Overview of GoldenFish for generating different types of transgenic constructs. (A) The BsmBI/BsaI restriction site contains a 6-bp recognition site (yellow/pink) and a cleavage site outside the recognition site (blue). (B) Generation of P–G–T construct. The small circles represent BsmBI-mediated overhangs. (C) Three modules are cloned into T-vectors conferring ampicillin resistance (Amp^R) and flanked by inward-facing BsmBI sites, which expose the designated 4-nt overhangs (colored boxes) after BsmBI digestion. The 3′ overhangs of the upstream module must be compatible with the 5′ overhangs of the subsequent module for correct assembly. (D) GoldenFish recipient vector containing ccdB or LacZ cassette exposes overhangs matching with the 5′ overhang of Module P and the 3′ overhang of Module T. The ccdB or LacZ cassette is cut off from the vector, enabling negative or blue–white screening. (E) The P–G–T construct is assembled in a user-specified order. Only a predetermined construct eliminating all BsmBI sites can stably exist. (F) Tg(β-actin:hKikGR1) confocal image shows the whole body expressing light conversion fluorescent protein hKikGR1. Under the irradiation of a 405-nm laser, the focused region was photo-converted from green to red. Scale bar, 50 μm. (G) Generation of P–G1.G2…Gn–T construct. The final construct has one gene selection T(1), two gene selection T(2), etc. (H) Confocal images of Tg(lfabp:loxP-mCherry-STOP-loxP-eGFP) identified with the background of Tg(hsp70l:Cre-cryaa:Venus). After heat-shock, Cre-mediated recombination resulted in most of the mCherry⁺ hepatocytes beginning to express eGFP. Scale bar, 50 μm. (I) Step 1: modules generate complete TUs flanked by newly generated BsaI sites (NNNN) and get kanamycin resistance (Kan^R) via a BsmBI Golden Gate reaction. Diamonds represent BsaI-mediated overhangs. (J) Step 2: TUs are further assembled into (P–G–T)n construct via a BsaI Golden Gate reaction. TUs with corresponding numbers select vectors and terminators with corresponding numbers. For example, TU1 selects Vector 1 and T1. But the last TU always selects Tn (black dotted line box). (K) A triple-transgenic line was generated by GoldenFish, which expresses different fluorescent proteins in the pancreatic β cells, liver, and whole body simultaneously. Scale bar, 50 μm.