Integration site analysis of remobilized SB transposons. (a) Schematic representation of the 8F donor locus showing the predicted orientation of the trimeric concatemer in scaffold 57. This injection-mediated integration event occurred by a non-canonical mechanism. (Not to scale.) (b) Schematic representation of the novel integration event in the remobilized tadpole shown in Figure 4b. EPTS LM-PCR was used to clone the sequence flanking the 5' end of the pT2 βGFP transposon and the 5' and 3' flanking sequences were verified using PCR primers designed to the scaffold sequence. The novel integration event occurred on the same scaffold (57 at position 2544323 bp) of the Joint Genome Institute X. tropicalis genome sequence assembly v4.1 as the 8F transposon donor locus (57:2456981) and represented a local hop. The sequence of the SB transposon and genomic DNA junctions (arrows) indicated that the remobilization event occurred via a canonical transposition reaction. The pT2 βGFP transposon is flanked by the expected TA dinucleotide target site duplication (TSD; bold underlined), and the transposon is inserted precisely, without any flanking plasmid sequence from the donor site. The genomic DNA sequence of scaffold 57 is capitalized and the transposon sequence is in lowercase italics. (Not to scale.) (c) The preferred sequence for SB transposon re-integration in the X. tropicalis genome. Weblogo analysis http://weblogo.berkeley.edu for the five base pair sequence flanking the TA target site. The relative size of the letters indicates the strength of the information on the y-axis, with the maximum indicated by two bits. The table shows the base distribution of the pT2 βGFP transposon re-integration target sites. EPTS LM-PCR: extension primer tag selection linker-mediated polymerase chain reaction; PCR: polymerase chain reaction; SB: Sleeping Beauty.