1. N-end(50bp)GFPC-end(50bp)GFPpMOD4FLAGrpsLTetAompF pMOD4 RT-G (4615 bp) pMOD4 GFP FLAG pMOD4-FLAG-GFP ( 4029bp) N-end(50bp)GFPC-end(50bp)GFPFLAGGalkN-end(50bp)CTAPN-end(50bp)NTAPC-end(50bp) CTAPc-end(50bp)NTAPpMOD4 pMOD4) UNC-119 Lox P pLoxP-UNC-119 ( 7846bp) N-end(50bp)GFPC-end(50bp)GFPpMOD4FLAGGalk 2548bp cassette for RT recombineering 1381bp cassette for GalK recombineering 1612 bp cassette for GalK recombineering pMOD4GalK-G (3462 bp) pMOD4GalK-GT (3693 bp) Vectors

2. High fidelity PCR, Dpn I digestion and gel–recovery 50 bp homology arm of G.O.I 50 bp of G.O.I Culture of Fosmid clone of Gene of Interest (G.O.I) Extraction of G.O.I Fosmid DNAs Transformation of G.O.I Fosmid DNAs into SW106 Preparation of SW106 Competent cells of G.O.I Fosmid DNAs with induction in water bath G.O.I 1 st recombineering with RT positive selection (Tet+Chl+) G.O.I Original RT recombineering 2 d 2.5 d 3.5 d pBAC-RT rpsL TetA 50 bp homology arm of G.O.I PCR and gel-recovery TAG (GFP or TAP) rpsL TetA 50 bp of G.O.I TAG ( GFP or TAP) rpsL TetAompF rpsL TetAompF rpsL TetAompF Preparation of Competent cells 2 nd recombineering with RT negative selection on hypotonic media (Str+Chl+) Lox P

3. 50 bp homology arm of G.O.I 50 bp of G.O.I Culture of Fosmid clone of Gene of Interest (G.O.I) Extraction of G.O.I Fosmid DNAs Transformation of G.O.I Fosmid DNAs into SW106 Preparation of SW106 Competent cells of G.O.I Fosmid DNAs with induction in water bath G.O.I 1 st recombineering with RT positive selection (Tet+Chl+) or TAG ( GFP or TAP) G.O.I pMOD4UNC-119 Lox P pLoxP-UNC-119 pLoxP / Cre recombineering with 0.1% arabinose (AMP+Chl+) TAG ( GFP or TAP) G.O.I UNC-119 + Modified RT recombineering 2 d 2.5 d 3.5 d 2 d pMOD4RT-G pMOD4 rpsLTetA rpsLTetA rpsLTetA 50 bp of G.O.I TAG ( GFP or TAP) GFP rpsL TetAompF rpsL TetAompF rpsL TetAompF Preparation of Competent cells 2 nd recombineering with RT negative on hypotonic media (Str+Chl+) High fidelity PCR and gel–recovery ampR Lox P

4. 50 bp homology arm of G.O.I 50 bp of G.O.I Culture of Fosmid clone of Gene of Interest (G.O.I) Extraction of G.O.I Fosmid DNAs Transformation of G.O.I Fosmid DNAs into SW106 Preparation of SW106 Competent cells of G.O.I Fosmid DNAs with induction in water bath 2 nd recombineering with 2-Deoxy-galactose negative selection (Chl+) 1 st recombineering with galactose positive selection (Chl+) or pMOD4UNC-119 Lox P pLoxP-UNC-119 pLoxP / Cre recombineering with 0.1% arabinose (AMP+Chl+) G.O.I UNC-119 + gal K recombineering Verifying on MacConkey Agar individually Preparation of Competent cells 2 d 3.5 d 5 d 2 d pMOD4GalK TAG (GFP or TAP) G.O.I TAG ( GFP or TAP) 50 bp of G.O.I TAG (GFP or TAP) pMOD4GalK-GT High fidelity PCR and gel–recovery Lox P ampR Lox P