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. 2012 Aug 23;30(1):88–99. doi: 10.1093/molbev/mss202

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© The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

This is an Open Access article distributed under the terms of the Creative Commons Non-Commercial Attribution License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 2. — Schematic of the L1 and Alu constructs. A representation of the basic components of the constructs is shown. The L1 constructs contain the codon-optimized ORF1 and ORF2 separated by the L1_RP inter-ORF sequence (Wagstaff et al. 2011) or the wild-type sequence of the L1PA8 ORFs. Two types of L1 constructs were built, which differ only at their 3′-end: 1, untagged containing the SV40 polyadenylation signal (pA) and 2, tagged with the neomycin indicator cassette designed for retrotransposition assays (mneoI). The individual ORF1 (blue) and ORF2 (purple) sequences were cloned downstream of the CMV promoter of the expression vector pBudCE4.1 (Invitrogen). The ORF1 was cloned, so that the protein will contain a myc-his tag (myc) at the carboxy terminus. The Alu subfamily (yellow) constructs are tagged with the neomycin indicator cassette (neo^TET) designed for retrotransposition assays. RNA transcription is performed by the CMV promoter (L1) or the internal pol III promoter of the Alu enhanced by the upstream sequence of the 7SL gene (shown as gray arrows). The retrotransposition indicator cassettes (mneoI or neo^TET) contain an inverted neomycin resistance gene disrupted by an intron that will splice only from a transcript generated by the CMV or Alu promoter. The neo^TET contains a modification of the ribozyme from Tetrahymena (represented as a looped line) that functions as a self-splicing intron (Esnault et al. 2002). Only retrotransposed copies of the spliced RNA will confer G418 resistance. Some of the unique restriction sites used in the construction of the vectors are shown.