Supplementary Information

Transcription

1 Supplementary Information Fluorescent protein choice The dual fluorescence channel ratiometric promoter characterization plasmid we designed for this study required a pair of fluorescent proteins with the following properties:. Bright fluorescence signal.. Spectrally distinguishable excitation and emission. 3. Similar maturation rates. Similar sequence locally to promoter The requirement for similar DNA sequence close to the upstream promoter stems from observations of interactions between transcript and promoter sequences. Differences in maturation times would affect the synchronization of concurrent measurements. Emission and excitation spectra must be distinguishable to avoid bleed-through between channels. To guide our choice of fluorescent proteins, we designed a single channel promoter characterization plasmid (Figure ). This design is identical to the ratiometric characterization plasmid presented in the main text except that there is no reference gene. In particular the promoter is fused to the same BioBrick ribosome binding sequence BBa_B3. Each of our six test promoters was cloned into this plasmid (Px) with one of GFPmut3, mcherry, EYFP or ECFP fluorescent protein coding sequences (FP), giving a total of plasmids. Promoter activity (P.A.) at peak growth was then estimated using the method described in the main text (Figure ). To enable comparison between measurements with different reporters we normalized these P.A.s by the activity of J3 measured with the same fluorescent protein. This gives the relative promoter activity, which is unity for promoter J3. All fluorescent proteins produced strong fluorescence signal with the promoters used in this study. We found that the rank order of promoter activities was conserved between mcherry, ECFP and EYFP, but not by GFPmut3. This may be due to interference of the GFPmut3 coding sequence with the RBS or promoter. We therefore rejected GFPmut3 as a suitable reporter. The remaining proteins are spectrally distinguishable; mcherry (ex. 587nm, em. 6nm), ECFP (ex. 3nm, em. 77nm) and EYFP (ex. 5nm, em. 57nm). However, we noted that ECFP and EYFP have very similar sequences (8 mismatched base-pairs and amino acid substitutions) and maturation halflives (ECFP:.min -. EYFP:.78min - ) 3. We therefore rejected mcherry in favor of ECFP and EYFP, which meet all four of the requirements listed above. Figure. Single channel promoter characterization plasmid design. Px is the promoter of interest, and FP is the fluorescent protein.

2 Figure. Promoter activity measured from single channel plasmids. Bars are labelled according to the pro- moter of interest, and colors indicate fluorescent protein: GFPmut3 (green), mcherry (red), ECFP (blue), and EYFP (yellow). Black bars are the measurements presented by Kelly et al.. All promoter activities are normal- ised to J3 (STD) measured with the same fluorescent protein.

3 Fluorescence was proportional to OD in exponential growth phase A R5 R R J35 J35 D J Glucose (6 replicates) R Mean Std. dev B R5 R R J35 J35 J Glycerol (6 replicates) R Mean Std. dev Figure 3. Fluorescence against OD during exponential phase. EYFP (red) and ECFP (blue) fluorescence in- tensities (y- axis) measured from ratiometric plasmids (see main text) were well correlated with culture optical density (OD, x- axis). Three replicates from two separate colonies for each plasmid, lines show regression with R at right of figure. Culture growth media were (A) M9 +.% glucose, (B) M9 +.% glycerol, supplemented with.% casamino acids and kanamycin for selection.

4 A R5 R R J35 J35 J Chloramphenicol (6 replicates) R Mean Std. dev B R5 R R J35 J35 J Rifampicin (6 replicates) R Mean Std. dev Figure. Fluorescence against OD during exponential phase. As figure 3, with culture growth media: M9 +.% glucose +.% casamino acids + (A) μg/ml chloramphenicol, (B) μg/ml rifampicin, with kanamycin for selection.

5 Single channel promoter characteristics Statistics of single channel promoter characteristics estimated from ratiometric plasmids for reference ECFP promoter (αc) and promoter of interest driving EYFP (αy) are shown in Table. Plasmid Mean(αy) Std(αy) CV(αy) Mean(αc) Std(αc) CV(αc) pr5.63x.39x..67x.57x.5 pr.65x.373x. 3.9x.3x.3 pr.6x.x x.33x. pj35.5x.x.69 3.x.5x.7 pj35.777x.369x.75 3.x.63x. pj3.6x.59x. 3.6x.3x.73 Table. Statistics of promoter characteristics estimated from measurement of dual- channel plasmids. CV=coefficient of variation. Ratiometric characterization in LB rich medium The measurement of ratiometric plasmids described in the main text was repeated with cultures growing in LB rich medium (Figure 5). LB medium exhibits strong background fluorescence readings in the ECFP (53/nm) channel, which limits the accuracy of promoter activity estimates (error bars in Figure 5), however ratiometric characteristic ρ shows similar values to those from cultures grown in M9 minimal media (see main text).

6 Figure 5. Promoter characterisation from cells with ratiometric plasmids, growing in LB rich medium at 37 degrees centigrade. See main text for explanation of characterisation procedure. Error bars show one standard deviation. Single cell EYFP and ECFP fluorescence were correlated To assess single cell variation in fluorescence we grew cultures carrying each of the six ratiometric plasmids overnight in M9 +.% casamino acids +.% glucose and kanamycin selection for approximately 6 hours. Cultures were then run them through a BeckmanCoulter MoFlo MLS highspeed cell sorter to measure approximately, cells. ECFP was excited by a 7/3nm and EYFP by a 5nm laser line, and emission filtered at 7/nm and 56/nm. Untransformed host strain (ECG) was measured as a control, and gating performed using FlowJo software (FlowJo Enterprise). EYFP and ECFP fluorescence were correlated (R>.7) for all plasmids.

7 R5 R R EYFP (AU) R=.7 R=.7 R=.7 J35 J35 J3 R=.7 R=.7 R=.7 ECFP (AU) Figure 6. Flow cytometry of cells with ratiometric plasmids. Density maps with correlation coefficient (R) shown inset, red line illustrates linear regression. Sequence information Listed below are sequences of the dual channel characterization plasmids described in the main text (pr5, pr, pr, pj3, pj35, and pj35). Promoters R5, R, R, J3, J35, and J35 in red, reference promoter J3 in blue, EYFP in yellow, RBS B3 in purple, B5 terminator in green and ECFP in cyan. pr5 taacaccgtgcgtgttgactattttacctctggcggtgataatggttgctactagagaaagaggagaaatactagatggtgagcaaggg cgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgaggg cgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgacc accttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggcta cgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaa ccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacg tctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcg ccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgag caaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtac aagtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaa cgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaaa cgggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcg ctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatgctctgccagtgt tacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatt tttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccg actcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatcc ggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgca accggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccgggga tcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagt

8 ctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgat agattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcga gcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattttta tcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacta taaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaagg cccagtctttcgactgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtgatcccggcg gcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcg ggcagcagcacggggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgtggcggat cttgaagttggccttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgtgccccaggat gttgccgtcctccttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggtcttgtag ttgccgtcgtccttgaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcg gggtagcggctgaagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagat gaacttcagggtcagcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgccgtccagctc gaccaggatgggcaccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctagcataatacct aggactgagctagctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacct acaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttctt cacgaggcagacctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcag gagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcg ttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgaga gggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaaccc gacaggactataaagataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgct gttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgt tcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccact ggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaag ccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagat tacgcgcagaccaaaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggattttg gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaactt ggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgt agataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatca gcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatggtgccacct gacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagc tttcgctaaggatgatttctggaattcgcggccgcttctagag pr aattgtgagcggataacaattgacattgtgagcggataacaagatactgagcacatactagagaaagaggagaaatactagatggtg agcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtcc ggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc ctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgccc gaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacac cctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaaca gccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagc gtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagt ccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgg acgagctgtacaagtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg tttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtcc ggcaaaaaaacgggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcact gactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatg ctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattat caataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcgg tctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtg acgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatca

9 ctcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacagg aatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgt tttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtca gccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttccca tacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatc gcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgat gatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaagga tcagatcactataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataa aacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtg atcccggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggta gtggttgtcgggcagcagcacggggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgt tgtggcggatcttgaagttggccttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgt gccccaggatgttgccgtcctccttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgc gggtcttgtagttgccgtcgtccttgaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgct tcatgtggtcggggtagcggctgaagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccgg tggtgcagatgaacttcagggtcagcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgc cgtccagctcgaccaggatgggcaccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctag cataatacctaggactgagctagctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaac tggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagca acaccttcttcacgaggcagacctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttc atgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctac gctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacaggg aagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtgg cgaaacccgacaggactataaagataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgt cattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacga accccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcag cagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgct cctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagag caagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaag ggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgag taaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcccc gtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccag atttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatgg tgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaa tccttagctttcgctaaggatgatttctggaattcgcggccgcttctagag pr tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaagaggagaaatactagatggtgag caagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccgg cgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctc gtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccga aggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccct ggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagcc acaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtg cagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccg ccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg agctgtacaagtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttg tcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggc aaaaaaacgggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgact cgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatgctct gccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaa taccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctg

10 cgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacga ctgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgc atcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatc gaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttc ccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagcc agtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatac aatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcg gcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgata tatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcag atcactataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaac gaaaggcccagtctttcgactgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtgatc ccggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtg gttgtcgggcagcagcacggggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgt ggcggatcttgaagttggccttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgtgcc ccaggatgttgccgtcctccttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggt cttgtagttgccgtcgtccttgaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcat gtggtcggggtagcggctgaagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggt gcagatgaacttcagggtcagcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgccgtc cagctcgaccaggatgggcaccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctagcat aatacctaggactgagctagctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactgg tccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaaca ccttcttcacgaggcagacctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgt ggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctc ggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagt gagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaa acccgacaggactataaagataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattc cgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaacccc ccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagcc actggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctcc aagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaag agattacgcgcagaccaaaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaa cttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcg tgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatggtgcca cctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatcctt agctttcgctaaggatgatttctggaattcgcggccgcttctagag pj3 tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatggtgagcaagggcgaggagctgttc accggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgcc acctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacgg cctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgca ccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctga agggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccg acaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaac gagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaataatact agagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactag agtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaaacgggcaaggtgt caccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctcggtcgttcgg ctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaatta

11 accaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagcc gtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaaca tcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatgg caaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattc attcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcagg aacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtga gtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcat ctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacc tgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcc cgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgta acatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcactataaacgcagaaa ggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgac tgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtgatcccggcggcggtcacgaact ccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggcagcagcacg gggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgtggcggatcttgaagttggcc ttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgtgccccaggatgttgccgtcctcc ttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggtcttgtagttgccgtcgtcctt gaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcggggtagcggctg aagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagatgaacttcagggtc agcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgccgtccagctcgaccaggatggg caccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctagcataatacctaggactgagcta gctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctc tcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagac ctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggc tgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcg agcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaa agccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataa agataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgttt gtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgct gcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttaga ggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggtt caaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagacca aaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatc aaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgat acgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccag ccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatggtgccacctgacgtctaagaa accattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat gatttctggaattcgcggccgcttctagag pj35 tttacggctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatggtgagcaagggcgaggagctgttc accggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgcc acctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacgg cctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgca ccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctga agggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccg acaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaac gagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaataa tactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctac tagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaaacgggcaagg

12 tgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctcggtcgttc ggctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaat taaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaag ccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaa catcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaat ggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttat tcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgca ggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggt gagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatct catctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgc acctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtt tcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaat gtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcactataaacgcaga aaggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcg actgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtgatcccggcggcggtcacgaa ctccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggcagcagcac ggggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgtggcggatcttgaagttggc cttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgtgccccaggatgttgccgtcctc cttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggtcttgtagttgccgtcgtcc ttgaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcggggtagcggct gaagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagatgaacttcagggt cagcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgccgtccagctcgaccaggatgg gcaccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctagcataatacctaggactgagct agctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagct ctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcaga cctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaagg ctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcg agcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaa agccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataa agataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgttt gtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgct gcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttaga ggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggtt caaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagacca aaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatc aaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgat acgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccag ccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatggtgccacctgacgtctaagaa accattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat gatttctggaattcgcggccgcttctagag pj35 ttgatggctagctcagtcctaggtacaatgctagctactagagaaagaggagaaatactagatggtgagcaagggcgaggagctgttc accggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgcc acctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacgg cctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgca ccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctga agggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccg acaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaac

13 gagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaataatact agagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactag agtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaaacgggcaaggtgt caccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctcggtcgttcgg ctgcggcgagcggtatcagctcactcaaaggcggtaatctcgagtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaatta accaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagcc gtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaaca tcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatgg caaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattc attcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcagg aacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtga gtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcat ctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacc tgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcc cgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgta acatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcactataaacgcagaaa ggcccacccgaaggtgagccagtgtgactctagtagagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgac tgagcctttcgttttatttgatgcctggctctagtattattacttgtacagctcgtccatgccgagagtgatcccggcggcggtcacgaact ccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggcagcagcacg gggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgtggcggatcttgaagttggcc ttgatgccgttcttctgcttgtcggcggtgatatagacgttgtggctgatgtagttgtactccagcttgtgccccaggatgttgccgtcctcc ttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggtcttgtagttgccgtcgtcctt gaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcggggtagcggctg aagcactgcacgccccaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagatgaacttcagggtc agcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgttcacgtcgccgtccagctcgaccaggatggg caccaccccggtgaacagctcctcgcccttgctcaccatctagtatttctcctctttctctagtagctagcataatacctaggactgagcta gctgtaaagcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctc tcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagac ctcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggc tgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcg agcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaa agccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataa agataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgttt gtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgct gcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttaga ggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggtt caaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagacca aaacgatctcaagaagatcatcttattaaggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatc aaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgat acgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccag ccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattccatggtgccacctgacgtctaagaa accattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat gatttctggaattcgcggccgcttctagag Listed below are maps of the dual channel characterization plasmids described in the main text (pr5, pr, pr, pj3, pj35, and pj35).

14 BBa_R5..9 BBa_B EYFP prefix BBa_B BBa_B T7 TE 9..9 BBa_B T7 TE p5a pr5 Suffix bp BBa_J BBa_B ECFP 3..8 KanR T7 TE BBa_B T7 TE BBa_B 7..9 BBa_B 6..7 Figure 7. Plasmid map for pr5 vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and R5 promoter is shown in red.

15 BBa_R..5 BBa_B EYFP 8..8 prefix BBa_B BBa_B T7 TE BBa_B 9..9 T7 TE p5a pr Suffix bp BBa_J BBa_B ECFP KanR T7 TE 6..9 BBa_B T7 TE BBa_B BBa_B Figure 8. Plasmid map for pr vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and R promoter is shown in red.

16 BBa_R..5 BBa_B EYFP prefix BBa_B BBa_B T7 TE BBa_B 9..9 T7 TE p5a pr Suffix bp BBa_J BBa_B ECFP KanR T7 TE 6..8 BBa_B T7 TE 8..6 BBa_B BBa_B 65.. Figure 9. Plasmid map for pr vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and R promoter is shown in red.

17 BBa_J BBa_B3..55 EYFP prefix BBa_B BBa_B T7 TE BBa_B T7 TE p5a pj3 Suffix bp BBa_J BBa_B ECFP KanR T7 TE..9 BBa_B T7 TE 6..3 BBa_B BBa_B 6..3 Figure. Plasmid map for pj3 vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and J3 promoter is shown in red.

18 BBa_J BBa_B3..55 EYFP prefix BBa_B BBa_B T7 TE BBa_B T7 TE p5a pj35 Suffix bp BBa_J BBa_B ECFP KanR T7 TE..9 BBa_B T7 TE 6..3 BBa_B BBa_B 6..3 Figure. Plasmid map for pj35 vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and J35 promoter is shown in red.

19 BBa_J BBa_B3..55 EYFP prefix BBa_B BBa_B T7 TE BBa_B T7 TE p5a pj35 Suffix bp BBa_J BBa_B ECFP KanR T7 TE..9 BBa_B T7 TE 6..3 BBa_B BBa_B 6..3 Figure. Plasmid map for pj35 vector. P5A replication origin and kanamycin resistance gene are shown in blue, EYFP reporter is shown in yellow, ECFP reference reporter is shown in cyan, ribosome binding sites B3 are shown in purple, reference promoter J3 is shown in black and J35 promoter is shown in red.

20 Step-by-Step guide for dual-channel promoter characterization - Clone your promoter of interest into the ratiometric plasmid maintaining the same flanking sequences across the entire set of plasmids to be measured (Fig 3). We recommend Gib- son Assembly since it produces seamless assemblies. A computational tool, such as Gibthon, can be used for primer design ( Figure 3. Schematic view of promoter cloning into dual ratiometric plasmids. Flanking sequences to the promoter of interest are shown to facilitate primer design. - Transform each of the characterization plasmids, containing the promoters to be meas- ured, into competent E. coli cells, and incubate them overnight in LB agar plates containing kanamycin (5 μg ml ) for selection. 3- Select two colonies of each of these transformations and inoculate each of them separate- ly into 5 ml of M9 media containing kanamycin (5 μg ml ),.% casamino acids and.% glucose. - Grow these cultures overnight in a shaking incubator at 37 C and rpm for approximate- ly 6 hours. 5- Dilute these cultures : into fresh M9 media containing kanamycin (5 μg ml ),.% casamino acids and.% glucose. 6- Add μl of each of these diluted cultures to three different wells (technical replicates) of a black 96- well micro- plate with clear bottom. 7- Measure optical density at 6 nm and fluorescence every ~ minutes in a plate reader. Maintain temperature at 37 C during the measurement assays and use orbital shaking ( rpm) between reads to avoid cell aggregation and precipitation. Use excitation filter 3/nm and emission filter 8/nm for ECFP measurements; and excitation filter 5/nm and emission filter 53/nm for EYFP measurements.

21 8- Export data as spreadsheets, download our matlab analysis software (github.com/haselofflab/platypus), and follow the analysis procedure given in the file ex- ample.m. References. Lou, C., Stanton, B., Chen, Y.-J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology 3, 37 ().. Kosuri, S. et al. Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proceedings of the National Academy of Sciences, 9 (3). 3. Gordon, A. et al. Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nature Methods, 75 8 (7).. Kelly, J. R. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 3, (9).