Schematic representation of expression constructs for PBase variants, PBase fusion proteins, and the dual fluorescent <i>UGm</i> transposon used in this study.

<p>(A) The mPB and NP-mPB coding sequences of mouse codon-optimized PBase were cloned into the <i>pTriEx-HTNC</i> plasmid by replacing the <i>Cre</i> cassette between <i>Spe</i>I and <i>Xho</i>I. The mPB and NP-mPB coding sequences were preceded by a hexa-histidine encoding sequence (6× His tag). NP-mPB included an additional nucleolus-predominant (NP) signal peptide. (B) The <i>pTriEx-mPB-tGFP</i> and <i>pTriEx-NP-mPB-tGFP</i> constructs expressed tGFP-fused PBases. The tGFP moiety allowed real-time imaging of the PBase variant subcellular distributions. The <i>pTriEx-mPB-2A-eGFP</i> and <i>pTriEx-NP-mPB-2A-eGFP</i> plasmids expressed PBase variants, which were linked to eGFP by the self-cleaving 2A peptide. All protein-encoding cassettes were transcriptionally regulated by a hybrid promoter composed of the CMV immediate early enhancer fused to the chicken β-actin promoter (CAG), and followed by a polyadenylation signal sequence (pA). (C) Flanking the 5′ and 3′ inverted terminal repeats (ITRs) (empty arrows), the dual fluorescent transposon (<i>pXL-T3-Neo-UGm-cHS4X</i>; <i>UGm</i>) carried a human ubiquitin C (UBC) promoter-driven <i>H2B-eGFP-2A-mCherry-GPI</i> (Gm) transgene that labeled the transposed cells with a characteristic chromatin EGFP and membrane mCherry dual fluorescence. Additional abbreviations: Neo^r, a neomycin phosphotransferase expression cassette providing resistance to G418 selection; 2× Ins, two copies of the insulator sequence from chicken β-globin.