Heterologous and native Plasmodium falciparum genes can be successfully assembled into pfYC vectors using all three cloning strategies. (A) The firefly luciferase gene (FL = 1.65 kb) was cloned into the pfYC1 and pfYC3 series (Additional file 3) using either yeast HR or Gibson assembly. Topological mapping with HindIII digestion yields three fragments, as FL and the selection markers do not contain HindIII sites. A 3.9 kb fragment is released from the pfYC1 series whether FL is present or not (Figure 2). The fragments containing cassettes A and B from pfYC10x:FL plasmids are (1.5 kb + FL) = 3.2 kb and (1.7 + selection marker size) kb, respectively. Similarly, the fragments containing cassettes A and B from pfYC1x0:FL plasmids are (1.5 + selection marker size) kb and (1.7 + FL size) = 3.4 kb, respectively. The sizes of the different selection markers are: nptII (0.8 kb); hdhfr (0.6 kb); bsd (0.4 kb) and ydhodh (0.95 kb). (B) Two native P. falciparum genes, ama1 (apical membrane antigen 1; PF3D7_1133400; 1.87 kb) and trxR (thioredoxin reductase; PF3D7_0923800.1; 1.85 kb) were cloned in parallel using restriction/ligation, Gibson assembly and yeast HR, and the same PCR products and digested pfYC120 vector. Successful gene insertion is expected to yield three HindIII digestion products that include: a backbone fragment (denoted as C); cassette B with the ama1 or trxR gene inserted (denoted as B when no insert is present and B′ when containing the proper insert); and cassette A containing the bsd gene (denoted as A). As a reference, the parent pfYC120 plasmid yields products denoted as A, C and B upon HindIII digestion. The asterisk in the yeast HR trxR panel denotes sample degradation that occurred during storage prior to analysis by gel electrophoresis.