugated donor beads, respectively. An intact complex luminesced strongly, whereas after CASP3 cleavage and dissociation of the protein fragments, the luminescence was abolished or reduced

    Techniques Used: Screening Assay, Clone Assay, Polymerase Chain Reaction, Amplification, Ligation, Modification, Plasmid Preparation, Construct

    2) Product Images from "RNA‐mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus"

    Article Title: RNA‐mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus

    Journal: Molecular Plant Pathology

    doi: 10.1111/j.1364-3703.2010.00666.x

    A flow chart showing the construction of a hairpin RNA (hpRNA)‐expressing RNA interference (RNAi) vector using pSGate1. A DNA fragment from a target gene was amplified by polymerase chain reaction (PCR) with primers containing the att B1 and att B2 sites. The PCR product was cloned into the plasmid pDONR221 to create an entry clone by BP reaction. The insert in the entry clone then replaced the ccdB genes of pSGate1 by LR reaction, resulting in an RNAi construct expressing the hpRNA of the target gene.
    Figure Legend Snippet: A flow chart showing the construction of a hairpin RNA (hpRNA)‐expressing RNA interference (RNAi) vector using pSGate1. A DNA fragment from a target gene was amplified by polymerase chain reaction (PCR) with primers containing the att B1 and att B2 sites. The PCR product was cloned into the plasmid pDONR221 to create an entry clone by BP reaction. The insert in the entry clone then replaced the ccdB genes of pSGate1 by LR reaction, resulting in an RNAi construct expressing the hpRNA of the target gene.

    Techniques Used: Flow Cytometry, Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay, Construct

    3) Product Images from "From Gateway to MultiSite Gateway in one recombination event"

    Article Title: From Gateway to MultiSite Gateway in one recombination event

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-7-46

    pDONR-R4-R3 . BP recombination of the att R4- att R3 Gateway cassette flanked by att B1 and att B2 sites with a linear Gateway donor vector pDONR221 (Invitrogen) which leads to the creation of the vector pDONR-R4-R3 and a cytotoxic by-product.
    Figure Legend Snippet: pDONR-R4-R3 . BP recombination of the att R4- att R3 Gateway cassette flanked by att B1 and att B2 sites with a linear Gateway donor vector pDONR221 (Invitrogen) which leads to the creation of the vector pDONR-R4-R3 and a cytotoxic by-product.

    Techniques Used: Plasmid Preparation

    4) Product Images from "Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins"

    Article Title: Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156718

    Study overview. cDNA clones encoding the E3 ubiquitin ligases (E3s) used in this study were divided into two categories. The clones in category B could be used directly for split-primer PCR, but those in category A were inserted into vectors that cannot be used directly for split-primer PCR and thus were transferred into the pDONR221 vector by using the Gateway system. The names of the vectors and the origins of the clones are indicated.
    Figure Legend Snippet: Study overview. cDNA clones encoding the E3 ubiquitin ligases (E3s) used in this study were divided into two categories. The clones in category B could be used directly for split-primer PCR, but those in category A were inserted into vectors that cannot be used directly for split-primer PCR and thus were transferred into the pDONR221 vector by using the Gateway system. The names of the vectors and the origins of the clones are indicated.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation

    5) Product Images from "Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis"

    Article Title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

    Journal: The Scientific World Journal

    doi: 10.1155/2016/2597376

    Schematic representation of Nod2 and RICK, and sequencing chromatograms of Nod2 plasmids containing the BS/EOS-associated mutations, and protein syntheses. (a) Schematic representations of biotinylated wild-type and mutant Nod2. C-terminal biotinylated full-length wild-type Nod2 (Nod2-WT-Btn), full-length R334W-mutated Nod2 (Nod2-R334W-Btn), full-length N670K-mutated Nod2 (Nod2-N670K-Btn), and tandem CARD1- and CARD2-domains-only Nod2 (Nod2-CARDs) are indicated. (b) Schematic representations of FLAG-tagged wild-type and CARD-domain-only RICK. N-terminal FLAG-tagged full-length RICK (FLAG-RICK-FL) and CARD-domain-only RICK (FLAG-RICK-CARD) are indicated. The caspase recruitment domain (CARD) is indicated by black boxes. The nucleotide-binding oligomerization domain-containing protein (Nod) is indicated by grey boxes. Leucine-rich repeats are indicated by striped boxes. Amino acid sequence number and mutated amino acids are indicated under each schema. (c) Sequencing chromatograms of Nod2 and mutated-Nod2 plasmids. The wild-type and mutated-Nod2 plasmids pDONR221-Nod2-WT, pDONR221-Nod2-R334W, and pDONR221-Nod2-N670K were sequenced to confirm (from CGG to TGG corresponding to R334W in the right panel; from AAC to AAA corresponding to N670K in the left panel) mutations at the appropriate site. (d) Western blotting analysis of biotinylated Nod2 and its mutants. A total of 1.5 μ g of synthetic protein was subjected to SDS-PAGE followed by Western blotting. Protein detection on the membranes was performed using HRP-conjugated streptavidin. Molecular weights are indicated at right. (e) Western blotting analysis of FLAG-tagged RICK and CARD-domain-only RICK. A total of 1.5 μ g of synthetic protein was subjected to SDS-PAGE followed by Western blotting. Protein detection on the membranes was performed using anti-FLAG mAb M2. Molecular weights are indicated at right.
    Figure Legend Snippet: Schematic representation of Nod2 and RICK, and sequencing chromatograms of Nod2 plasmids containing the BS/EOS-associated mutations, and protein syntheses. (a) Schematic representations of biotinylated wild-type and mutant Nod2. C-terminal biotinylated full-length wild-type Nod2 (Nod2-WT-Btn), full-length R334W-mutated Nod2 (Nod2-R334W-Btn), full-length N670K-mutated Nod2 (Nod2-N670K-Btn), and tandem CARD1- and CARD2-domains-only Nod2 (Nod2-CARDs) are indicated. (b) Schematic representations of FLAG-tagged wild-type and CARD-domain-only RICK. N-terminal FLAG-tagged full-length RICK (FLAG-RICK-FL) and CARD-domain-only RICK (FLAG-RICK-CARD) are indicated. The caspase recruitment domain (CARD) is indicated by black boxes. The nucleotide-binding oligomerization domain-containing protein (Nod) is indicated by grey boxes. Leucine-rich repeats are indicated by striped boxes. Amino acid sequence number and mutated amino acids are indicated under each schema. (c) Sequencing chromatograms of Nod2 and mutated-Nod2 plasmids. The wild-type and mutated-Nod2 plasmids pDONR221-Nod2-WT, pDONR221-Nod2-R334W, and pDONR221-Nod2-N670K were sequenced to confirm (from CGG to TGG corresponding to R334W in the right panel; from AAC to AAA corresponding to N670K in the left panel) mutations at the appropriate site. (d) Western blotting analysis of biotinylated Nod2 and its mutants. A total of 1.5 μ g of synthetic protein was subjected to SDS-PAGE followed by Western blotting. Protein detection on the membranes was performed using HRP-conjugated streptavidin. Molecular weights are indicated at right. (e) Western blotting analysis of FLAG-tagged RICK and CARD-domain-only RICK. A total of 1.5 μ g of synthetic protein was subjected to SDS-PAGE followed by Western blotting. Protein detection on the membranes was performed using anti-FLAG mAb M2. Molecular weights are indicated at right.

    Techniques Used: Sequencing, Mutagenesis, Binding Assay, Western Blot, SDS Page

    Related Articles

    Transduction:

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual. .. Twenty-four hours after transfection the medium was exchanged and the supernatants containing retroviral particles were harvested after 24 and 48 h. For retroviral transduction 2 ml of the supernatants were added per well to Mefs seeded in 6-well plates with 50% confluency.

    Clone Assay:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Paragraph title: Cloning of AQP5-GFP fusion constructs for analysis in HEK293 cells ... Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: The reverse transcription–polymerase chain reaction (RT-PCR) procedure was 94°C for 4 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 35 cycles; and 72°C for 10 min. RT-PCR products were gel-purified and cloned into pMD19-T vector (TaKaRa, Dalian, China), and then transformed into E. coli DH5α competent cells and sequenced (Sangon, Shanghai, China). .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
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    Article Title: Reproducible gene targeting in recalcitrant Escherichia coli isolates
    Article Snippet: The Gateway® Technology (Invitrogen) is a universal cloning method providing a rapid and efficient way to move DNA sequences into multiple vector systems. .. The attB sites are used for a site-specific BP-recombination reaction with the attP sites of the pDONR221 vector, using the Gateway® technology (Invitrogen).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: .. Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen). .. The destination fragments then were introduced into the plant pEarleyGateway 102 , pEarleyGateway 103 , and pEarleyGateway 203 binary vectors ( ) using LR Clonase (Invitrogen).

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: .. The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen). .. The resulting pDONR221-ZIP s entry clones were transformed into DH5α E. coli competent cells and then sequenced at the Beijing Genomics Institute (BGI, China).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct. .. Primers used for cloning are summarized in . pBdEF1α:ZmFBX92 was introduced into maize cultivar B104 by Agrobacterium tumefaciens transformation of immature embryos as described before ( ). pCaMV35S:ZmFBX92 , p35S:AtFBX92 , p35S:AtFBX92 del , p35S:AtFBX92 -amiRNA and pAtFBX92:GFP:GUS constructs were transformed into A. tumefaciens strain C58C1 RifR harboring the plasmid pMP90, followed by transformation into Arabidopsis Col-0 using the floral dip protocol ( ).

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. The L4440 unc-22 plasmid, pLT61, was provided by Andy Fire (Carnegie Institution of Washington, Baltimore).

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: .. The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the unc-47 promoter (kind gift from Dr. Erik Jorgensen, University of Utah), the unc-54 3′UTR plasmid pCM5.37 (Dr. Geraldine Seydoux, Johns Hopkins, Addgene plasmid 17253) and the destination vector pCFJ150 to create unc-47 ::TDP-43 and unc-47 ::FUS expression vectors.

    Amplification:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: .. The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. Donor sequence was mobilized into pDEST-17 and constructs were verified via DNA sequencing (GENEWIZ).

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: KOD polymerase was used in PCR amplification of the AQP cDNA. .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). .. The mixture of BP reaction was transformed into DH5α competent cells and sequenced (Sangon, Shanghai, China).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: .. Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: The mutated full-length PID D205A ( mPID ) coding sequence was amplified using a site-directed mutagenesis method. .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen).

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: .. The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen). .. The resulting pDONR221-ZIP s entry clones were transformed into DH5α E. coli competent cells and then sequenced at the Beijing Genomics Institute (BGI, China).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct. .. Primers used for cloning are summarized in . pBdEF1α:ZmFBX92 was introduced into maize cultivar B104 by Agrobacterium tumefaciens transformation of immature embryos as described before ( ). pCaMV35S:ZmFBX92 , p35S:AtFBX92 , p35S:AtFBX92 del , p35S:AtFBX92 -amiRNA and pAtFBX92:GFP:GUS constructs were transformed into A. tumefaciens strain C58C1 RifR harboring the plasmid pMP90, followed by transformation into Arabidopsis Col-0 using the floral dip protocol ( ).

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. The L4440 unc-22 plasmid, pLT61, was provided by Andy Fire (Carnegie Institution of Washington, Baltimore).

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: .. The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the unc-47 promoter (kind gift from Dr. Erik Jorgensen, University of Utah), the unc-54 3′UTR plasmid pCM5.37 (Dr. Geraldine Seydoux, Johns Hopkins, Addgene plasmid 17253) and the destination vector pCFJ150 to create unc-47 ::TDP-43 and unc-47 ::FUS expression vectors.

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The IRES-eGFP coding sequence was amplified with the primers IRES-eGFP-for/IRES-eGFP-rev containing the attB2/attB3 attachment sites and the EF1α-Promotor was amplified with primers EF1α-for-B4/EF1α-rev-B1 containing the attB4/attB1 attachment sites. .. The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual.

    Polymerase Chain Reaction:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. This created expression vectors with the cycle 3 mutant of the GFP gene at the carboxy-terminus of the AQP gene of interest, which were subsequently expressed as fusion proteins.

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). .. The mixture of BP reaction was transformed into DH5α competent cells and sequenced (Sangon, Shanghai, China).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. PCR product was isolated and cloned into pENTR to generate Gateway entry clone using Gateway® BP Clonase® II enzyme mix (Invitrogen, catalog no. 11789-020).

    Article Title: Reproducible gene targeting in recalcitrant Escherichia coli isolates
    Article Snippet: The resulting PCR-fragment comprises homology regions A and B, flanked by attB -sites, and has a unique PmeI restriction enzyme site situated between the two homology regions. .. The attB sites are used for a site-specific BP-recombination reaction with the attP sites of the pDONR221 vector, using the Gateway® technology (Invitrogen).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: .. Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The Gateway system was used to introduce the obtained PCR fragments via recombination into pDONr221 (Invitrogen Life Technologies), followed by recombination via the att Latt R sites into binary vector pK7GW2 ( https://gateway.psb.ugent.be ) into which a cassette containing the seed-specific napin promoter ( ) driving GFP was introduced, further indicated as pK7GW2napin , to allow the selection of transgenic seeds based on GFP expression in the seed. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. The L4440 unc-22 plasmid, pLT61, was provided by Andy Fire (Carnegie Institution of Washington, Baltimore).

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: .. The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the unc-47 promoter (kind gift from Dr. Erik Jorgensen, University of Utah), the unc-54 3′UTR plasmid pCM5.37 (Dr. Geraldine Seydoux, Johns Hopkins, Addgene plasmid 17253) and the destination vector pCFJ150 to create unc-47 ::TDP-43 and unc-47 ::FUS expression vectors.

    Construct:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. Donor sequence was mobilized into pDEST-17 and constructs were verified via DNA sequencing (GENEWIZ).

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Paragraph title: Cloning of AQP5-GFP fusion constructs for analysis in HEK293 cells ... Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. For the generation of UAS-GFP-CncC , the coding sequence of CncC was retrieved from total RNA from S2R+ cells using the RNeasy Plus minikit (Qiagen, catalog no. 74134), and cDNA was constructed using Transcriptor Universal cDNA Master (Roche Applied Science, catalog no. 05 893 151 001).

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: To generate pEarleyGateway-PID-CFP , pEarleyGateway-PID-GFP , pEarley Gateway-myc-PID , and pEarleyGateway-myc-mPID constructs, the full-length PID ORF with attB sites was amplified from Col cDNA. .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct. .. Primers used for cloning are summarized in . pBdEF1α:ZmFBX92 was introduced into maize cultivar B104 by Agrobacterium tumefaciens transformation of immature embryos as described before ( ). pCaMV35S:ZmFBX92 , p35S:AtFBX92 , p35S:AtFBX92 del , p35S:AtFBX92 -amiRNA and pAtFBX92:GFP:GUS constructs were transformed into A. tumefaciens strain C58C1 RifR harboring the plasmid pMP90, followed by transformation into Arabidopsis Col-0 using the floral dip protocol ( ).

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: A Gateway (Invitrogen)-compatible L4440 vector was constructed by ligating Gateway Cassette C into Sma I-digested L4440. .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway.

    Incubation:

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. For the experiments shown in , 2-ml cultures in LB/carbenicillin were started with a 1:10 dilution of an overnight culture (grown in LB/carbenicillin plus tetracycline) and incubated for 90 min at 37°C with shaking.

    Infection:

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: Sugarcane ScAPX6 gene isolation and gateway entry vector construction The sequence of a putative APX6 unigene (ScAPX6 ) from our previous transcriptome data of sugarcane in response to SrMV infection was used to design the cloning primer APX6-1F/1R (Table ). .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Expressing:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. This created expression vectors with the cycle 3 mutant of the GFP gene at the carboxy-terminus of the AQP gene of interest, which were subsequently expressed as fusion proteins.

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). .. The positive plasmid pDONR221-ScAPX6 was achieved and then used for the constructions of prokaryotic expression vector and eukaryotic expression vector.

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. Entry clone was then used to generate expression clone (pPW-DmKeap1 and pPGW-DmKeap1) using Gateway® LR Clonase® II enzyme mix (Invitrogen, catalog no. 11791-100).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The Gateway system was used to introduce the obtained PCR fragments via recombination into pDONr221 (Invitrogen Life Technologies), followed by recombination via the att Latt R sites into binary vector pK7GW2 ( https://gateway.psb.ugent.be ) into which a cassette containing the seed-specific napin promoter ( ) driving GFP was introduced, further indicated as pK7GW2napin , to allow the selection of transgenic seeds based on GFP expression in the seed. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the unc-47 promoter (kind gift from Dr. Erik Jorgensen, University of Utah), the unc-54 3′UTR plasmid pCM5.37 (Dr. Geraldine Seydoux, Johns Hopkins, Addgene plasmid 17253) and the destination vector pCFJ150 to create unc-47 ::TDP-43 and unc-47 ::FUS expression vectors.

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual. .. Final expression vectors were generated by a LR-recombination reaction between pDest-Super, pDONR™P4-P1R-EF-1α, the pDONR™221 entry vectors containing the proteasome-subunit inserts and pDONR™P2R-P3-IRES-eGFP according to manufacturer's instructions resulting in the expression vectors pEX-EF-1α-β5-Flag-IRES-eGFP, pEX-EF-1α-LMP7-Flag-IRES-eGFP, pEX-EF-1α-proLMP7mβ5-Flag-IRES-eGFP and pEX-EF-1α-proβ5mLMP7-Flag-IRES-eGFP, respectively.

    Touchdown PCR:

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: The touchdown PCR procedure was 94°C for 4 min; 94°C for 30 s, 70°C for 30 s and then each loop drop 0.5°C, 72°C for 1 min and 30 s, 35 cycles; and 72°C for 10 min. .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Modification:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. The mutant constructs S156E and S156A were amplified using the well-established, modified QuikChange procedure (Stratagene), as previously described [ ] and according to their manual.

    Article Title: Reproducible gene targeting in recalcitrant Escherichia coli isolates
    Article Snippet: The components of the lambda recombination system were modified to improve the specificity and efficiency of the system. .. The attB sites are used for a site-specific BP-recombination reaction with the attP sites of the pDONR221 vector, using the Gateway® technology (Invitrogen).

    Transformation Assay:

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). .. The mixture of BP reaction was transformed into DH5α competent cells and sequenced (Sangon, Shanghai, China).

    Article Title: Reproducible gene targeting in recalcitrant Escherichia coli isolates
    Article Snippet: The attB sites are used for a site-specific BP-recombination reaction with the attP sites of the pDONR221 vector, using the Gateway® technology (Invitrogen). .. After transformation to CaCl2 -competent E. coli DH5α cells [ ], clones were selected on LB medium containing kanamycin.

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen). .. The resulting pDONR221-ZIP s entry clones were transformed into DH5α E. coli competent cells and then sequenced at the Beijing Genomics Institute (BGI, China).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The generated constructs, pCaMV35S:AtFBX92 and pCaMV35S:AtFBX92 del , were subsequently transformed into Arabidopsis. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Derivative Assay:

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: The cDNAs derived from Zn- or Fe-deficient leaves and roots of trifoliate orange served as templates (see below). .. The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen).

    Transfection:

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: Restriction fragments (≈400-700 bp) from the targeted cDNAs (central marker B2, GenBank accession no. ; central marker B10, GenBank accession no. ; gut marker D14, GenBank accession no. ; photoreceptor marker E30, GenBank accession no. ) were ligated into the double T7 vector pL4440 double T7 script II ( ) and transfected into competent DH10B cells. .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway.

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual. .. The expression vectors were packed into ecotropic retroviral particles by transient transfection of Phoenix E cells using CaPO4 precipitation.

    Ligation:

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The proβ5mLMP7 or proLMP7mβ5 were generated by blunt-end ligation of the respective fragments using T4 Ligase (Fermentas) at 16°C overnight. .. The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual.

    Introduce:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: This was followed by five bases (bold) to introduce a Kozak sequence upstream and to keep the sequence in frame with the AQP coding sequence. .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The Gateway system was used to introduce the obtained PCR fragments via recombination into pDONr221 (Invitrogen Life Technologies), followed by recombination via the att Latt R sites into binary vector pK7GW2 ( https://gateway.psb.ugent.be ) into which a cassette containing the seed-specific napin promoter ( ) driving GFP was introduced, further indicated as pK7GW2napin , to allow the selection of transgenic seeds based on GFP expression in the seed. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Generated:

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. To isolate a flower-specific promoter region, PANS was generated by PCR with tobacco genomic DNA as previously described [ ].

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The generated constructs, pCaMV35S:AtFBX92 and pCaMV35S:AtFBX92 del , were subsequently transformed into Arabidopsis. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Transgenic lines were created by microinjection of unc-119(ed3) worms, multiple lines were generated and strains behaving similarly were kept for further analysis.

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The proβ5mLMP7 or proLMP7mβ5 were generated by blunt-end ligation of the respective fragments using T4 Ligase (Fermentas) at 16°C overnight. .. The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual.

    DNA Sequencing:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. Donor sequence was mobilized into pDEST-17 and constructs were verified via DNA sequencing (GENEWIZ).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: .. Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: The reverse transcription–polymerase chain reaction (RT-PCR) procedure was 94°C for 4 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 35 cycles; and 72°C for 10 min. RT-PCR products were gel-purified and cloned into pMD19-T vector (TaKaRa, Dalian, China), and then transformed into E. coli DH5α competent cells and sequenced (Sangon, Shanghai, China). .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Recombinant:

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. The resulting recombinant plasmids were introduced into competent ( ) HT115(DE3) cells that lack RNase III and can be induced to express T7 polymerase in the presence of isopropyl β- d -thiogalactoside (IPTG) ( , ).

    Cellular Antioxidant Activity Assay:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Finally 18-25bp of the AQP sequence without the stop codon were added to create the carboxy-terminal forward primers 5′-GGG G AC CAC TTT GTA CAA GAA AGC TGG GT C –AQP5(18-25bp)-3′. .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Immunofluorescence:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: Fluorescence microscopy localization assays Localization of CT695 and TarP was determined via indirect immunofluorescence using CT695-specific antibodies or α-TarP [ ]. .. The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221.

    Fluorescence:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: Paragraph title: Fluorescence microscopy localization assays ... The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221.

    Mutagenesis:

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. This created expression vectors with the cycle 3 mutant of the GFP gene at the carboxy-terminus of the AQP gene of interest, which were subsequently expressed as fusion proteins.

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: The mutated full-length PID D205A ( mPID ) coding sequence was amplified using a site-directed mutagenesis method. .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen).

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: Transgenic TDP-43 and FUS worms Human cDNAs for wild type and mutant TDP-43[A315T], and wild type and mutant FUS-TLS[S57Δ] were obtained from Dr. .. The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen).

    Isolation:

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: Paragraph title: Sugarcane ScAPX6 gene isolation and gateway entry vector construction ... The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. PCR product was isolated and cloned into pENTR to generate Gateway entry clone using Gateway® BP Clonase® II enzyme mix (Invitrogen, catalog no. 11789-020).

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: Paragraph title: 4.1. Gene Isolation and Vector Construction ... Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing.

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen). .. The ZIP genes isolated from trifoliate orange were designated PtZIP genes.

    Microscopy:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: Paragraph title: Fluorescence microscopy localization assays ... The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221.

    Purification:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. His-Tagged CT695 was expressed in E . coli BL21-Al (Invitrogen), and protein was purified to homogeneity via passage of lysates over TALON affinity resin (Clontech, Mountain View, CA).

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. This created expression vectors with the cycle 3 mutant of the GFP gene at the carboxy-terminus of the AQP gene of interest, which were subsequently expressed as fusion proteins.

    Sequencing:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: .. The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. Donor sequence was mobilized into pDEST-17 and constructs were verified via DNA sequencing (GENEWIZ).

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: Finally 18-25bp of the AQP sequence without the stop codon were added to create the carboxy-terminal forward primers 5′-GGG G AC CAC TTT GTA CAA GAA AGC TGG GT C –AQP5(18-25bp)-3′. .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen).

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: Sugarcane ScAPX6 gene isolation and gateway entry vector construction The sequence of a putative APX6 unigene (ScAPX6 ) from our previous transcriptome data of sugarcane in response to SrMV infection was used to design the cloning primer APX6-1F/1R (Table ). .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: .. For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. PCR product was isolated and cloned into pENTR to generate Gateway entry clone using Gateway® BP Clonase® II enzyme mix (Invitrogen, catalog no. 11789-020).

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: The mutated full-length PID D205A ( mPID ) coding sequence was amplified using a site-directed mutagenesis method. .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen).

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: The IRES-eGFP coding sequence was amplified with the primers IRES-eGFP-for/IRES-eGFP-rev containing the attB2/attB3 attachment sites and the EF1α-Promotor was amplified with primers EF1α-for-B4/EF1α-rev-B1 containing the attB4/attB1 attachment sites. .. The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual.

    Activated Clotting Time Assay:

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: The fly stocks ( UAS-CncC/CyO, UAS-Dmkeap1-IR , UAS-keap1, GstD1-GFP/Fm7c , FRT82 , keap1 Δ 036 (all kindly provided by D. Bohmann ( )), atg61 and pmCh-Atg8a (gift from E. Baehrecke ( )), ref(2)P03993 , atg13 Δ 81 , h sp70-Flp ; UAS-Dicer ; r4-mCh :: Atg8a , and act > CD2 > GAL4 , UAS-GFPnls (gift from T. P. Neufeld ( )), atg8ad4 (gift from G. Juhasz ( )), FRT82 , RFP (gift from H. Stocker), UAS-MitFEA (termed MitFDN ) ( ) (gift from F. Pignoni), en-Gal4,UAS-RFP/CyO , atg8a-IRTRIP.JF02895 , CncC-IRTRIP.HMS00650 , UAS-atg8aEP362 , atg9-IRTRiP.HMS01246 , UAS-ref(2)P-IRTRiP.HMS00551 ) were obtained from the Bloomington Stock Center. .. For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017).

    Plasmid Preparation:

    Article Title: Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis
    Article Snippet: .. The coding sequence for C . trachomatis L2 CT695 was amplified using Q5 DNA polymerase and primers sets (5’-GGGGACAAGTTTGTACAAAAAA GCAGGCTTCAG TAGCATAAGCCCTATAGGGGGG-3’ and 5’-GGGGACCACTT TGTACAAGAAAGCTGG GTCCTATTAGATATTCCCAACCGAAGAAGG-3’) for transfer into the GATEWAY (Life Technologies) entry vector pDONR-221. .. Donor sequence was mobilized into pDEST-17 and constructs were verified via DNA sequencing (GENEWIZ).

    Article Title: Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways
    Article Snippet: .. Samples were heated to 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 68°C for 3 min and then 68°C for 7 min. Purified PCR products were sub-cloned into the pDONR221™ entry vector (Invitrogen) using the att B1 and att B2 sites in a reaction with Gateway™ BP Clonase™ enzyme mix (Invitrogen). pDONR221™ vectors containing the required sequences were recombined with the pcDNA-DEST47 Gateway™ vector using the att L and att R reaction with Gateway™ LR Clonase™ enzyme mix (Invitrogen). .. This created expression vectors with the cycle 3 mutant of the GFP gene at the carboxy-terminus of the AQP gene of interest, which were subsequently expressed as fusion proteins.

    Article Title: A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane
    Article Snippet: .. The PCR amplification products were gel-purified and transformed into the Gateway@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). .. The mixture of BP reaction was transformed into DH5α competent cells and sequenced (Sangon, Shanghai, China).

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: .. For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017). .. PCR product was isolated and cloned into pENTR to generate Gateway entry clone using Gateway® BP Clonase® II enzyme mix (Invitrogen, catalog no. 11789-020).

    Article Title: Reproducible gene targeting in recalcitrant Escherichia coli isolates
    Article Snippet: .. The attB sites are used for a site-specific BP-recombination reaction with the attP sites of the pDONR221 vector, using the Gateway® technology (Invitrogen). .. After transformation to CaCl2 -competent E. coli DH5α cells [ ], clones were selected on LB medium containing kanamycin.

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: .. Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: Phosphorylation and negative regulation of CONSTITUTIVELY PHOTOMORPHOGENIC 1 by PINOID in Arabidopsis
    Article Snippet: .. The target fragments were cloned into the gateway vector pDONR-221 using BP Clonase (Invitrogen). .. The destination fragments then were introduced into the plant pEarleyGateway 102 , pEarleyGateway 103 , and pEarleyGateway 203 binary vectors ( ) using LR Clonase (Invitrogen).

    Article Title: Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)
    Article Snippet: .. The amplified att B-PCR fragments were then cloned into the entry vector pDONR221 (Invitrogen) to generate entry clones by BP recombination reaction with the Gateway BP Clonase II enzyme (Invitrogen). .. The resulting pDONR221-ZIP s entry clones were transformed into DH5α E. coli competent cells and then sequenced at the Beijing Genomics Institute (BGI, China).

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct. .. Primers used for cloning are summarized in . pBdEF1α:ZmFBX92 was introduced into maize cultivar B104 by Agrobacterium tumefaciens transformation of immature embryos as described before ( ). pCaMV35S:ZmFBX92 , p35S:AtFBX92 , p35S:AtFBX92 del , p35S:AtFBX92 -amiRNA and pAtFBX92:GFP:GUS constructs were transformed into A. tumefaciens strain C58C1 RifR harboring the plasmid pMP90, followed by transformation into Arabidopsis Col-0 using the floral dip protocol ( ).

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway. .. The L4440 unc-22 plasmid, pLT61, was provided by Andy Fire (Carnegie Institution of Washington, Baltimore).

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: .. The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Multisite Gateway recombination was performed with the pDONR TDP-43 and FUS clones along with clones containing the unc-47 promoter (kind gift from Dr. Erik Jorgensen, University of Utah), the unc-54 3′UTR plasmid pCM5.37 (Dr. Geraldine Seydoux, Johns Hopkins, Addgene plasmid 17253) and the destination vector pCFJ150 to create unc-47 ::TDP-43 and unc-47 ::FUS expression vectors.

    Article Title: The Proteasome System in Infection: Impact of ?5 and LMP7 on Composition, Maturation and Quantity of Active Proteasome Complexes
    Article Snippet: .. The proteasome subunit inserts were first subcloned into the entry vector pDONR™221 (Invitrogen), the IRES-eGFP insert into pDONR™P2R-P3 (Invitrogen) and the EF1α-promotor into pDONR™P4-P1R using the BP-recombination reaction according to the MultiSite Gateway® Three-Fragment Vector Construction Kit Manual. .. Final expression vectors were generated by a LR-recombination reaction between pDest-Super, pDONR™P4-P1R-EF-1α, the pDONR™221 entry vectors containing the proteasome-subunit inserts and pDONR™P2R-P3-IRES-eGFP according to manufacturer's instructions resulting in the expression vectors pEX-EF-1α-β5-Flag-IRES-eGFP, pEX-EF-1α-LMP7-Flag-IRES-eGFP, pEX-EF-1α-proLMP7mβ5-Flag-IRES-eGFP and pEX-EF-1α-proβ5mLMP7-Flag-IRES-eGFP, respectively.

    Irradiation:

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen). .. Transgenes were integrated by UV irradiation and lines were outcrossed to wild type N2 worms 5 times before use.

    Selection:

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: The Gateway system was used to introduce the obtained PCR fragments via recombination into pDONr221 (Invitrogen Life Technologies), followed by recombination via the att Latt R sites into binary vector pK7GW2 ( https://gateway.psb.ugent.be ) into which a cassette containing the seed-specific napin promoter ( ) driving GFP was introduced, further indicated as pK7GW2napin , to allow the selection of transgenic seeds based on GFP expression in the seed. .. For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Transgenic Assay:

    Article Title: F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana
    Article Snippet: Paragraph title: Cloning and generation of transgenic plants ... For analysis of the AtFBX92 promoter, a 1,362 bp fragment upstream of the ATG start codon was amplified with Phusion High-Fidelity DNA polymerase (Thermo Fischer Scientific) from Arabidopsis Col-0 genomic DNA, cloned into pDONR™221 (Invitrogen Life Technologies) and transferred to the pFAST-G04 binary vector ( ) ( https://gateway.psb.ugent.be ) to generate the pAtFBX92:GFP:GUS construct.

    Article Title: Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans
    Article Snippet: Paragraph title: Transgenic TDP-43 and FUS worms ... The cDNAs were amplified by PCR and cloned into the Gateway vector pDONR221 following the manufacturer's protocol (Invitrogen).

    Concentration Assay:

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *
    Article Snippet: Flies were cultivated at 25 °C on our standard laboratory fly medium consisting of (per liter) 32.7 g of dried potato powder, 60 g of sucrose, 27.3 g of dry yeast, 7.3 g of agar, 4.55 ml of propionic acid, and 2 g of nipagin, giving a final concentration of 15.3 g/liter protein and 6 g/liter sugar. .. For the generation of UAS-DmKeap1 and GFP-Dmkeap1 fly lines, the coding sequence of Dmkeap1 was retrieved from Dmkeap1 cDNA from pUASt-DmKeap1-HA plasmid (Berkeley Drosophila Genome Project) with the help of primers containing the recombination site for Gateway entry vector pENTR (Invitrogen, catalog no. 12536-017).

    Marker:

    Article Title: Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter
    Article Snippet: Amplified DNA products were cloned into the Gateway entry vector pDONR221 (Invitrogen, Carlsbad, CA, USA) through PCR with the att recombination adapter primers according to the manufacturer’s instructions, and clones were verified by DNA sequencing. .. Subsequently, the mPAP1 and B-peru genes were inserted into the destination vector pB7WG2D, in which the CaMV 35S promoter drives gene expression [ ] and contains the Bar gene encoding phosphinothricin acetyltransferase as a selectable marker.

    Article Title: Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians
    Article Snippet: Restriction fragments (≈400-700 bp) from the targeted cDNAs (central marker B2, GenBank accession no. ; central marker B10, GenBank accession no. ; gut marker D14, GenBank accession no. ; photoreceptor marker E30, GenBank accession no. ) were ligated into the double T7 vector pL4440 double T7 script II ( ) and transfected into competent DH10B cells. .. Clone H.108.3a (GenBank accession no. ) was cloned into the resulting L4440 Gateway vector by PCR amplification and cloning into pDONR221 (Invitrogen), and subsequent transfer by using an LR cloning reaction into L4440 Gateway.

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  • 99
    Thermo Fisher pdonr221
    Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using <t>pDONR221</t> and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.
    Pdonr221, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pdonr221 - by Bioz Stars, 2020-04
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    92
    Thermo Fisher mef2d pdonr221 vector
    Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using <t>pDONR221</t> and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.
    Mef2d Pdonr221 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mef2d pdonr221 vector - by Bioz Stars, 2020-04
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    99
    Thermo Fisher pdonr221 vector
    Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using <t>pDONR221</t> and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.
    Pdonr221 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pdonr221 vector/product/Thermo Fisher
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    Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using pDONR221 and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.

    Journal: PLoS ONE

    Article Title: Novel gateway binary vectors for rapid tripartite DNA assembly and promoter analysis with various reporters and tags in the liverwort Marchantia polymorpha

    doi: 10.1371/journal.pone.0204964

    Figure Lengend Snippet: Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using pDONR221 and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.

    Article Snippet: The PCR-amplified DNA fragments were transferred to the donor vector, pDONR221, by a BP recombination (Thermo Fisher Scientific).

    Techniques: Clone Assay, Construct, Marker, Selection

    Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using pDONR221 and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.

    Journal: PLoS ONE

    Article Title: Novel gateway binary vectors for rapid tripartite DNA assembly and promoter analysis with various reporters and tags in the liverwort Marchantia polymorpha

    doi: 10.1371/journal.pone.0204964

    Figure Lengend Snippet: Outline of cloning procedure using the R4pMpGWB system. Promoter entry clones are constructed by a BP reaction between pDONR P4-P1R and an attB4 -promoter- attB1 fragment. The cDNA entry clones are constructed by the BP reaction using pDONR221 and the attB1 -cDNA- attB2 fragment. The libraries of promoters and cDNAs can be used as resources for construction of chimeric fusions. Promoter and cDNA entry clones and R4pMpGWB vectors are used in a tripartite LR reaction to form a C-terminal fusion of cDNA-encoded protein and a reporter or tag. Note: as pDONR P4-P1R has been discontinued, pENTR 5′-TOPO (Thermo Fisher Scientific) can be used as an alternative for promoter entry clone production. Arrowheads, T-DNA border sequences; B1 , att B 1 ; B2 , att B2; B4 , att B4; P4 , att P4; P1R , att P1R; L1 , att L1; L2 , att L2; L4 , att L4; R1 , att R1; R2 , att R2; Km r , kanamycin-resistant marker; Cm r , chloramphenicol-resistant marker; Spec r , spectinomycin-resistant marker; ccdB , negative selection marker used in bacteria.

    Article Snippet: The PCR-amplified DNA fragments were transferred to the donor vector, pDONR221, by a BP recombination (Thermo Fisher Scientific).

    Techniques: Clone Assay, Construct, Marker, Selection