(A) Denaturing acrylamide gel showing casposase-catalysed disintegration of a DNA flap from fork-3 DNA. Reactions lasted 1 h and contained 25 nM of fork-3 DNA and 150 nM of casposase wild-type or mutant proteins as indicated. (B) The cartoon illustrates the integration assay and its outcomes. The agarose gel is a representative summary of Cy5-labelled single-end (nicked) and double-end (linear) integration products formed from integration into pACYC-Duet of substrates (100 nM) indicated above each lane, by 150 nM of casposase or inactive casposase mutant D254A for 1 h. (C) Measurement of relative integration activities of casposase (150 nM) on ssDNA or dsDNA substrates (100 nM) into pACYC-Duet (150 ng). Reactions were in triplicate to quantify integration relative to the dsLE30 substrate, showing distribution of standard error from the calculated mean. The DNA integration substrates are shown labelled and the box highlights the presence of the significant cytosine at the -3 position from the DNA 3′ end. (D) Average integration efficiency of DNA-bla with or without TIRs into pACYC-Duet catalysed by casposase. Results were from three independent repeats and full data were shown in Supplementary Figure S6. Error bars standard error of the mean. Unpaired t test showed the difference between mini-casposon integration and DNA-bla blunt was significant (P=0.0131); *: P-value<0.05. (E) Location of integration sites of mini-casposon (black arrows) and DNA-bla without TIRs (red arrow) in pACYC-Duet. Ten mini-casposon integrated pACYC-Duet plasmids were sequenced and one for DNA-bla. Direction of arrows indicates the direction of insertion of the 3′-OH of the left end nucleophilic attack strand into the plus strand or the minus strand. (F) DNA logo plot of pre-integration site generated from 10 sequenced mini-casposon integration sites in pACYC-Duet. Nucleotides 1–15 were upstream sequence and nucleotides 16–30 were target site which would be duplicated after integration. Nucleotides 31–45 were downstream sequence. The A. boonei T469 casposon insertion site (NC 013926.1) was shown below the DNA logo for comparison.