Author: Schwarz, Megan C.; Sourisseau, Marion; Espino, Michael M.; Gray, Essanna S.; Chambers, Matthew T.; Tortorella, Domenico; Evans, Matthew J.
Title: Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone Document date: 2016_9_28
ID: 0i1abjfa_24
Snippet: However, all derived clones carried nonsense or frameshift mutations, suggesting that the ZIKV open reading frame still induced toxicity in these bacteria. We chose a plasmid with a frameshift mutation created by a single G nucleotide insertion after nucleotide position 3170 for further cloning. A gBlock bearing 15 nucleotides before and after the SalI and XhoI sites, respectively, was synthesized with a silent G3127A mutation in the MR766 coding.....
Document: However, all derived clones carried nonsense or frameshift mutations, suggesting that the ZIKV open reading frame still induced toxicity in these bacteria. We chose a plasmid with a frameshift mutation created by a single G nucleotide insertion after nucleotide position 3170 for further cloning. A gBlock bearing 15 nucleotides before and after the SalI and XhoI sites, respectively, was synthesized with a silent G3127A mutation in the MR766 coding sequence followed immediately by the synthetic intron from plasmid pHTN (Promega, Madison, WI) (GenBank accession no. JF920304), which is based on the human beta-globin intron. In-Fusion cloning of this DNA into the above plasmid resulted in a plasmid that was stably propagated in bacteria. We termed this plasmid pCDNA6.2 ATCCMR766 Intron3127 HDVr.
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