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FastCloning, a reliable cloning technique for plasmid construction, is a widely used protocol in biomedical research laboratories. Only two-step molecular manipulations are required to add a gene (cDNA) of interest into the desired vector. However, parallel cloning of the gene into multiple vectors is still a labor-intensive operation, which requires a range of primers for different vectors in high-throughput cloning projects. The situation could even be worse if multiple fragments of DNA are required to be added into one plasmid. Here, we describe a high-throughput FastCloning (HTFC) method, a protocol for parallel cloning by adding an adaptor sequence into all vectors. The target gene and vectors were PCR amplified separately to obtain the insert product and linear vectors with 18-base overlapping at each end of the DNAs required for FastCloning. Furthermore, a method for generating polycistronic bacterial constructs based on the same strategy as that used for HTFC was developed. Thus, the HTFC technique is a simple, effective, reliable, and low-cost tool for parallel cloning. [ABSTRACT FROM AUTHOR]