Cloning and Isolation of HzTransib Transposase

The full length or an N-terminal truncated fragment (residues 17–507) of HzTransib transposase fused to a C-terminal His₆ tag or an N-terminal maltose-binding protein (MBP) tag were cloned into pFastBac1 expression vector (ThermoFisher Scientific) between BamHI and HindIII restrictive sites. pB-5’/3’TIR, a derivative of pBR322 containing the TIR substrate for ProtoRAG transposases, was described previously^{33 (link)}. To generate TIR substrate for HzTransib transposases, the ProtoRAG 5’TIR and 3’TIR of pB-5’/3’TIR were substituted by the first 51 bp and 50 bp of HzTransib transposon 5’TIR and 3’TIR, respectively, using In-Fusion cloning (Clontech). The PCR amplified and linearized HzTransib substrate contains a HzTransib 5’TIR and 3’TIR separated by 411 bp between their tips, 126 bp of DNA flanking 5’TIR and 276 bp of DNA flanking 3’TIR. The whole substrate was depleted of 5’-CAC-3’ sequence instances except for those contained in 5’TIR and 3’TIR regions.