*2.1. Nb23 Sequence Inferences*

The Nb23 construct characterized here consists of 136 amino acids, including an initial methionine residue introduced as a start codon and therefore referred to as Met0, and a (His)<sup>6</sup> tag at the C-terminus of the protein for expression in *E. coli* and purification, amounting to a molecular weight of 15.1 kDa. There are two cysteines at position 22 and 96 which form the disulfide bond between the two β-sheets of the expected immunoglobulin domain. Nb23 and Nb24 are of equal lengths with 71% identity, and 75% positive identity. This level of homology indicates structural and functional similarity [13]. The fact that the main variation in sequences between Nb23 and Nb24 coincides with the CDRs (located between residues 26–32, 52–57, and 100–116), together with a general consensus on the typical structural similarity of the framework regions of immunoglobulin variable domains, suggests that the frameworks of both nanobodies are similar.
