Fig. 6

Assembly fidelity evaluation of vectors linearized by restriction digestion. a Schematic assembly of SalI-digested pUC19-UMCS-S-EGFP and mCherry CDS with 9-, 12-, and 15-bp homologous linkers. b The five possible plasmid products that can be observed after the transformation of vectors and inserts mentioned in (a). Among them, if the 3′-terminus of mCherry CDS was replaced by part of the SalI recognition sequence (“TCGAC”), the stop codon of the mCherry protein would then be destroyed, resulting in the expression of the mCherry-EGFP fusion protein with a characteristic yellow color that can be counted accurately. c Colonies with green, orange, and yellow colors can be observed when cells express different fluorescent proteins. Note that cells transformed with the pUC19 plasmid (colorless) were also spread onto the plate as a reference. d The orange and yellow colonies counted after the transformation of vectors and inserts mentioned in (a). Method fidelity can be calculated by one minus the number of yellow colonies divided by the number of orange colonies (n = 4). e-h The other experiments and results following the same procedure described in (a-d), except the vector was linearized by EcoRV digestion