Objective: To synthesize a gene (Homo sapiens) with 50,245 bp in length, located on chromosome Y.
Strategy: (a) Divided the sequence into 10 x 5 kb fragments containing Ascl restriction sites at 5’ and 3’ ends with 200 bp overlap regions (Fig. 1); (b) all fragments were cloned in pUC57 vector and then transformed in yeast for assembly; (c) after screening (Fig. 2), selected clones were subjected to restriction enzyme digestion and NGS sequencing for final validation.
Results: Results from the gel electrophoresis of digested gene (Fig. 3) and also NGS sequencing (Fig. 4) demonstrated that the 50 kb gene was synthesized without any error and at full length.
Fig. 1 Final construct and assembly strategy.
Fig. 2 Colony screening
Fig. 3 Plasmid gel electrophoresis (lane 1) and digested products by SalI (lane 2).
Fig. 4 NGS sequencing for final validation.