Fig. 2.

Optimization of the PS-Brick reaction conditions. a seamless fusion of mCherry gene through TA cloning or blunt-end ligation based PS-Brick assembly. b The transform efficiency of BmrI digested pOB vector and MlyI digested pOM vector. c, d The second cutting efficiency of SphI was investigated through the assembly efficiency and correct ligation rate. The vector backbones of the BmrI cleaved pOB and MlyI cleaved pOM were digested with SphI from 15 min to 180 min, and then linked to PCR products digested by SphI for 180 min. The CFUs after transformation were recorded as assembly efficiency, and the percentage of the correct colonies by DNA sequencing among the total colonies was calculated as assembly accuracy. e Comparison of the efficiency and accuracy between PS-Brick and the traditional Type IIP RE-based clone method with XbaI and SphI. The effect 5′-phosphorylation of the non-cut PCR end on the efficiency and accuracy of PS-Brick. Data shown are mean values from three biological replicates, and the standard deviations are presented