Pcdna6 v5 his a expression vector

pMOI-GFP and pMOI-PARP12 (Homo sapiens) expression plasmids were purchased from GeneCopoeia and described previously (51 (link)). The viral RNA of the GZ01/2016 strain was isolated and used in reverse transcription PCR experiments to obtain the complementary DNA (cDNA) sequences of ZIKV nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Each ZIKV nonstructural gene was cloned into the pcDNA6/V5-His A expression vector (Invitrogen) using standard molecular cloning techniques and verified by sequencing. DsRed-NS1, DsRed-NS3, HA- or EGFP-PARP12, and PARP12 mutants were cloned using standard molecular cloning and oligo-nucleotide mutagenesis methods. HA-ubiquitin, HA-K48, and HAK63 plasmids were provided by S. Wang (52 (link)). Briefly, WT, K48, and K63 ubiquitin were cloned into pCMV-HA vector to generate HA-ubiquitin, which was subsequently mutated at the indicated residue to generate HA-K48 and HA-K63 plasmids. To create a stable cell line for PARP12 expression, PARP12 was cloned into the pMXsIG-IgkFLAG vector and cotransfected into A549 and MEF cells with VSV glyco-protein and pCpG helper plasmids. The cells were collected 72 hours after transfection, and the PARP12-overexpressing cells were then sorted by fluorescence-activated cell sorting (FACS).