Assembly of a full-length SARS-CoV cDNA clone in a BAC (refer to Materials and Methods section for a detailed description of construction steps). (A) Arrows symbolize positions of PCR fragments on the SARS-CoV genome. These were cloned in subgenomic plasmids. (B) Subgenomic plasmids pA1 - pF. Plasmids are either based on pSMART (identified by an "S" symbol within the respective clones) or on pCR2.1 (no symbol). Squares on each plasmid symbolize the approximate positions of erroneous mutations from initial cloning corrected by fragment-extension technique before assembly to higher-order clones. Small extension-PCR symbols above clones pB and pF indicate mutations introduced into plasmids to facilitate subsequent construction steps (deletion of an Mlu I-site in pB) or to fill in the 45 nt deletion in ORF 7b in pF. (C) Assembly of quarter clones. Circles represent plasmids, ovals represent BACs. Bold grey arrows symbolize essential BAC-encoded genes reconstituted during BAC ligation, in order to achieve high cloning efficiency. Restriction digestion steps are symbolized by thin arrows. The utilized restriction enzymes are identified next to the arrows. PCR primer symbols (small arrows) next to plasmids indicate that these plasmids were first amplified with primers introducing restriction sites (identified next to primer symbols) before the resulting products were double-digested as indicated. The large horizontal arrows below plasmids pA1 and pA2 indicate that these fragments were joined by overlap-extension PCR with primers eliminating a Bgl I restriction site as symbolized by a square on both of the parental plasmids. In each construction, fragment ends shown in close proximity were first ligated in-vitro. The ligation products were then purified, ligated at sites drawn in greater distance, and transformed in E. coli.