Fig. 4

Short code elements, higher order Lox interactions, transient Cre activation, and distance dependent Lox-Lox complex formation. a Possible size-stable barcodes if m<5 bp. b Cassette with 17 elements and m=4 bp that attains an effective code diversity of 19,716 barcodes, requiring that the minimal Lox interaction distance is greater than 80 bp. c Higher order Lox interactions, where two or more pairs of Lox sites recombine simultaneously, can lead to unexpected recombination products. d Estimated barcode distribution if up to two Lox pairs can interact simultaneously (blue). The distribution becomes flatter at the lower end, which implies that rare codes are more likely compared to a scenario in which recombination events occur sequentially only (black). e Mimicking a short Cre activation pulse in a population of a million cells carrying a 13 element Lox cassette, the number of recombination events is assumed Poisson distributed with mean 1. The main graph shows code abundance after the pulse, where almost 10⁴ distinct barcodes are generated, with circa 30 % being generated only once. The inset, similar to Fig. 3 e, gives the number of 99 %-unique barcodes as a function of induced and discarded barcodes. f Distance dependent Lox-Lox interactions. Assuming that the likelihood for two productive Lox sites to form a complex is inversely proportional to their distance, interactions between sites that are close-by are favored over interactions of sites that lie further apart (see inset for the distribution over distance dependent (red) and uniform (gray) Lox-Lox interactions). The difference in barcode probabilities is relatively small between the distance dependent (red) and uniform (gray) scenario, their distribution being more homogeneous if close-by sites form complexes more frequently