Fig. 2

Barcode stability and code diversity. a The size-stability of barcodes with a single element depends on the length of the sequence between the flanking sites and their relative orientation. b Critical distances of barcodes of different sizes from a cassette with inverted flanking sites. The dotted line show the critical distance if flanking sites are oriented in the same direction. c Stability of barcodes from 2 to 5 elements for a Lox barcode cassette with inverted flanking sites. If the critical distance surpasses the minimal distance of 82 bp, stable codes (green) become unstable (gray). Barcodes of size three and four are unstable if m≥24 and m≥5 respectively, while codes of size five are always unstable. The gray interval illustrates potential uncertainty in the estimate of the minimal interaction distance. d Sequences between Lox cleavage sites represent the fundamental building blocks of the Lox barcode cassette. There are two with inverted Lox repeats (red, green) and two direct Lox repeats (blue) types of blocks. In the example, code elements are of size 7 bp and N denotes an arbitrary base. e For a cassette with inverted flanking sites pointing at each other and 5≤m<24, there are four possible block compositions ({k _r ,k _g ,k _b }): two for barcodes of size three (three: {1,0,2} and one: {2,1,0}), one for barcodes of size two (two: {1,0,1}) and one for barcodes of size one (one: {1,0,0}). f) Operations (moves) on odd and even elements possible via Cre Lox recombination, in cassettes with five, six and seven elements. Each move inverts the orientation of the respective element