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Construction of the full-length infectious SA14-14-2 cDNA as a BAC.

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posted on 31.07.2014, 03:24 by Sang-Im Yun, Byung-Hak Song, Jin-Kyoung Kim, Gil-Nam Yun, Eun-Young Lee, Long Li, Richard J. Kuhn, Michael G. Rossmann, John D. Morrey, Young-Min Lee

(A) Structure of the SA14-14-2 genomic RNA (GenBank accession no. JN604986). NCR, non-coding region; ORF, open reading frame. (B) Diagram of a panel of four overlapping SA14-14-2 cDNAs contained in pBAC/Frag-I to IV. SP6 promoter and an artificial run-off site are shown. An asterisk indicates a pre-existing XbaI site at nucleotide 9131 that was inactivated by introducing a silent point mutation, A9134→T. (C) Structure of the full-length SA14-14-2 cDNA (pBAC/SA14-14-2). (D) Functionality of pBAC/SA14-14-2. After in vitro transcription with SP6 RNA polymerase, RNA transcripts were electroporated into BHK-21 cells, and infectious plaque centers were determined (RNA infectivity). At 22 and 40 hpt, supernatants from RNA-transfected cells were harvested for virus titration on BHK-21 cells (Virus yield).