## 2 Materials and Methods
### Construction of Semi-Synthetic VNA Library  
The method of constructing a semi-synthetic VNAR library was modified based on the research of [PERSON] et al. [35]. Using HEL-5A7 gene as PCR template, sets of NNK saturation-mutated DNA cassettes were introduced into the CDR3 loop by splice-overlap polymerase chain reaction (PCR). The first round of PCR used For-1 st as forward primer, with Rev12-X3 CX2 CX5, Rev13-X6 CX1 CX4, Rev16-X6 CX3 CX5, Rev18-X6 CX4 CX6, Rev20-X5 CX5 CX8, or Rev23-X6 CX5 CX10 as the reverse primer to create six various CDR3 loop lengths (12, 13, 16, 18, or 20 and 23 amino acids) with 10, 11, 14, 16, or 18 and 21 randomized residues. The primer sequences used in this study were listed in Table 1. Fifty microliters of the first PCR components are 11.5 \(\upmu\)L H\({}_{2}\)O, 10 \(\upmu\)L 5 \(\times\) Q5 buffer, 10 \(\upmu\)L 5 \(\times\) Q5 enhancer, 4 \(\upmu\)L 2.5 mM dNTP, 5 \(\upmu\)L 1 mM forward primer, 5 \(\upmu\)L 1 mM reverse primer, 4 \(\upmu\)L 2.5 ng/\(\upmu\)L HEL-5A7 DNA template, and 0.5 \(\upmu\)L 2 U/\(\upmu\)L Q5 DNA polymerase (New England Biolabs, Ipswich, MA, USA). The amplification was carried out with an initial denaturing step of 98 \({}^{\circ}\)C for 30 s, 35 cycles of 98 \({}^{\circ}\)C for 5 s, 60 \({}^{\circ}\)C for 15 s, and 72 \({}^{\circ}\)C for 15 s. The second PCR with the first PCR product as a template to create 40 bp flanks was compatible with the yeast display vector, pHEL5A7-GFP, allowing cloning by yeast homologous recombination [36]. Fifty microliters of the second PCR components are 11.5 \(\upmu\)L H\({}_{2}\)O, 10 \(\upmu\)L 5 \(\times\) Q5 buffer, 10 \(\upmu\)L 5 \(\times\) Q5 enhancer, 4 \(\upmu\)L 2.5 mM dNTP, 5 \(\upmu\)L 1 mM forward primer (For-TU1 mod-40 fl), 5 \(\upmu\)L 1 mM reverse primer (Rev-TU58 mod-40 fl), 4 \(\upmu\)L 25 ng/\(\upmu\)L purified first PCR product, and 0.5 \(\upmu\)L 2 U/\(\upmu\)L Q5 DNA polymerase (New England Biolabs). The amplification was carried out with an initial denaturing step of 98 \({}^{\circ}\)C for 30 s, 35 cycles of 98 \({}^{\circ}\)C for 5 s, 68 \({}^{\circ}\)C for 15 s, and 72 \({}^{\circ}\)C for 15 s. The primers used in PCR were synthesized by Sangon Biotech, Shanghai, China. The primer sequences are shown in Table 1, and the PCR products were purified with QIAquick PCR purification kit (Qiagen) before proceeding to the next step. The pHEL5A7-GFP plasmid was digested with _Hind_ III and _Bam_ HI restriction enzymes before transformation.  
A yeast surface display library was generated by electroporation-mediated transformation of the second PCR product and the digested pHEL5A7-GFP vector into EBY100 yeast using the lithium acetate method [37]. The method of electroporation-mediated transformation was modified based on the protocol by [PERSON] et al. [38]. Each electroporation was performed using a MicroPulser electroporator (Bio-Rad), where 3 \(\upmu\)g of PCR product and 1 \(\upmu\)g of digested vector were added to 400 \(\upmu\)L of electrocompetent EBY100 yeast. The electroporation was performed at 2.5 kV, 25 \(\upmu\)F, and 200 \(\Omega\). After twelve electroporation rounds were finished, 10 mL of yeast recovery medium (1:1 mixed 1 M sorbitol and YPD medium) was added to each batch of electroporated yeast cells and incubated at 30 \({}^{\circ}\)C with shaking for 1 h. As the control, an electroporation reaction containing only the digested or undigested pHEL5A7-GFP vector was also performed. After incubation, the yeast cells in the recovery medium were recovered, and cells were resuspended in 1 L of SD-CAA medium. The yeast cells in SD-CAA medium were cultured at 30 \({}^{\circ}\)C with shaking for 36 h. Following cultivation, cells were cryopreserved and stored at \(-\)80 \({}^{\circ}\)C. Each cryopreservation tube contained about 1.1 \(\times\) 10\({}^{10}\) cells.