Fig. 2.

A broad range of gene modifications using the intronic attB. a Strategy for Bxb1 integrase-mediated recombination to produce diverse changes to a target gene. The pLN-attP plasmid carries the attP element, the downstream intronic sequence and desired downstream modifications. bsd, neo and hdhfr are selectable markers; int represents Bxb1 integrase gene as expressed from the pINT helper plasmid. Transfection of the GOI-attB parasite replaces the downstream ORF (red arrow) with a desired modification (yellow arrow) of the gene of interest (GOI). Recombination between attB and attP elements yields attL and attR sites. b Example modifications. Top row shows a site-directed mutation, an insertion, an epitope tag, and the glmS riboswitch in the 3’ untranslated region (left to right, respectively). Second row shows production of the TFLC3 parasite expressing two full-length CLAG3 proteins (Dd2 and 3D7 isoforms) with distinct epitope tags (HA and mSG-F) under a single clag3 promoter; the two proteins are separated during translation through insertion of a viral skip peptide (T2A, [52]). c Gene replacement approach showing insertion of a full-length gene with promoter and terminator sequences after the silent attB. This approach simultaneously disrupts the GOI and integrates a cloned gene downstream of the target site