Table 1 List of missing positions of the Vip3Af1(WT) protein which could not be tested. Amino acid positions correspond to the Vip3Af1 protein sequence (NCBI accession No. CAI43275).

List of clones within the library with no expression of the modified-Vip3Af1 protein

Asn⁵, Ile¹⁷⁶, Asn¹⁸⁰, Phe¹⁸³, Phe²⁸⁶, Arn²⁹³, Asp³⁰², Tyr⁴⁰⁹, Phe⁴¹⁶, Tyr⁴²⁰, Glu⁴⁵⁸, Leu⁵¹¹, Leu⁵²⁰, Asn⁵⁴¹, Thr⁵⁶⁴, Gly⁵⁶⁹, Gly⁶³⁹, Ile⁷⁸⁰, Ser⁷⁸⁶

19

 First amino acid (start codon)

Met¹

1

 Gap at the N-terminus region

From Ser¹⁵ to Thr¹⁶⁵

151

 Alanine in the wt sequence (Ala^x)

x = 10, 12, 172, 189, 203, 216, 248, 252, 257, 280, 283, 285, 299, 336, 345, 351, 356, 375, 440, 462, 469, 496, 517, 554, 559, 572, 657, 670, 690, 725

30

 Failed mutagenesis

Lys⁷, Val¹⁷⁹, Lys²⁹⁴, Leu²⁹⁶, His³¹⁰, Leu³¹¹, Asn³¹², Lys³¹³, Glu³¹⁶, Tyr³³⁵, Gly³⁴⁰, Glu³⁷⁴, Asp³⁸⁶, Leu³⁹⁹, Pro⁴¹⁷, Glu⁴³⁷, Val⁴⁸⁰, Thr⁵¹⁸, Asp⁵¹⁹, Phe⁵⁸¹, Tyr⁵⁹⁵, Ile⁶⁰⁷, Leu⁶⁰⁹, Lys⁶²⁷, Asp⁶²⁸, Thr⁶⁴⁰, Leu⁶⁴⁹, Asn⁷⁰⁰, Thr⁷⁰⁶, Arg⁷⁰⁸, Gln⁷⁰⁹, Ser⁷¹⁵, Tyr⁷¹⁶, Ser⁷²⁰, Ile⁷²¹, Phe⁷²⁹, Arg⁷³⁴, Val⁷³⁹, Ser⁷⁴⁸, Ser⁷⁴⁹, Ser⁷⁵¹, Phe⁷⁵⁷, Asn⁷⁶², Asn⁷⁶³, Val⁷⁶⁸, Ser⁷¹¹, Phe⁷⁸², Glu⁷⁸³

48

 Total (missing positions)

230