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Figure S1. Plasmid pUA67.

Figure S2. Clones derived from AncS and AncD strains that were used in our experiment.

Figure S3. Nomenclature of the evolved lines based on the selection condition.

Figure S4. Fitness of the ancestral strains (AncS and AncD) in ampicillin + cefotaxime.

Figure S5. Fitness of the ancestral strains in imipenem.

Figure S6. Initial fitness measurement of the ancestral strains (AncS and AncD) in ampicillin + imipenem.

Figure S7. Growth curves of the ancestral strains (AncS and AncD).

Figure S8. Fitness of the evolved CA lines.

Figure S9. Fitness of the evolved N lines.

Figure S10. Fitness of the evolved A lines.

Figure S11. Fitness of the evolved A[UPWARDS ARROW] lines.

Figure S12. Fitness of the evolved C lines.

Figure S13. Fitness of the evolved I lines.

Figure S14. Fitness of the evolved IA lines.

Figure S15. Increase of plasmid copy number in the evolved lines.

Figure S16. Analysis of TEM-1 gene expression level in AncS, AncD, EvoS-CA3, and EvoD-CA3 lines using quantitative PCR.

Figure S17. Frequency of individuals retaining duplicate copies of the TEM-1 gene in the EvoD lines.

Figure S18. Analysis of the inserts in the plasmids isolated from the clones of the EvoD-CA1 line.

Figure S19. Change in population frequency of the T-53C mutation in the evolved lines over the rounds of the experiment.

Figure S20. Analysis of TEM-1 mRNA expression level of the reconstructed T-53C mutant and the wild-type control using quantitative PCR.

Figure S21. Change in the sequence diversities of the evolved lines over the course of the experiment.

Figure S22. Change in the sequence diversities in replicate lines.

Figure S23. Growth curves of the evolved lines EvoS-CA3 and EvoD-CA3.

Figure S24. Antibiotics concentrations used for selection experiments.

Figure S25. Correlation between the read coverage and the number of polymorphisms detected.

Figure S26. Change in frequency of the mutations that were present in the starting lines over the rounds of the experiment.

Figure S27. Average number of nucleotide differences between sequence reads in pairwise comparisons.

Figure S28. Ka/Ks ratio in the evolved lines.

Figure S29. Barcodes in the amplicons for 454 sequencing (not to scale).

Figure S30. Amplicons used for 454 sequencing and how they map to TEM-1 regions (not to scale).

Figure S31. Error rates in the amplicons from the next generation sequencing data.

Figure S32. Error rates in two sets of reads in the 454 sequencing data.

Table S1. Primer pairs for screening of individuals containing duplicate genes in the EvoD lines.

Table S2. Fitness measurements of the ancestral strains.

Table S3. Relative fitness values of the evolved lines.

Table S4. Fold-increase in plasmid copy number in the evolved lines.

Table S5. Percentage of individuals that retained duplicate genes in the EvoD lines.

Table S6. Average mutation rate in the mutator E. coli strain (TB90).

Table S7. Primers with barcodes for sample multiplexing in 454 sequencing.

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