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Gateway pet dest42 vector

Manufactured by Thermo Fisher Scientific
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The Gateway™ pET-DEST42 vector is a tool used for the expression of recombinant proteins in E. coli. It enables the transfer of DNA sequences from an entry clone to a destination vector through site-specific recombination. The vector includes a T7 promoter for high-level protein expression, a C-terminal V5 epitope tag, and a polyhistidine (6xHis) tag for detection and purification of the expressed protein.

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Lab products found in correlation

Hispur cobalt resin kit, by Thermo Fisher Scientific (3 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (3 mentions) Pgem 3z vector, by Promega (3 mentions) Bl21 codonplus de3 ripl, by Agilent Technologies (3 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (3 mentions) 4d nucleofector system, by Lonza (3 mentions) Gateway system, by Thermo Fisher Scientific (2 mentions) Scramble sirna and sirna targeting trim63, by Santa Cruz Biotechnology (2 mentions)

Close spelling variants of this product

PET-DEST42

3 protocols using gateway pet dest42 vector

1

Synthesis and Characterization of DMD ZnF Mutants

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The H19 sequence was synthesized by GenScript and cloned into the pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies). An expression vector encoding the wild-type sequence of the human DMD ZnF domain was purchased from the shRNA and ORF core of MD Anderson Cancer Center, and the coding regions were subcloned into the Gateway™ pET-DEST40 vector for mammalian expression and Gateway™ pET-DEST42 vector for prokaryotic expression (Invitrogen). All single-point and deletion mutations were generated using QuikChange™ Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). DMD ZnF Y/F-A mutant substitutes tyrosine (Y) or phenylalanine (F) to alanine (A) at sites Y3326, F3332, Y3334, and F3341. Recombinant DMD WT and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific). Plasmid transfections were performed using Lipofectamine3000 (Life Technologies) or electroporation using the 4D-Nucleofector™ System (Lonza) according to the manufacturer’s instructions.
Li Y., Zhang Y., Hu Q., Egranov S.D., Xing Z., Zhang Z., Liang K., Ye Y., Pan Y., Chatterjee S.S., Mistretta B., Nguyen T.K., Hawke D.H., Gunaratne P.H., Hung M.C., Han L., Yang L, & Lin C. (2021). Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects. Genome Medicine, 13, 137.
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2

Molecular Cloning and Expression of DMD

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H19 DNA sequences were synthesized by GenScript and cloned into pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies) or pMS2 vector for mammalian expression. The full-length human or mouse DMD and human DMD zinc finger domain sequence was obtained from MDACC shRNA and ORFeome Core and Addgene. Gateway™ pET-DEST42 vector (Invitrogen) was used for prokaryotic expression of human DMD c-termini. Mammalian full-length DMD, zinc finger domain of DMD, or H19 wild type and mutant vectors were constructed by subcloning the corresponding gene sequences into the SFB-tagged expression vector (provided by J. Chen, MD Anderson Cancer Center, USA) using the Gateway system (Life Technologies). All single-point and deletion mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Scramble siRNA and siRNA targeting TRIM63 were obtained from Santa Cruz Biotechnology. Plasmid transfections were performed using Lipofectamine3000 (Life Technologies), or electroporation using the 4D-Nucleofector™ System (Lonza) according to manufacturer’s instructions. Recombinant DMD wild type and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific).
Zhang Y., Li Y., Hu Q., Xi Y., Xing Z., Zhang Z., Huang L., Wu J., Liang K., Nguyen T.K., Egranov S.D., Sun C., Zhao Z., Hawke D.H., Li J., Sun D., Kim J.J., Zhang P., Cheng J., Farida A., Hung M.C., Han L., Darabi R., Lin C, & Yang L. (2020). LncRNA H19 Alleviates Muscular Dystrophy Through Stabilizing Dystrophin. Nature cell biology, 22(11), 1332-1345.
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3

Molecular Cloning and Expression of DMD

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H19 DNA sequences were synthesized by GenScript and cloned into pGEM-3Z vector (Promega) for in vitro transcription and into the pcDNA3.1 (+) vector (Life technologies) or pMS2 vector for mammalian expression. The full-length human or mouse DMD and human DMD zinc finger domain sequence was obtained from MDACC shRNA and ORFeome Core and Addgene. Gateway™ pET-DEST42 vector (Invitrogen) was used for prokaryotic expression of human DMD c-termini. Mammalian full-length DMD, zinc finger domain of DMD, or H19 wild type and mutant vectors were constructed by subcloning the corresponding gene sequences into the SFB-tagged expression vector (provided by J. Chen, MD Anderson Cancer Center, USA) using the Gateway system (Life Technologies). All single-point and deletion mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Scramble siRNA and siRNA targeting TRIM63 were obtained from Santa Cruz Biotechnology. Plasmid transfections were performed using Lipofectamine3000 (Life Technologies), or electroporation using the 4D-Nucleofector™ System (Lonza) according to manufacturer’s instructions. Recombinant DMD wild type and mutants were expressed in the E.coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using a HisPur Cobalt Resin Kit (Thermo Scientific).
Zhang Y., Li Y., Hu Q., Xi Y., Xing Z., Zhang Z., Huang L., Wu J., Liang K., Nguyen T.K., Egranov S.D., Sun C., Zhao Z., Hawke D.H., Li J., Sun D., Kim J.J., Zhang P., Cheng J., Farida A., Hung M.C., Han L., Darabi R., Lin C, & Yang L. (2020). LncRNA H19 Alleviates Muscular Dystrophy Through Stabilizing Dystrophin. Nature cell biology, 22(11), 1332-1345.
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