Assembly of chimeric rabbit/human “fragment antigen binding” (Fab) libraries in phagemid pC3C. Shown is a schematic of the workflow for the generation of a phagemid pC3C-based chimeric rabbit/human (rb/hu) Fab library, starting with a first PCR step in which rbV_κ-, rbV_λ-, and rbV_H-encoding cDNAs are amplified from reverse-transcribed mRNA of a naive or immune rabbit antibody repertoire. Diverse primer pairs (14 × 5 for rbV_κ, 8 × 1 for rbV_λ, and 11 × 2 for rbV_H) are used in 100 separate PCRs for each individual rabbit antibody repertoire. Subsequently, a second PCR step fuses the rbV_κ and rbV_H pools and the rbV_λ and rbV_H pools via a separately amplified invariable cDNA fragment that comprises huC_κ and huC_λ, respectively; a downstream ribosome binding site; and pelB. The flanking primers in the second PCR step also introduce the asymmetric SfiI (a) (5′-GGCCC_AGG^CGGCC-3′) and SfiI (b) (5′-GGCC_CCG^TCGGCC-3′) restriction sites, which are cut and ligated into the correspondingly cut phagemid pC3C. Electroporation of the ligation mixture into Escherichia coli completes the phagemid library, which typically comprises 10⁸–10¹⁰ independent transformants. Addition of helper phage to the host bacterial cells converts the phagemid-encoding Fab library to a phage-encoding and displaying Fab library. In pC3C, a lac promoter (black circle) drives bicistronic ompA-V_L-C_L and pelB-V_H-C_H1-HA-ΔpIII expression cassettes, with ompA and pelB serving as signal peptides, HA (hemagglutinin) as a detection tag, and ΔpIII, which is a C-terminal segment of the minor coat protein pIII of filamentous phage. Shine–Dalgarno sequences are indicated as black triangles, and a transcriptional terminator sequence is depicted as a reverse black triangle. Annotated features of pC3C are discussed in detail elsewhere (see Overview: The pComb3 Phagemid Family of Phage Display Vectors [Rader 2024]).