The P{lexAop-Raeppli-NLS} construct is a multicolor live imaging lineage-tracing tool. It allows whole-tissue labelling such that the descendents of the majority of cells in a single organ are labelled, and can be followed simultaneously relative to one another. It contains the sequences necessary for marking cells with one of four possible fluorescent proteins: E2-Orange, mKate2, mTFP1 and TagBFP. Each fluorescent protein is tagged with a nuclear localisation sequence (Tag:NLS(Unk)). The coding sequence for each fluorescent protein is downstream of an identical attP site that may be recombined to the single attB site of the construct using phiC31:int integrase. This single attB site is placed as far as possible upstream from the attP sites to minimise any bias in fluorescent protein selection. The expression of the selected fluorescent protein is driven by lexAop regulatory sequences.

Two steps are required in order for one of the four fluorescent proteins to be selected and expressed from the starting P{lexAop-Raeppli-NLS} construct. First, phiC31:int expression must be induced from sequences present in the construct. In the starting P{lexAop-Raeppli-NLS} construct a full Hsp70 promoter is separated from the phiC31:int coding region by a FRT cassette containing a stop codon. To activate phiC31:int expression, this cassette must be excised using a source of FLP recombinase, after which the phiC31:int coding region is expressed either under the control of the Hsp70 promoter, or under the control of the lexAop regulatory sequences present in the construct (which are further upstream of the phiC31:int coding region than the Hsp70 promoter sequences). phiC31:int promotes site-specific recombination between the upstream attB site and one of the four attP sites. Recombination excises the intervening region, bringing one of the four fluorescent proteins under the control of the lexAop regulatory sequences (only the fluorescent protein brought closest to the regulatory sequences is expressed as a stop codon is present after each fluorescent marker coding region). In all cases, the phiC31:int coding region is excised during the recombination event, making the color choice irreversible (in addition to the irreversibility of the attB attP recombination event). The end product is a construct that expresses a single fluorescent protein under the control of lexAop regulatory sequences.