Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here we report a flexible and versatile DNA assembly strategy that allows rapid, modular and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation friendly ligase cycling reaction (LCR) method and the high fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to pre-assemble basic genetic parts into gene expression cassettes or pre-assemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all pre-assembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4 kb, 24 kb, and 44 kb were rapidly constructed with the fidelity of 100%, 88% and 71%, respectively. The yeast strain harboring constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids.