The pLA513 plasmid was co-transformed with the phsp-pBac helper plasmid, which served as a source for the piggy Bac transposase.

Transcription of the DsRed2 protein begins at the Drosophila melanogaster actin 5c promoter and terminates at the D. melanogaster dorsomycin 3' untranslated region. The promoter drives expression of the fluorescent protein marker, which causes an accumulation of soluble protein within the cells.

Transcription of the tetracycline‐transcriptional activator variant (tTAV) begins at the D. melanogaster heat shock protein 70 promoter and terminates at the D. melanogaster DNA-binding protein K10 3' untranslated region (poly-adenylation signal). The transcript initially includes a D. melanogaster alcohol dehydrogenase intron at the 5' end of the transcript to enhance expression of tTAV. Immediately adjacent to the tTAV cassette is a tetracycline operator, which acts as a repressible switch. In the presence of tetracycline, tTAV preferentially binds tetracycline rather than the operator sequences. Thus, transcription remains at a basal level and repressed. In the absence of tetracycline, tTAV binds the operator sequences and stimulates transcription. Thus, under these conditions, transcription of tTAV is expected to be strong and with production of the tTAV protein occurring at elevated levels.

Please note:
- tTAV is a synthetic construct and contains sequences from the Escherichia coli tetracycline repressor and the Human herpesvirus 1 viral protein 16 transactor
- DNA deletions in the piggyBac sequences prevent mobility of the transposon. Additionally, the construct does not introduce a transposase. Thus, re-mobilization is not expected.
- Southern blot analysis confirmed a single insertion into the mosquito genome occurred
- Inverse PCR and sequencing suggested that the insertion does not interrupt an open reading frame
- The plasmid backbone contains an ampicillin resistance gene, which was not detected in the transformants