Figure 2. Design of primers for Gibson assembly.

(A) Scheme of forward and reverse primers used for Gibson assembly. (B) Diagram of Bam HI-digested pYD1 vector, barcode adapter-ligated fragment (DNA fragments), and primers. (C) Forward (For) and reverse (Rev) primers used to amplify barcode adapter-ligated fragments for Gibson assembly (see pairing regions in B). The sequences in orange correspond to a ~20 bp vector sequence complementary to the pYD1 vector, the black letters are the Bam HI restriction sites, and the ~20 bp nanopore barcode adapter annealing sequences are in green. F and R: forward and reverse primers, respectively, that pair to the vector flanking the cloning site and are used to amplify cloned fragments by PCR for cloning validation or sequencing.