Extended Data Fig. 4 | Reconstruction of synSARS-CoV-2-GFP and TAR
cloning of full-length synthetic fragments 5 and 7 in yeast. a, Genome
organization of the synSARS-CoV-2-GFP and 19 viral fragments used for TAR
cloning (F1–F10; F12–F15). Fragments 5 and 7 were split in four (5A–5D) and
three (7Aa, 7Ab, 7B) DNA parts, respectively. Viral ORFs, the ORF for GFP and
sequence elements at the 5′ UTR and 3′ UTR are indicated. Primers used to
generate the fragments are listed in Supplementary Table 1. J2–J12 and J14
represent the junctions, that is, overlapping regions, between the subgenomic
fragments. J1 and J13 represent junctions with the TAR vector. Gel images show
the results of two multiplex PCRs designed to confirm the presence of
correctly recombined junctions. Multiplex PCR using set 1 primers (left)
detects junctions J1, J3, J5, J7, J9, J11 and J13, and multiplex PCR using set 2
primers (middle) detects junctions J2, J4, J6, J8, J10 and J12. The presence of the
GFP gene inserted in fragment 14 was confirmed (right). PCR-product sizes are
depicted and confirm the proper assembly of the synSARS-CoV-2 full-length
genome in all four YAC clones analysed. b, TAR cloning of the full-length
synthetic fragment 5 in yeast. Four overlapping synthetic DNA fragments
(5A–5D) provided by Genscript were reconstructed as a YAC after
transformation in yeast. Correct reassembly was confirmed by multiplex PCR
over junctions J1–J5 for 9 out of the10 clones screened (clone 8 was considered
incorrect). c, TAR cloning of the full-length synthetic fragment 7 in yeast. Full-
length fragment 7 was assembled by TAR cloning using 3 synthetic dsDNA parts
(7Aa, 7Ab and 7B) provided by Genscript. Correct reassembly was confirmed by
multiplex PCR over junctions J6–J9 for 5 out of 6 clones (clone 6 is considered
incorrect). Cloning experiments shown in a–c have been performed once. pT7,
T7 RNA polymerase promoter; An, poly(A) tail; M, GeneRuler 100-bp plus DNA
marker (Thermo Scientific).