Transposition with <EGFP-V> (A) The transposon, <EGFP-V>, is flanked by 19 bp inverted repeats, the MEs. The EGFP coding region is positioned such that when <EGFP-V> inserts between the codons of a target gene, a fusion protein will be produced. <EGFP-V> also carriesKanr. There is a stop codon in the 5' end of the Kanr cassette in the same frame as the EGFP coding sequence, so if the transposon lands in an open reading frame, in the correct orientation and frame, a truncated, EGFP-tagged, protein will initially be produced. Removal of theKanr cassette by Srf I digestion and re-ligation produces a reading frame that extends across the entire transposon. (B) The target plasmid, αsEE in pcDNA1/Amp, encodes an epitope tagged version of the G protein subunit αs. (C) Transposed plasmids carry Ampr and Kanr. <EGFP-V> insertions within the target gene produce a PCR product when <EGFP-V> is inserted in the correct orientation, and the size of the PCR product reveals which <EGFP-V> insertions are in the coding sequence.
Sheridan et al. BMC Neuroscience 2002 3:7 doi:10.1186/1471-2202-3-7