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. 2020 Apr 13;27(5):841–848.e3. doi: 10.1016/j.chom.2020.04.004

Figure 1.

Figure 1

Assembly of a Full-Length SARS-CoV-2 Infection cDNA Clone

(A) Genome structure SARS-CoV-2. The open reading frames (ORFs) from the full genome are indicated.

(B) Strategy for in vitro assembly of an infectious cDNA clone of SARS-CoV-2. The nucleotide sequences and genome locations of the cohesive overhangs are indicated. The WT full-length (FL) cDNA of SARS-CoV-2 (IC WT) was directionally assembled using in vitro ligation.

(C) Diagram of the terminal sequences of each cDNA fragment recognized by BsaI and Esp3I.

(D) Gel analysis of the seven purified cDNA fragments. Individual fragments (F1–F7) were digested from corresponding plasmid clones and gel purified. Seven purified cDNA fragments (50–100 ng) were analyzed on a 0.6% native agarose gel. The 1-kb DNA ladders are indicated.

(E) Gel analysis of cDNA ligation products. About 400 ng of purified ligation product was analyzed on a 0.6% native agarose gel. Triangle indicates the FL cDNA product. Circles indicate the intermediate cDNA products.

(F) Gel analysis of RNA transcripts. About 1 μg of in-vitro-transcribed (IVT) RNAs were analyzed on a 0.6% native agarose gel. DNA ladders are indicated. Because this is a native agarose gel, the DNA size is not directly corelated to the RNA size. Triangle indicates the genome-length RNA transcript. Circles show the shorter RNA transcripts.