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. 2023 Dec 22;14:1009–1033. doi: 10.18632/oncotarget.28546

Figure 9. One-step SARS-COv2 genome assembly in yeast S. cerevisiae using transformation-associated recombination (TAR) in yeast [120].

Figure 9

Step 1: Genomic DNA of SARS-CoV-2 was divided into 12 overlapping fragments. F1 fragment was fused with T7 promoter at its 5′ terminus (in green and in light brown rectangle) and F12 fragment was fused with poly(A) (pA) at its 3′ terminus (in dark brown). TAR vector has targeting hooks (in light brown and in dark brown) having homology to T7 promoter (the light brown part) and the sequence fused with F12 (in dark brown). Step 2: One-step delivery of 12 fragments and the linearized TAR vector into yeast cells and following assembly of the viral full-length cDNA by homologous recombination. Step 3: The EagI site at the 5′ end of the fragment F12 was cleaved to linearize the molecule. The linearized molecule was used as the template to synthesize the viral infectious full-length RNA of SARS-Cov2 using T7 RNA polymerase.