A monomeric red fluorescent protein -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Campbell et al. (2002) Proc. Natl. Acad. Sci. USA 99 (12), 7877–7882. (10.1073/pnas.082243699)

Table 3. Codon replacements used in the directed evolution of the dimer2 and mRFP1

Library	Primer pairs for fragment amplification	Positions	Codon*	Possible substitutions	Genetic diversity
D1	1. PRSET_BamH1_FW/I161X_K163X_RV 2. I161X_K163X_FW/S179X_RV 3. S179X_FW/S197X_A_RV 4. S197X_A_FW/PRSET_EcoR1_RV	I161 K163 S179 S197	VYC MWS NCC NCC	T/I/A/V/P/L Q/H/L/K/N/M/I A/S/T/P A/S/T/P	~800 sequences
D3	1. PRSET_BamH1_FW/S197X_B_RV 2. S197X_B_FW/T217X_RV 3. T217X_FW/PRSET_EcoR1_RV	K70^† S197 T217	ARG NNK DYC	K/R All 20 a.a. A/T/S/V/I/F	~400 sequences
D4	1. PRSET_BamH1_FW/C117X_V127T_RV 2. C117X_V127T_FW/PRSET_EcoR1_RV	C117 F118 F124 V127	RVS NTS NTS ACC	T/K/S/N/R/A/E/G/D L/M/V/I/F L/M/V/I/F T	~800 sequences
D5	1. PRSET_BamH1_FW/I161X_K163X_RV 2. I161X_K163X_FW/S179X_RV 3. S179X_FW/S197X_RV 4. S197X_FW/H222G_F224G_RV	I161 K163 S179 S197 H222 F224	VYC MWS NCC NCC GGC GGC	T/I/A/V/P/L Q/H/L/K/N/M/I A/S/T/P A/S/T/P G G	~400 sequences
M1	1. PRSET_BamH1_FW/E144X_A164X_RV 2. E144X_A164X_FW/H222G_F224G_RV	E144 A145 S146 I161 H162 K163 A164 H222 F224	VVS VVS ASC NHC ARG NWS ARG GGC GGC	G/R/A/P/T/E/Q/K/S/D/H/N G/R/A/P/T/E/Q/K/S/D/H/N S/T N/D/H/Y/T/A/P/S/I/L/V/F K/R K/E/Q/M/V/L/N/D/H/Y/I/F K/R G G	~500,000 sequences
M3	1. PRSET_BamH1_FW/C117X_S131X_RV 2. C117X_S131X_FW/H162X_A164X_RV 3. H162X_A164X_FW/Y192A_Y194A_RV 4. Y192A_Y194A_FW/H222G_F224G_RV	C117 F124 V127 S131 H162 K163 A164 Y192 Y194 H222 F224	RVS NTS ACC YCC ARG MWS ARG GCC GCC GGC GGC	T/K/S/N/R/A/E/G/D L/M/V/I/F T S/P K/R Q/H/L/K/N/M/I K/R A A G G	~1,600,000 sequences with 4 templates shuffled in 4 fragments
M4	1. PRSET_BamH1_FW/K70X_K83X_RV 2. K70X_K83X_FW/Y192X_S197X_RV 3. Y192X_S197X_FW/T217X_L225X_RV	K70 V71 K83 Y192 Y194 V195 S197 T217 L223 L225	ARG GYC WWK RMS RMS RYC RYC DCC RMS RMS	K/R V/A N/K/I/M/Y/F/L E/D/A/K/N/T E/D/A/K/N/T T/I/A/V T/I/A/V S/T/A E/D/A/K/N/T E/D/A/K/N/T	~6,000,000 sequences
M6	1. PRSET_BamH1_FW/L174X_I180X_RV 2. L174X_I180X_FW/PRSET_EcoR1_RV	L174 V175 F177 I180	VVC NYC NYC AYC	A/D/G/H/N/P/R/S/T P/T/S/A/L/I/F/V P/T/S/A/L/I/F/V I/T	~4,600 sequences with 2 templates shuffled in 2 fragments
M7	1. PRSET_BamH1_FW/A145X_V156X_RV 2. A145X_V156X_FW/PRSET_EcoR1_RV then 3.V1a_del_FW/H222S_REV	V1a A145 R149 L150 R153 V156 H222	Deletion SCC CRS NTS VAS RBC TCC	Deletion A/P Q/H/R/R L/M/V/I/F E/Q/D/H/K/N V/A/T/I/S/G S	~2,300 sequences

*The standard mixed base definitions are R = A/G, Y = C/T, M = A/C, K = G/T, S = C/G, W = A/T, H = A/C/T, B = C/G/T, V = A/C/G, D = A/G/T, N = A/C/G/T.
^†Multiple templates were used to introduce these K70 substitutions.