The transcriptional stop cassette present in PBac{UAS(B3RT.B2)BoNTA} has been removed via B3R recombinase, leaving behind a single B3RT site (that is innocuous). As a result, UAS regulates expression of a BacTrace toxin construct (BoNT/A) in which the native receptor-binding domain (RBD) of Cbot\botA has been exchanged for a single-chain anti-GFP nanobody (VHH(GFP4)) followed by the extracellular portion of a transmembrane protein (Tag:M(rCd2)). The N-terminus has the C. elegans PAT3 signal peptide sequence for endoplasmic reticulum targeting, while the C-terminus has the Kir2.1 ER export signal to improve trafficking to the plasma membrane. TEV Tag:CS(TEVp) and thrombin cleavage sites have been introduced between the toxin light and heavy chains and between VHH(GFP4) and Tag:M(rCd2), allowing release of the toxin from the membrane after cleavage. (GenBank MN658777.)