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. 2020 Sep 2;21(17):6370. doi: 10.3390/ijms21176370

Figure 4.

Figure 4

High-throughput reporter insertion in new species D human adenovirus (HAdV-D). (a) Identification of conserved sequences in the adenovirus early gene 3 (E3). Genome sequences of four new HAdV-Ds (Ad70, Ad73, Ad74, and Ad75) were analyzed by ClustalW Multiple alignment in BioEdit Sequence Alignment Editor, the conserved sequences on the left and right end of E3 was used as homologous arm (E3-HAL and E3-HAR) for reporter insertion. (b) Diagram shows the GLN reporter cassette insertion into the E3 genomic region. GLN reporter cassette expresses turbo Green fluorescent protein (GFP), nanoLuciferase (Nluc), and neomycin resistance (neo) under the control of the CAG promoter (CMV enhancer, chicken beta-Actin promoter, and rabbit beta-Globin splice acceptor site). (c) AhdI restriction check of the reporter insertion. SM, selection marker used for GLN insertion. M, 1 Kb DNA marker. (d) GFP images showing rescue of reporter-labeled adenoviruses in HEK 293 cells. Scale bar 500 µm.