Selected article for: "DNA polymerase and expression vector"
Author: Wanchao Yin; Chunyou Mao; Xiaodong Luan; Dan-Dan Shen; Qingya Shen; Haixia Su; Xiaoxi Wang; Fulai Zhou; Wenfeng Zhao; Minqi Gao; Shenghai Chang; Yuan-Chao Xie; Guanghui Tian; He-Wei Jiang; Sheng-Ce Tao; Jingshan Shen; Yi Jiang; Hualiang Jiang; Yechun Xu; Shuyang Zhang; Yan Zhang; H. Eric Xu
Title: Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir
  • Document date: 2020_4_9
    • ID: 7v7pzclb_18
    Snippet: The gene for the full-length SARS-CoV-2 nsp12 encompassing 932 residues was chemically synthesized with codon optimization (General Biosystems). The gene was cloned into a modified pFastBac baculovirus expression vector containing a 5' ATG starting sequence and Cterminal Tobacco Etch Virus (TEV) protease site followed by a His8 tag. The plasmid construction contains one residue (methionine) to the N-terminus and GGSENLYFQGHHHHHHHH to the C-termin.....
    Document: The gene for the full-length SARS-CoV-2 nsp12 encompassing 932 residues was chemically synthesized with codon optimization (General Biosystems). The gene was cloned into a modified pFastBac baculovirus expression vector containing a 5' ATG starting sequence and Cterminal Tobacco Etch Virus (TEV) protease site followed by a His8 tag. The plasmid construction contains one residue (methionine) to the N-terminus and GGSENLYFQGHHHHHHHH to the C-terminus of the protein. The genes for SARS-CoV-2 nsp7 encompassing the 83 residues and SARS-CoV-2 nsp8 encompassing the 198 residues were respectively cloned into the pFastBac vector containing a 5' ATG starting sequence. All constructs were generated using the Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co.,Ltd) and verified by DNA sequencing. All constructs were expressed in Spodoptera frugiperda (Sf9) cells. Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 2-3 million cells per ml and then infected with three separate baculoviruses at a ratio of 1:2:2 for nsp12, nsp7 and nsp8 at a multiplicity of infection (m.o.i.) of about 5. The cells were collected 48 h after infection at 27 °C and cell pellets were stored at − 80 °C until use.

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