E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions and grown on antibiotic-selective LB medium.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
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