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Figure 1 | BMC Biotechnology

Figure 1

From: A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins

Figure 1

Two new transposons for generating multicolored GFP fusion protein libraries. In-frame insertions of the Either-Or transposon, <EYOR> (A), within another coding sequence initially create truncated C-terminal YFP fusions. Alternate restriction digestion with Asc I or Srf I removes one of the fluorescent proteins and the KanR. Subsequent re-ligation produces identical full-length YFP and CFP fusion proteins. The fluorescent protein is flanked by 9 amino acid linkers encoded by the Tn5 MEs and restriction sites. The Double Barrel transposon, <DBT> (B), encodes green and red fluorescent proteins (GFP and DsRed) antiparallel to one another. Therefore, <DBT> insertions with another coding sequence have a 1:3 chance of being in-frame regardless of orientation. The GFP coding sequence uses the same reading frame through the Tn5 MEs as YFP in <EYOR>. DsRed however, has been shifted by 1 bp to use a different reading frame through the Tn5 MEs, doubling the number of usable insertion sites within a target coding sequence (Note that the reading frame shown in <DBT> is that used for DsRed , translated from the lower DNA strand, and is read from right to left).

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