Fig. 1

Cloning procedure using the An2 reporter and the TRV2-LIC vector adapted from Dong et al. [12]. The TRV2-LIC vector was digested with PstI and treated with T4 DNA polymerase and dTTP to generate sticky ends. The gene of interest was amplified by PCR using gene specific primers (GSP) and LIC (Gene_lic_F) and An2 adaptors (Gene_An2_R) using cDNA, and then treated with T4 DNA polymerase. An2 was amplified using specific primers with an An2 adaptor (An2_F) and an LIC adaptor (An2_lic_R). The An2 adaptor sequence annealed to both the gene and An2 fragments and a subsequent PCR using the LIC adaptor-attached primers fused the two fragments. The following PCR fragments were treated with T4 DNA polymerase and dATP to generate complementary sticky ends to anneal the ends of the linearized vector without DNA ligase. A mixture of both fragments was then transformed into E. coli DH5α