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www.sciencemag.org/cgi/content/full/science.1249252/DC1
Supplementary Materials for
Total Synthesis of a Functional Designer Eukaryotic Chromosome
Narayana Annaluru, Héloïse Muller, Leslie A. Mitchell, Sivaprakash Ramalingam, Giovanni Stracquadanio, Sarah M. Richardson, Jessica S. Dymond, Zheng Kuang, Lisa
Z. Scheifele, Eric M. Cooper, Yizhi Cai, Karen Zeller, Neta Agmon, Jeffrey S. Han, Michalis Hadjithomas, Jennifer Tullman, Katrina Caravelli, Kimberly Cirelli, Zheyuan
Guo, Viktoriya London, Apurva Yeluru, Sindurathy Murugan, Karthikeyan Kandavelou, Nicolas Agier, Gilles Fischer, Kun Yang, J. Andrew Martin, Murat Bilgel, Pavlo
Bohutski, Kristin M. Boulier, Brian J. Capaldo, Joy Chang, Kristie Charoen, Woo Jin Choi, Peter Deng, James E. DiCarlo, Judy Doong, Jessilyn Dunn, Jason I. Feinberg, Christopher Fernandez, Charlotte E. Floria, David Gladowski, Pasha Hadidi, Isabel
Ishizuka, Javaneh Jabbari, Calvin Y. L. Lau, Pablo A. Lee, Sean Li, Denise Lin, Matthias E. Linder, Jonathan Ling, Jaime Liu, Jonathan Liu, Mariya London, Henry Ma, Jessica Mao, Jessica E. McDade, Alexandra McMillan, Aaron M. Moore, Won Chan Oh, Yu
Ouyang, Ruchi Patel, Marina Paul, Laura C. Paulsen, Judy Qiu, Alex Rhee, Matthew G. Rubashkin, Ina Y. Soh, Nathaniel E. Sotuyo, Venkatesh Srinivas, Allison Suarez, Andy
Wong, Remus Wong, Wei Rose Xie, Yijie Xu, Allen T. Yu, Romain Koszul, Joel S. Bader, Jef D. Boeke,* Srinivasan Chandrasegaran*
*Corresponding author. E-mail: jef.boeke@nyumc.org (J.D.B.); schandra@jhsph.edu (S.C.)
Published 27 March 2014 on Science Express DOI: 10.1126/science.1249252
This PDF file includes:
Materials and Methods Supplementary Text Figs. S1 to S19 Tables S1 to S9 References
Supporting Online Material for
Total Synthesis of a Functional Designer Eukaryotic Chromosome Narayana Annaluru†, Héloïse Muller†, Leslie A. Mitchell, Sivaprakash Ramalingam, Giovanni Stracquadanio, Sarah M. Richardson, Jessica S. Dymond, Zheng Kuang, Lisa Z. Scheifele, Eric M. Cooper, Yizhi Cai, Karen Zeller, Neta Agmon, Jeffrey S. Han, Michalis Hadjithomas, Jennifer Tullman, Katrina Caravelli‡, Kimberly Cirelli‡, Zheyuan Guo‡, Viktoriya London‡, Apurva Yeluru‡, Sindurathy Murugan, Karthikeyan Kandavelou, Nicolas Agier, Gilles Fischer, Kun Yang, J. Andrew Martin, Murat Bilgel‡, Pavlo Bohutski‡, Kristin M. Boulier‡, Brian J. Capaldo‡, Joy Chang‡, Kristie Charoen‡, Woo Jin Choi‡, Peter Deng‡, James E. DiCarlo‡, Judy Doong‡, Jessilyn Dunn‡, Jason I. Feinberg‡, Christopher Fernandez‡, Charlotte E. Floria‡, David Gladowski‡, Pasha Hadidi‡, Isabel Ishizuka‡, Javaneh Jabbari‡, Calvin Y.L. Lau‡, Pablo A. Lee‡, Sean Li‡, Denise Lin‡, Matthias E. Linder‡, Jonathan Ling‡, Jaime Liu‡, Jonathan Liu‡, Mariya London‡, Henry Ma‡, Jessica Mao‡, Jessica E. McDade‡, Alexandra McMillan‡, Aaron M. Moore‡, Won Chan Oh‡, Yu Ouyang‡, Ruchi Patel‡, Marina Paul‡, Laura C. Paulsen‡, Judy Qiu‡, Alex Rhee‡, Matthew G. Rubashkin‡, Ina Y. Soh‡, Nathaniel E. Sotuyo‡, Venkatesh Srinivas‡, Allison Suarez‡, Andy Wong‡, Remus Wong‡, Wei Rose Xie‡, Yijie Xu‡, Allen T. Yu‡, Romain Koszul, Joel S. Bader, Jef D. Boeke* and Srinivasan Chandrasegaran*
†These authors contributed equally to this work. ‡Build-A-Genome course students. *Corresponding authors (boekej01@nyumc.org; schandra@jhsph.edu) This PDF file includes: Materials and Methods References Figures S1 to S18 Tables S1 to S9
Supporting Online Material
Table of Contents
Section Page Supplementary Text 4
Figure S1. Map of synIII with common ORF names. 16 Figure S2. Map of synIII with systematic ORF names. 18 Figure S3. Hierarchy and nomenclature for synIII. 19 Figure S4. Schematic diagram of USER and Gibson isothermal assembly methods. 20 Figure S5. PCRTag analysis of synIII gDNA. 21 Figure S6. PCRTag analysis of wild-type gDNA (BY4742). 22 Figure S7. Incorporation of the synthetic version of YCP4 into synIII. 23 Figure S8. Karyotypic analysis of synIII and intermediate assembly strains by pulsed- field gel electrophoresis.
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Figure S9. Validation of the left and right arm telomere ends of synIII. 25 Figure S10. Growth curves for wild type (BY4742) and synIII strains in absence of estradiol induction.
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Figure S11. Colony sizes of wild type (BY4742) and synIII strains at different temperatures and on various media types.
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Figure S12. SynIII strain cell morphology. 29 Figure S13. Transcript profiling of wild-type (BY4742) and synIII strains. 30 Figure S14. ChIPseq analysis of synIII strain. 31 Figure S15. Replication origins of synIII. 33 Figure S16. Conditional genome instability (SCRaMbLE) of synIII strain. 35 Figure S17. SUP61 essential tRNA complementation by a synthetic tRNA gene. 36 Figure S18. SCRaMbLE leads to a gain of mating type a behavior in synIII heterozygous diploids.
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Table S1. SynIII minichunk plasmids. 38 Table S2. Summary of eleven iterative one-step assemblies and replacements of native chromosome III with synthetic fragments needed to construct synIII
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Table S3. Yeast strains used in this study. 44 Table S4. Sequence variants in the synIII chromosome. 46 Table S5. Wild type and synthetic PCRTags and the expected size of the amplicons. 47 Table S6. SynIII restriction sites that were removed. 60 Table S7. SynIII restriction site “landmarks” that were added. 63 Table S8. PCRTags for non-essential segments analysis. 65 Table S9. Replication origins in native chromosome III (from SGD) and synIII. 67 References 68
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Supplementary Text Expanded Materials and Methods SynIII Design Yeast chromosome III (12) that is well studied and characterized genetically (27-30), was redesigned extensively based on the version of III available as of [01/12/2006] on SGD; which is from S288C-derived strains (31). SynIII was produced in silico using the genome editing suite BioStudio (manuscript in preparation; chromosome version chr03.3_41). Systematic edits introduced globally to native III include TAG/TAA stop-codon replacements, PCRTag watermark sequences and insertion of loxPsym sites (Figures S1 and S2). Once completed genome-wide in the final Sc2.0 strain, elimination of all TAG stop codons by recoding to TAA will free the TAG codon for future genetic code expansion and could serve as a mechanism of reproductive isolation and control (32). Sc2.0 design principles prioritize a wild-type phenotype and a high level of fitness despite the incorporated modifications; synIII has a sequence alteration approximately every 500 bp, ~2.5% of total sequence is altered, and carries 98 loxPsym sites. The design principles used to guide introduction of changes were: (i) The change should confer a (near) wild-type phenotype and fitness; (ii) The synthetic chromosome should lack destabilizing elements such as tRNA genes or transposons; and (iii) The synthetic chromosome should incorporate genetic flexibility to facilitate future studies Designed synIII comprises 272,907 bp, a ~13.8 % overall reduction in size from native III. Ten transfer RNA genes, 21 Ty elements and/or derived long terminal repeats (LTRs), the silent mating loci HML (3,967 bp) and HMR (4,818 bp), and subtelomeric sequences lying to the left of YCL073C (6,099 bp) and the right of YCR098C (16,880 bp) were removed. The auxotrophic marker LEU2 (1,095 bp) was removed to serve as a selectable marker during assembly. The telomeres were specified by a designer “universal telomere cap” (UTC) comprising 305 bp of T(G)1-3 sequence. Systematic edits introduced globally to native III include TAG/TAA stop-codon replacements, PCRTag sequences and insertion of loxPsym sites (Figures S1 and S2). PCRTags are short pairs of recoded sequences unique to either the wild-type or synthetic genome, used to verify both introduction of synthetic sequence and removal of native sequence using PCR. We inserted loxPsym sites 3 bp after each nonessential gene stop codon and at major genomic landmarks, such as sites of LTR and tRNA deletion or flanking the centromere (Figures S1& S2 and Supplementary Text). Overall, the synIII designed sequence has a total of 5,410 bp base changes (PCRTags, RE sites, stop codon changes, etc) compared to the native yeast III and is shorter than the native sequence by 43,710 bp owing to the removal of 47,841 bp, inclusion of 99 loxPsym sites (3,366 bp) and added strategically positioned restriction enzyme (RE) sites. Native III encodes seven intron-containing protein-coding genes; introns from these genes were systematically removed in synIII. A single copy tRNA gene on III, SUP61, encoding tRNASer (CGA) is essential and therefore was encoded in trans on a centromeric plasmid allowing deletion of the gene (32). Version control Each editing step was assigned a new version number to enable rollbacks in case of errors made during the design process. A systematic nomenclatural hierarchy was developed to name all DNA elements used in the project (see Hierarchy and nomenclature). The versions consisted of
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three “global edits” named chr3.0_00 (wild-type) through chr3.3_00, which are as follows: global edit 1: insert loxPsym sites 3 bp downstream of the stop codon of all nonessential genes; global edit 2: exchange TAG stop codons for TAA (Stop codon swapping); global edit 3: insert PCRTags. “Global” genome editing was performed using custom scripts that eventually were incorporated into the genome editing application “BioStudio” (manuscript in preparation). Subsequently, “local editing” versions were generated (i.e. modifications made to chr 3 only) starting with chr3.3_01 and ending with chr3.3_41. [Dymond et al. 2010, (2) for the underlying design rules of the global edits]. Version chr3.3_42 represents the actual physical version of the sequence. After these global edit steps the left and right arms of native chr III were subdivided into contiguous sequence blocks of approximately 10 kb, and each of these 10 kb “chunks” was bounded by rare-cutting restriction enzyme sites (Fig. S1 and S2). Recoding to create or eliminate restriction sites (Table S6, S7) was only permitted within coding regions and was performed using GeneDesign (13). Subsequent rounds of local editing were performed to precisely delete introns, delete silent cassette information, delete tRNA genes and transposon sequences (regions lying between tRNAs and transposons or between clusters of transposon sequences were also deleted), removed LEU2 sequence and adjacent sequences removed in the construction of designer deletion leu20 (33), flanked the CEN3 sequence with loxPsym sites, and deleted repetitive subtelomeric sequences. With the exception of intron deletions, deletions were accompanied by insertion of a loxPsym site. All genes distal to GIT1 on the right arm were deleted, as very similar sequences to these are found in other telomeres. Moreover all these genes are absent from ring chromosome III, which reportedly has no growth defect (34). Special design features used in synIII Several design alterations were required to synthesize synIII. Unlike the previously designed synIXR, synthesized as a circular chromosome (2), synIII was designed to be linear and thus requires telomeres. A UTC was designed that could define each chromosome end, with T(G)1-3 exposed to serve as a telomere seed sequence. The UTCs used at each end of synIII are identical but in opposite orientations and specify precisely structured telomeres. Secondly, unlike chromosome IXR, native III encodes seven intron-containing protein-coding genes. These seven introns were systematically removed following the rules of RNA splicing, in the hope that it will be possible to eventually assemble a version of Sc2.0 lacking introns altogether. Finally, a single copy tRNA gene on III, SUP61, encoding tRNASer (CGA) is known to be essential based on genetic studies, as it alone decodes UCG codons (32). Consistent with prior work, deletion of this tRNA from the genome was impossible; we initially encoded it in trans on a centromeric plasmid. The synthetic SUP61S tRNA gene design consisted of the S. cerevisiae tRNA coding region (less its 18-bp intron) flanked by sequences from the corresponding tRNA locus in Eremothecium (Ashbya) gossypii. Universal Telomere Cap (UTC) Design A 305 bp UTC1 sequence conforming to the consensus (C1-3A)n, was designed by randomly concatenating sequences conforming to the consensus in silico. The UTC1 sequence was flanked by a NotI site on the centromere proximal end and a BsaI site on the centromere distal end. Cleavage with BsaI releases the telomere end with a 4 bp 5’ overhang within the (C1-3A)n
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sequence, providing a telomere seed sequence that can be extended by telomerase. This insert was synthesized and cloned into plasmid pUC19 to form pUTC1. Synthetic SUP61S Design The essential SUP61 gene was converted to a synthetic version SUP61S by removing the intron, and then, to avoid introducing S. cerevisiae repetitive sequences (such as LTR and Ty sequences), substituting sequences (500 bp 5´ and 20 bp 3´) flanking the corresponding isoacceptor tRNA from Eremothecium gossypii, a related species that retains a high degree of synteny with S. cerevisiae. The E. gossypii sequence was chosen in part because it lacks recognizable transposable element derived sequences, unlike native S. cerevisiae tRNA flanking regions. Furthermore, although the E. gossypii primary DNA sequence is distinct, there exists recognizable synteny with S. cerevisiae chromosomes and promoter and terminator sequences have been shown to function well in S. cerevisiae (35, 36). This synthetic SUP61S plasmid, tagged with the HIS3 selectable marker, was introduced prior to the step in which the native tRNA would have been deleted by incorporation of the relevant synIII segment (Table S1). As expected, although the CEN plasmid could readily be lost by nonselective growth prior to swapping in the SUP61-less fragment, once that portion of synIII had been introduced, the SUP61S plasmid became essential. Hierarchy and Nomenclature Hierarchy and an example A diagram of the hierarchy from oligonucleotide to intact chromosome is shown in Fig. S3. A Sample name for a specific BB, 3L.3_23.B2.07 and an oligonucleotide (o) within it are shown. The chromosome arm is IIIL – hence 3L. Chromosome versions The version in the example is 3_23 (global version 3, chromosome-specific version 23). Version 3_41 is the final version merging two separate designs originally made for the left and right arms separately. Thus it represents the “designed” version. Note that version 3_42 represents the experimentally determined sequence from strain HMSY011. Chunks and BBs The chunk number is B2. B is the ~30 kb “megachunk” and 2 is the second of three ~10 kb chunks in B. The Building Block (BB) is 07. There are typically 10-18 BBs per ~10 kb chunk. The oligo number is 07. This is the 7th oligo in BB11. A typical BB has 18 oligos. The small letter “o” indicates oligonucleotide. Nomenclature for BB clones 3L.3_23.B2.07.c05: This is the 5th clone (“c”) picked for BB07 in chunk B2 on the left arm of chromosome III. Nomenclature for systematic Sanger sequencing reads 3L.3_23.B2.07.c05.f1 for the forward read 3L.3_23.B2.07.c05.r1 for the reverse read Subsequent re-sequencing reads are labeled f2, etc.
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SynIII synthesis and assembly The designed synIII chromosome sequence was divided into 367 BBs with an average size of 0.75 kb, of which 133 BBs corresponded to synIIIL, including a single BB [3L.3_23. D3.10] spanning CEN3; 234 BBs encoded synIIIR. At the next higher level of organization, the synIII chromosome was divided into 127 minichunks, ranging from ~2-4 kb in size, of which 43 minichunks corresponded to synIIIL, 83 minichunks corresponded to synIIIR, and one minichunk, encoded by pNA044, spanned the centromere CEN3 (Table S1). Each minichunk consisted of 3 to 5 BBs with a single BB overlap specified for adjacent minichunks. The wet-bench workflow consisted of three major stages: 1) The 750 bp building blocks (BBs) were produced starting from overlapping 60- to 79-mer oligonucleotides designed to have compatible melting temperatures using GeneDesign (13) and assembled using standard polymerase chain assembly methods (14). All BBs were verified by sequencing (17). This critical step produced most of the starting material for the project and was carried out by undergraduate students in the Build-A-Genome class at Johns Hopkins University (Fig. 2A). 2) The 133 synIIIL (+ CEN) BBs were assembled into 44 overlapping minichunks of ~2-4 kb using one of two methods: (i) Uracil Specific Excision Reaction (USER) (16) or (ii) the “Gibson” isothermal assembly reaction (19) with a 40 bp overlap between adjoining BBs. The 234 synIIIR BBs were assembled into 83 overlapping DNA minichunks of ~2-4 kb in size using either the isothermal assembly reaction or by direct homologous recombination in yeast (Fig. 2B) (18, 19). The minichunks were also verified by sequencing. 3) All adjacent minichunks for synIII were designed to overlap one another by one BB, thus enabling further assembly in vivo by homologous recombination in yeast (20, 21). Using an average of 12 minichunks and alternating selectable markers in each experiment, the native sequence of S. cerevisiae III was systematically replaced by its synIII counterpart in eleven successive rounds of transformation (Table S2). The iterative replacement of native genomic segments of III with pools of overlapping synthetic DNA minichunks encoding alternating genetic markers enabled the total synthesis of the first functional designer eukaryotic chromosome, synIII, in vivo in yeast (Fig. 2C). Synthesizing Building Blocks (BBs) from Oligonucleotides The first step of the synthesis process, referred to as “templateless” PCR (T-PCR), involves assembling ~18 overlapping oligonucleotides that comprise the ~750 bp building block (BB), a method also known as polymerase chain assembly. Oligonucleotides in these reactions serve as both template and primers; the product appears as a smear when examined by agarose gel electrophoresis. The desired full-length ~750 bp BB is obtained by the “finish” PCR (F-PCR), using a dilution of the T-PCR product as template and the two outermost 5’ and 3’ primers (15, 39). Problematic BBs were assembled using a “touchdown” PCR protocol in the F-PCR step, in which successively lower annealing temperatures are used during each cycle in the early phase of the PCR. Lowering the extension temperature of the reaction from 72°C to 68°C helps to amplify through A-T rich templates that are less stable at the higher temperature. Each full-length ~750 bp BB was ligated to the pGEM-T vector (Promega), which allows for blue-white screening when transformed to E. coli. Twelve to twenty-four white colonies per BB were analyzed for insert size by colony PCRs, using the T7 and SP6 priming sites available within the pGEM-T vector, and 18 full-length inserts were sequenced, which yielded at least one with the desired sequence. 338 BBs were produced by undergraduate students in the Build-A-Genome class, while the remaining 29 BBs were purchased from commercial vendors (GeneArt,
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Regensburg, Germany; Celtek Genes, Nashville TN; and Epoch Life Science, Missouri City, TX). Sequence Verification of BBs by CloneQC CloneQC software that performs quality control on the sequenced clones is available as a free webserver (http://cloneqc.org) and as BSD-licensed source code. This software allows analysis of user-specified DNA reference sequences. CloneQC was used internally as an integral component of the Build-A-Genome course to analyze the sequences of BBs (38). Assembly of 2-4 kb Minichunks Three different assembly methods were used to make the 127 minichunks needed for the construction of the synIII chromosome: 1) USER assembly (31 minichunks); 2) Isothermal assembly (57 minichunks); and 3) Yeast assembly (39 minichunks). A brief description of each method is given below. USER Assembly USER method was used to assemble 31 synIIIL minichunks both in vitro (using Phusion polymerase/Taq ligase (New England Biolabs, Beverley, MA)) and in vivo (using E. coli) (Fig. S4). A detailed protocol for USER fusion assembly reaction is described elsewhere (16). Gibson Isothermal Assembly The isothermal assembly technique with 40 bp overlap between adjoining BBs was used to generate several synIIIR 2-4 kb minichunks using a blend of three enzymes (T5 exonuclease (Epicentre, Madison, WI), Phusion DNA polymerase (New England Biolabs, Beverley, MA) and Taq DNA ligase (New England Biolabs) in a single step assembly reaction (Fig. S4) (17). The enzyme reagent mix (readily stored at -20°C) was combined with overlapping BBs and vector; the reaction was incubated at 50°C for 15 min. A detailed protocol for the “Gibson” isothermal assembly reaction is described in detail elsewhere (40). Yeast Assembly Alternatively, we used a yeast-based homologous recombination based DNA assembly (Fig. 2B). It can efficiently assemble 3 to 5 BBs and a shuttle vector (pJHU2) with 40 bp terminal overlaps in a single transformation event. This approach is fully compatible with fragments that have been made for Gibson assembly. A detailed protocol for the yeast assembly reaction is described in detail elsewhere (21, 41). Plasmid Isolation from Yeast Cells were harvested from 3 ml overnight culture in YPD and resuspended in 250 µl of buffer P1 (QIAprep miniprep kit). After addition of 100 µl of glass beads, cells were broken by vortexing for 5 minutes. Following this treatment, plasmids were isolated using the standard alkaline lysis and a Qiagen miniprep spin column to isolate the DNA. Aliquots of the shuttle plasmid were used to transform E. coli. See Muller et al. (2012) for full description of this protocol (21). Direct Replacement of Native Chromosome III with Synthetic Minichunks in Yeast All consecutive minichunks were designed to have overlap by one BB to enable further assembly and replacement of native S. cerevisiae DNA in vivo by homologous recombination. This
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iterative replacement strategy is likely to be limited to organisms with an efficient HR repair mechanism. Equimolar amounts (~ 70 fmol each) of four to twenty-seven overlapping minichunks were co-transformed into yeast, along with a linker composed of an auxotrophic marker (URA3 or LEU2) fused to the “right end” terminal minichunk (Fig. 2), as well as a sequence of about 1000 bp of the native chromosome sequences adjacent to the right end-most minichunk introduced (Fig. 2), using a modified version of the yeast LiOAc transformation protocol. The protocol was modified as follows to improve transformation efficiency: Following heat shock, cells were centrifuged at 4000 rpm for 5 min, washed in 5 mM CaCl2 and incubated at RT for 10 min. Cells were centrifuged at 4000 rpm for 5 min, then resuspended in water and plated on the appropriate selective medium. The penultimate transformation involved selection on SC–URA medium, and 5-FOA resistance selection was used in the final step yielding a final synthetic chromosome lacking a selectable marker. Complete set of PCRTags analyses of synIII and wild type strains. Upon completion of synIII, as well as at each of the intermediate construction steps, PCRTag analysis (Table S5) revealed the presence of synIII synthetic PCRTags and absence of native PCRTags, excepting a single PCRTag, YCR004c.1 (Fig. S5). As expected, PCRTag analysis of the wild type (BY4742), showed the presence of only wild type and no synthetic PCRTags (Fig. S6). We were able to subsequently incorporate the synthetic version of YCP4 (YCR004c) into synIII by first deleting the native gene from the otherwise synthetic chromosome and then incorporating the synthetic version of YCR004c (Fig. S7). Yeast Colony PCR for Rapid Screening of WT and SYN PCRTags Yeast cells from single colonies were resuspended in 5 µL of 20 mM NaOH and heated at 95°C for 5 min in a thermocycler prior to adding regular PCR mix and performing PCR cycles. A detailed protocol for yeast colony PCR for PCRTag screening can be found elsewhere (21). Yeast Genomic DNA Preparation for PCRTags Analysis Yeast cells were isolated from 100 μL of saturated overnight culture by spinning at 2000 rpm for 2 min in a centrifuge at RT. The yeast pellet was resuspended in 200 μL breaking buffer (50 mM Tris pH8, 100 mM NaCl, 1% SDS, 2% TX100, and 1 mM EDTA). (Alternatively, resuspended cells were scooped with a sterile stick directly from a plate, in a 1.5 mL microfuge tube containing 200 μL breaking buffer). An equal volume of glass beads (Sigma G8772) and 200 μL of Phenol-Chloroform-Isoamyl alcohol (PCI) (25:24:1) were added and vortexed for ~10 min at RT and then centrifuged at 15000 rpm for 10 min. 75 μL of the top, aqueous layer, was transferred to a labeled microfuge tube containing 1 mL 100% ethanol and then inverted 5 times, and centrifuged for 20 min at 4°C. The supernatant was discarded and the DNA pellet washed by adding 500 μL of 70% ethanol, centrifuged at RT for 5 min. The genomic DNA was air-dried at RT and then resuspended in 50 μL 10 mM Tris (pH 7.4) buffer, and stored at −20°C. For each 5 μL PCR reaction, 0.1 μL of genomic DNA was used as template. PCRTags Analysis Reaction Conditions Amplification of PCRTags was performed using GoTaq Hot Start Polymerase (Promega, Madison, WI), 400 nM each of forward and reverse primers, and either BY4741 or synIII genomic DNA in a 5 µL final reaction volume. Touchdown PCR was performed as follows: 95°C/3 min, 20 cycles of (95°C /30 sec, 64°C /20 sec/-0.3°C /cycle, 72°C /30 sec), followed by
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15 cycles of (95°C /30 sec, 58°C /20 sec, 72°C /30 sec) and a final extension of 72°C /5 min. Detection of PCRTags was performed by diluting samples to 25 µL in H2O and using a Caliper LabChip GXII (Perkin Elmer, Waltham, MA) and the HT DNA 5K LabChip Kit, Version 2. Virtual gel images were generated using LabChip GX software version 4.0.1418.0. Yeast Genomic DNA Preparation for DNA Sequencing Yeast cells were isolated from 2 mL overnight culture by spinning at 3000 rpm in a centrifuge. The yeast pellet was resuspended in 200 μL breaking buffer. An equal volume of glass beads (Sigma G8772) and 200 μL of PCI were added and then vortexed. 200 μL of TE pH 8 (10 mM Tris pH 8, 1 mM EDTA pH8), mixed thoroughly and then centrifuged at 15000 rpm at 4oC for 10 min. The aqueous layer was transferred to another microfuge tube and 4 μL of RNAse A (10 mg/mL) was added and incubated at 37˚C for 1 h. 400 μL PCI was added to the reaction mixture, vortexed for 1 min and then centrifuged at 15000 rpm at 4˚C for 10 min. The aqueous layer was transferred to another tube. The genomic DNA was precipitated by adding ~35 μL 3M NH4OAc and 1 mL ethanol (−20oC) and kept at −80˚C for 1 h. The DNA was pelleted by spinning at 15000 rpm at RT for 20 min. The precipitate was then washed with 500 μL 70% ethanol and dried at 50˚C for 5 min. The genomic DNA was then resuspended in 25 μL TE pH 8. RNA Isolation from Yeast for RNA Sequencing A single yeast colony was grown in a 10 mL culture in YPD until the OD600 was 0.8 to 1.2. The cells were pelleted by spinning at 2000 rpm at RT for 5 min. The pellet was resuspended in 0.5 mL of RNAse-free water (DEPC water) and transferred to a microfuge tube. The cells were pelleted again by spinning briefly and discarding the supernatant. The cells were resuspended in 300 µL RNA lysis buffer (0.5 M NaCl, 0.2 M Tris-HCl [pH 7.5], 10 mM EDTA). 200 µL glass beads and 300 µL PCI was then added to the cells and vortexed twice for 1 min. The cells were kept on ice for 1 min between vortexes. Centrifuge at 15000 rpm at RT for 1 min. The aqueous layer (~300 µL) was then transferred into another microfuge tube; an equal amount of PCI was added, mixed well and centrifuged at 15000 rpm at RT for 4 min. The aqueous layer was then extracted with an equal volume of chloroform. The RNA was precipitated by adding 600 µL of 100% ethanol and incubating the tube at −80˚C for 1 h or overnight. The RNA was pelleted by centrifuging at 15000 rpm at 4˚C for 15 min. The RNA pellet was then washed with 500 µL of 70% ethanol, air-dried and then resuspended in 20 µL DEPC water. Nucleotide Sequence Analysis of synIII Single-end whole genome sequencing of strain HMSY011 was performed using an Illumina HiSeq using TruSeq preparations kits; 195,598,768 raw reads were obtained and used for downstream analysis. A set of high-quality reads was created from the raw data by removing adapters using TRIMMOMATIC and then quality filtering; reads with average quality less than Phred score 15, containing “N”s were filtered out, reducing the initial set of reads by ~1%. Filtered reads were mapped using BOWTIE2 with default sensitivity settings; a reference genome was constructed starting with the sequence for strain BY4742, in which chromosome III had been replaced with the synthetic version. 97.83% of reads were mapable; 81.28% map uniquely to the genome whereas 16.55% mapped to > 1 region. The average fold coverage on chromosome III was 554X with a standard deviation of 55.3 (window size: 500 bp), a minimum
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of 372X and a maximum of 816X, characterized by a skewness of -0.348 and kurtosis of 0.888, resulting in a good fit with the expected normal coverage distribution. Base changes and short indels relative to the designed reference synIII chromosome sequence where detected using UNIFIEDGENOTYPER of the Genome Analysis Toolkit (GATK) using standard parameters; large deletions were detected by checking depth of coverage at each location. The final sequence has three synonymous changes, three frameshifts, two non-synonymous coding changes and one change not in a coding region, 277 bp from the YCR042C gene (Table S4); furthermore, a loxPsym site was lost at position 59583. RNA-Seq Analysis of synIII Single-end non-strand-specific RNA-seq of the synIII stain (HMSY011) and BY4742 were performed using Illumina MiSeq and standard TruSeq preparations kits. Total counts of 100 bp single-end reads were 84,859,272 for the wild type strain and 105,038,509 for the synIII strain. Adapters and low quality were trimmed using TRIMMOMATIC (42), reducing the read count by about 2.5%. Reads were mapped using TOPHAT (43) to the reference S. cerevisiae BY4742 genome. For each gene, read counts were computed using HTSEQ (44) and analyzed for differential expression using DESEQ (45), with standard parameters and following the no replicates scenario. For each gene, we obtained a raw p-value and adjusted p-value using the standard Benjamini–Hochberg procedure. Genes were assessed for statistical significance by rejecting the null hypothesis if the adjusted p-value was <0.01 and if the raw p-value fell below the threshold of the 5% Family Wise Error Rate (FWER) after Bonferroni correction (threshold=7.02eX10-06). We obtained two genes with a significant change in expression: HSP30 on the synIII chromosome (p-value=1.76X10-06, p-adjusted=0.00132, log2 fold change=-4.25) and PCL1 on chromosome XIV (p-value=4.63X10-06, p-adjusted=0.00313, log2 fold change=4.05). SCRaMbLE Yeast cells were transformed with the URA3-tagged plasmid pLM158 expressing the Cre-EBD fusion protein under the control of the SCW11 promoter (2, 46) into wild-type and synIII strains, and plated on SC–Ura to select for the plasmid. Isolated colonies were inoculated into 5 mL SC–Ura for overnight culture at 30°C. Cultures were diluted to an OD600 of 0.1 in 200 mL SC–Ura plus or minus estradiol to a final concentration of 1 µM (Sigma E2257). As ethanol is used as a solvent for the stock solution of estradiol (10 mM), the same volume of ethanol 100% (20 μL) was added to the control cultures minus estradiol. Samples were taken before induction (T0) and at various time points after induction, adjusted to an OD600 of 0.1, and then 5 µL of 10-fold serially diluted cultures were spotted onto SC–Ura or YPD plates. OD600 of cultures was recorded over 48h of induction. Cell Morphology Cells were grown to mid-log phase in synthetic complete medium with 2% dextrose at 30°C. DIC Images were collected using a Zeiss Microscope Axioskop 2 mot Plus (63X) with a Zeiss AxioCam HRc Camera.
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Pulsed Field Gels Chromosome-sized DNAs were prepared as described elsewhere (22) . Identity of the chromosomes was inferred from the known molecular karyotype of WT (BY4742) that was run on the same gel. Samples were run on a 1.0% agarose gel in 0.5x TBE (pH 8.0) for 24 h at 14°C on a CHEF apparatus. The voltage was 6 V/cm, at an angle of 120° and 60-120 s switch time ramped over 24 h. Southern Blot Probe Preparation Probes were prepared using the Prime-It II kit (Stratagene), and hybridized using Ultrahyb hybridization solution (Ambion) according to manufacturer’s instructions. SUP61S Integration SUP61S essential tRNA complementation was done in two steps: First, synIII/III heterozygous diploid strains (yLM108 to yLM117) with SUP61S in HIS3 vector pRS413 were constructed. Second, the colonies were screened for the loss of HIS3-plasmid (yLM098 to yLM107), and transformed with SUP61S flanked by 500-bp homologous HO genomic sequences to integrate the donor at the HO locus to generate NAY002 to NAY011 strains (Fig. S17). Synthetic YCP4S Integration into synIII strain PCRTags analysis revealed that the synthetic version of YCP4 (YCP4S) was not originally assimilated into the synIII strain HMSY011, which was confirmed by sequencing of synIII. However, we were able to subsequently incorporate YCP4S by first deleting native YCP4 and then incorporating YCP4S. This was done in two steps: First, we deleted YCP4 by integrating URA3 at the native YCP4 locus; Second, we removed URA3 by incorporating YCP4S at this locus (Strain - NAY001), using 5-FOA medium to screen for the loss of URA3 (Fig. S7). Serial dilution assay on various types of media Wild type (BY4742), synIIIL (yLM043), and synIII (yLM197) strains were grown overnight in rich medium (YPD) at 30°C with rotation. Cultures were serially diluted in 10-fold increments in water and plated onto each type of medium. All drugs (methyl methanosulfate (MMS; defective DNA repair) (Sigma, 129925), benomyl (Aldrich, 381586; microtubule inhibitor), camptothecin (Sigma, C9911; topoisomerase inhibitor), hydroxyurea (HU; defective DNA replication) (Sigma, H8627)) were mixed into YPD, except 6-azauracil (6-AU; Sigma, A1757; defective transcription elongation), which was mixed into synthetic complete (SC) medium containing 2% dextrose. YPGE (respiratory defects) was prepared with 2% glycercol and 2% ethanol. High and low pH plates (9.0 and 4.0; vacuole formation defects) were prepared using NaOH and HCl in YPD, respectively. Sorbitol plates (osmotic stress) were prepared by adding the appropriate quantity of sorbitol (Sigma, S1876) to YPD. For hydrogen peroxide (Fluka, 88597; oxidative stress) and cycloheximide (Sigma C7698; defective protein synthesis), overnight cultures were treated for 2 hours in drug, harvested by centrifugation and resuspended in water prior to plating the serial dilutions on YPD. Plates were incubated at 30°C, unless otherwise indicated in Fig. S11B, for 2 days (YPD, camptothecin, 6-azauracil, benomyl, pH4.0, pH9.0, cycloheximide, hydrogen peroxide) or 3 days (sorbitol, YPGE, HU, MMS). Subculture
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Six single synIII strain (yLM197) colonies were inoculated in 5 mL of YPD. After 24 hours, 5 µL of overnight culture was transferred to 5 mL of fresh YPD. This experiment was continued for 10 days to get 120 generations. Then cells were plated on YPD plates to get single colonies. Five single colonies were picked from each of six “mother clones” to inoculate 3 mL culture of YPD and next day gDNA prep was carried for all the 30 clones. PCRTag analysis (Table S8) was carried out to monitor the presence of 58 presumed non-essential segments (i.e. segments lacking known essential genes) flanked by loxPsym sites. All the 58 PCRTags amplified in each strain, indicating the presence of these segments. A-Like-Faker Assay Mating between MATα strains will occur when the MATα locus from either parental genotype is lost. Possible events include loss of the chromosome III, deletion, translocation or removal of the MATα locus. One mL of overnight culture of synIII strain and Tester α strain were mixed and kept at 30˚C for 4 hours (for mating). The cells were then washed twice with water by spinning at 2500 rpm for 2 minutes. The cells were resuspended in 500 µl of water and plated on SD plates. Overnight cultures were also plated on YPD plates to estimate the CFU. This experiment was carried out for ten independent synIII and BY4742 strains and the average loss rate of MATa calculated using the method of the median (47). The yeast strain, yLM197, was used for this experiment. SCRaMbLE Mating Type Assay Ten independent heterozygous diploid strians (NAY002 to NAY11) were transformed with HIS3 plasmid pLM006 expressing the Cre-EBD fusion protein under the control of the SCW11 promoter. We then inoculated isolated colonies into 5 mL YPD for overnight culture at 30°C. We then re-inoculated the overnight cultures into 5 mL YPD (1:10 ratio) medium with 1 µM estradiol (Sigma E2257). After 30 min incubation at 30°C, cells were plated in the absence of estradiol at varying number of cells (~20, ~200, and ~2000 cells, respectively) on solid YPD medium and the colonies were grown for two days at 30˚C. Mating assays were performed using mating type tester strain lawns with overnight mating at 30˚C and subsequently replica-plating onto SD medium. Loss of genomic segments containing the MATα region of synIII (i.e. deletion of synIII chromosome segments between two loxPsym sites that result in the loss of MATα) in these cells should lead to the development of a-type mating cells, detected by their ability to mate with a MATα tester strain lawn. Fourteen such clones were analyzed by 24 SYN PCRTags, each pair covering ~10 kb of the synIII chromosome, for the absence of SYN PCR products in the PCRTag reactions. These clones were similarly analyzed using all the MATα region-specific SYN PCRTags for the loss of MATα region on the synIII chromosome. Effect of Cre-EBD induction on synIII strain Subsequent to completion of synIII, we transformed a Cre-EBD plasmid into the haploid synIII strain to enable SCRaMbLEing of the chromosome (2, 3), which resulted in genome rearrangements of the synIII strain. Cre-EBD induction appears to greatly reduce the fitness of the synIII strain, but not the wild type (Fig. S16A). We also monitored growth curves of wild type and synIII strains throughout Cre-EBD induction time course, and as expected, synIII shows much decreased fitness as compared to the wild type under these SCRaMbLE-inducing conditions (Fig. 16B).
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ChIPseq Analysis BY4742 and synIII cells were grown to an A600 of 0.5, and nocodazole was added to a concentration of 15 µg/mL. After arrest for 2 h at 30˚C, ~50 OD cells were fixed in 1% formaldehyde at 25˚C for 2 h and quenched in 125 mM glycine at 25˚C for 10 min. Cells were pelleted and washed twice with 1X TBS buffer before freezing. The frozen pellet was resuspended in 0.45 ml ChIP lysis buffer (50 mM HEPES•KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µM leupeptin, 5 µM pepstatin A, and Roche protease inhibitor cocktail) and lysed by bead beating. Lysate from 50 OD of cells was split into two tubes each containing 280 µL lysate and sonicated for 20 cycles (30 sec on, 30 sec off, high output) using a Bioruptor (Diagenode, Denville, NJ). The supernatant of the sonicated lysate was pre-cleared. 50 µL lysate was saved as input. 1.5 µg Mcd1 antibody (the kind gift of Vincent Guacci and Doug Koshland) was used. After incubation overnight, 50 µl protein A Sepharose beads (GE Healthcare Life Sciences, Piscataway, NJ, 17-5280-01) resuspended in ChIP lysis buffer was added and incubated for 1.5 h at 4˚C. Beads were washed twice with ChIP lysis buffer, washed twice with ChIP lysis buffer containing 500 mM NaCl, twice with DOC buffer (10 mM Tris•Cl pH 8.0, 0.25 M LiCl, 0.5% deoxycholate, 0.5% NP-40, 1 mM EDTA) and twice with TE. 125 µL of TES buffer (TE, pH 8.0 with 1% SDS, 150 mM NaCl, and 5 mM dithiothreitol) was added to resuspend the beads. The supernatant was collected after incubation at 65°C for 10 min. A second round of elution was performed and the eluates were combined. Reverse crosslinking was performed by incubation for 6 h at 65°C. An equal volume of TE containing 1.25 mg/mL proteinase K and 0.4 mg/mL glycogen was added to the samples after reverseing the crosslinks and the samples were incubated for 2 h at 37°C. Samples were extracted twice with an equal volume of phenol and once with 25:1 chloroform:isoamyl alcohol. DNA was precipitated in 0.1 volume 3.0 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-cold ethanol at -20˚C overnight. Pellets were washed once with cold 70% ethanol and resuspended in 20 µL TE. Library construction and sequencing were performed following the Illumina protocol; DNA ends were repaired and A-tailed. Barcoded adaptors were ligated and the DNA was run on a 2% agarose gel. 150 - 300 bp DNA fragments were excised from the gel and used for PCR. PCR products were gel-extracted again and quantified on an Agilent Bioanalyzer. Sequencing was performed on an Illumina Miseq. Raw reads were mapped using BOWTIE (-5 3 -3 10 --best) to the reference genome (sacCer2) or the synIII genome (identical to sacCer2 except replacing chr03 with synIII sequence) and the peaks were visualized using the CISGENOME Browser. Determination of replication origins of synIII The protocol for mapping replication origins was adapted from Agier et al. (36) as follows. Strain yLM197 was inoculated in 500 mL YPD at OD600 = 0.15 and grown O/N for approx. 18 h. 8X1010 cells were harvested by centrifugation 5 min at 4000 rpm, washed in 100 mL 1xPBS buffer and resuspended in 50 mL l PBS buffer to enter a Beckman elutriation system (Avanti J26 XP centrifuge combined with a JE-5.0 elutriator rotor and a 40 mL elutriation chamber). From the original asynchronous culture, G1 cells were sorted by elutriation, and 109 G1 cells were resuspended in 500 ml of fresh YPD pre-warmed to 25˚C before beginning the time course experiment. Subsequent culture growth was carried out in a water bath at 25˚C with constant shaking. Aliquots of cells were harvested at T0 and then every 10 minutes after a lag phase of 2 h and 30 min to allow entry into S-phase. At every time point, 1 mL cells were fixed with 2.3 mL EtOH (100%) to monitor S-phase progression using flow cytometry, and 30 mL cells were fixed
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in 0.1% Na azide (final concentration) on ice, to be used for the replication origin determination. Cell morphology was also examined under the microscope, to check for bud formation. At the end of the time course, cells fixed in Na azide were washed twice with 20 mL of cold water (4˚C), and pellets were frozen at −80˚C until used for DNA extraction. After 12 h in EtOH at 4˚C, cell aliquots for FACS analysis were labeled with SYTOX® Green (Life Technologies) dye and analyzed with a MACSQuant® Analyzer. DNA extraction of the frozen pellets was performed using the Qiagen kit Genomic-tip 20/G, following manufacturer’s instructions. Each sample was then processed for Illumina sequencing using standard protocols and custom adapters, allowing multiplexing of the time points chosen for the experiment. All multiplexed time points were sequenced on a HiSEQ2000, with a single-end 50 base reads. For each time point, reads were mapped to the reference genome (BY4742 genome sequence with synIII instead of WT chromosome III) to determine replication origins, using the procedure described in Agier et al. (48).
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Figure S1: Map of synIII with common ORF names. Open reading frames (ORFs) - red, essential; dark blue, non-essential; purple, null mutation confers a slow growth phenotype; light blue, uncharacterized; white, dubious/pseudogene. Autonomously replicating sequences (ARSs), pale yellow. Loci marked with an “X” are present in the WT chromosome and are deleted in synIII. Green diamonds represent loxPsym sites embedded in the 3’ UTR of non-essential genes and at several other landmarks. Gray bars in ORFs symbolize PCRTag pairs. Fuchsia circles indicate synthetic stop codons (TAG recoded to TAA). Rare-cutting restriction enzyme sites bordering ~10 kb chunks are also shown.
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Figure S2: Map of synIII with systematic ORF names. Open reading frames (ORFs) - red, essential; dark blue, non-essential; purple, null mutation confers a slow growth phenotype; light blue, uncharacterized; white, dubious/pseudogene. Autonomously replicating sequences (ARSs) pale yellow. Loci marked with an “X” are present in the WT chromosome and are deleted in synIII. Green diamonds represent loxPsym sites embedded in the 3’ UTR of non-essential genes and at several other landmarks. Gray bars in ORFs symbolize PCRTag pairs. Fuchsia circles indicate synthetic stop codons (TAG recoded to TAA). Rare-cutting restriction enzyme sites bordering ~10 kb chunks are also shown.
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Figure S3: Hierarchy and nomenclature for synIII. A diagram of the hierarchy from oligonucleotide to intact chromosome is shown. See Supplementary Text for more details.
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Figure S4: Schematic diagram of USER and Gibson isothermal assembly methods.
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Figure S5: SynIII PCRTag analysis. The presence of synIII and absence of native chromosome III was verified by amplification of synthetic PCRTags (SYN) compared to wild-type PCRTags (WT). PCRTag analysis of synIII strain revealed the presence of SYN PCRTags and absence of WT PCRTags, with the exception of one single PCRTag, YCR004c.1 The synthetic version of YCR004c.1 (YCP4S) was subsequently incorporated by first deleting the native gene (YCP4) from the otherwise synthetic chromosome and then incorporating YCP4S (Fig. S6. Two SYN PCRTags (YCR067C.1 and YCR073W-A.1) did not yield amplicons under the PCR conditions used. Nucleotide sequence determination of synIII confirmed the presence of these two SYN PCRTags.
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Figure S6: PCRTag analysis of wild-type gDNA (BY4742). PCRTags that are short pairs of recoded sequences, unique to either the wild-type or synthetic genome, were used to rapidly verify the introduction of designer synIII synthetic sequence and the removal of native wild-type yeast chromosome III sequence by PCR. PCRTag analysis of wild-type yeast (BY4742) gDNA showed the presence of only WT PCRTags, demonstrating the specificity of all PCRTag primers (Fig. S4). Two WT PCRTags, namely YCL061C.2 and YCL045C.2, did not yield any products under the PCR conditions that we employed for the PCRTag analysis. SYN, synthetic PCRTags; WT, wild-type PCRTags.
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Figure S7: Incorporation of the synthetic version of YCP4 into synIII. PCRTags analysis revealed that YCP4 was not assimilated into the synIII strain, which was further confirmed by nucleotide sequence determination of synIII. However, we were able to subsequently incorporate the synthetic version of YCP4 (YCP4S) by first deleting the native gene and then incorporating the synthetic version. This was done in two steps: In the first step, we deleted the wild-type YCP4 by integrating URA3 at the YCP4 locus. In the second step, we removed URA3 by incorporating synthetic YCP4 S at this locus. Agarose gel profile of the PCRTag analysis around the region of YCP4 locus for synIII, YCP4∆::URA3 and YCP4S strains are shown: Lanes: 1, PCRTags for YCR003W.1; 2, YCR004C.1 (YCP4); and 3, YCR005C.1. SynIII strain shows the presence of the synthetic PCRTags for YCR003W.1 and YCR005C.1 loci and wild-type PCRTag for YCR004C.1 (YCP4). YCP4∆::URA3 strain has the wild-type PCRTag YCR004C.1 (YCP4) deleted. The YCP4S strain shows the presence of only synthetic PCRTags around the YCP4 region [synthetic sequence only for YCR003W.1, YCR004C.1 (YCP4) and YCR005C.1] and no wild-type PCRTags. YCP4S, synthetic version of YCP4.
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Figure S8: Karyotypic analysis of synIII and intermediate assembly strains by pulsed-field gel electrophoresis revealed the size reduction of synIII compared to native III. Synthetic intermediate strains are denoted by the amount of synthetic DNA integrated (in kb) at each step. Yeast chromosome numbers are indicated on the right side. Native and synthetic versions of chromosome III are marked with closed circles. Karyotype abnormalities pre-existing in the starting strain (ΔYCL069W) and those that arose at the 5th integration step (93) are indicated with closed and open arrowheads, respectively. Backcrossing of the synIIIL intermediate to BY4742 eliminated chromosomal abnormalities (compare 97 to 97*) and yielded a strain of the opposite mating type (MATa versus MATα). SynIII (272,871 bp) and native chromosome VI (270,148 bp) co-migrate in the gel.
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Figure S9: Validation of the left and right arm telomere ends of synIII. Southern blot analysis was used to verify and validate the left and right arm telomere ends of synIII (which had been specified by the UTC sequence) using arm-specific radiolabeled probes. (A) SynIII left arm telomere end has HML region deleted. Southern blot analysis of NotI/AlwNI digest of synIII swap strain yielded the expected 8.9 kb fragment as compared to 6.7 kb fragment for the wild type (BY4742) and ∆YCL069w strains confirming the deletion of HML from the left arm telomere end. (B) SynIII right arm telomere end has HMR region deleted. Southern blot analysis of BspEI digest of synIII swap strain yielded the expected 7 kb fragment as compared to the 28 kb fragment for the wild type strain (BY4742), confirming the deletion of the HMR from the right arm telomere end. The nucleotide sequence of the designer synIII chromosome further confirmed these results.
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Figure S10: Growth curves for wild type (BY4742) and synIII strains in absence of estradiol induction (A) YPD and (B) SC−Ura. pRS, control plasmid without Cre; pCRE, plasmid with Cre.
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Figure S11: SynIII and synIIIL phenotyping on various types of media. Ten-fold serial dilutions of saturated cultures of wild type (BY4742), synIIIL and synIII strains were plated on the indicated media and incubated at noted temperatures (A). YPD, yeast extract peptone dextrose; SC, synthetic complete medium; SD, synthetic dextrose; YPGE, yeast extract peptone glycerol ethanol and MMS, methyl methanosulfate. Growth of wild type (BY4742), synIIIL intermediate and synIII strains have identical colony sizes at different temperatures and on various media types, with only one condition out of 21 (high sorbitol) showing a subtle fitness defect for synIII (B).
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Figure S12: SynIII strain cell morphology. SynIII has a subtle morphological defect that arises as a specific integration intermediate that occurred on integration step synIII-219 kb. Arrowheads indicate cells with the morphological defect. Cells were grown to mid-log phase in SC medium at 30°C. DIC Images were collected using a Zeiss MicroscopeAxioskop 2 mot Plus (63X) with a Zeiss AxioCam HRc Camera.
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Figure S13: Transcript profiling of wild-type (BY4742) and synIII strains. RNASeq analysis of synIII strain as compared to the wild type is shown in a volcano plot. Genes deleted from synIII are labeled in blue letters. Genes with significantly altered expression are shown in red. The dashed line identifies the Family Wise Error Rate (FWER) threshold at 5% (threshold=7.02e-6).
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Figure S14: ChIPseq map of cohesin binding sites on native chromosome and synIII. (A) Sequences were aligned to the native reference sequence (SacCer2) or to synIII (SYNIII). Red arrows and bars indicate tRNA genes. (B) The panel shows that the pattern of cohesin peaks is identical for chromosome VI in the synIII and wild-type strains. (C) The panel shows the chromosome III data from A, mapped to the synIII reference sequence rather than to the “Saccer2” reference sequence.
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Figure S15: Replication origins of synIII. Replication could be affected by the pervasive changes in underlying sequence. SynIII maintains 12 of 19 ARSs (autonomously replicating sequences) present in native III in completely unaltered form; six ARSs were deleted (they were associated with the subtelomere and HM loci) and three ARS sequences suffered very minor sequence changes not expected to affect ARS function (Table S9). Also, many tRNA genes, which can in principle affect the movement of replication forks, were deleted. We compared the replication dynamics of synIII and native III. The chromosomal extremities most likely to be affected by the synIII design are already known to bear late replicating origins that are not major contributors to global replication dynamics (14), and little change was observed in these regions. (A) FACS analysis of asynchronous cells before elutriation and all the time points of the replication origin experiment. Time points T1 to T9 were processed for Illumina sequencing. (B) Graph of relative DNA content (R) during the time course experiment, which varies between 1 and 2, is calculated using the following formula: R= (G1+2*G2) / (G1+G2). (C) Replication program of chromosome III from strain synIII (yLM197) (this study), S. cerevisiae from Muller et al. 2012 (36) and Raghuraman et al. 2001 (37) are shown. For each study, replication data were plotted along the chromosome, after normalization of replication times or the relative copy number ranging between 0 and 1. Replication origins were detected on normalized data, as described in Agier et al. 2013 (35) which are indicated as diamonds on top of the replication curves. To be able to make a comparison between the WT chromosome III and synIII, the alignment between both chromosomes were considered for the projection of the replication data. (D) Correlation between replication data among the three studies. For each chromosome, we performed all pairwise comparisons by calculating the Spearman correlation coefficient (Rho). Each vertex corresponds to a pairwise comparison. E) Replication program of all the chromosomes of S. cerevisiae from this study, Muller et al. 2012 (35) and Raghuraman et al. 2001 (37) including chromosome III (C), are shown.
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A
B
Figure S16: Conditional genome instability (SCRaMbLE). (A) Induction of the SCRaMbLE system by Cre results in genome rearrangements of synIII. Cre induction greatly reduces the fitness of the synIII strain, but not the wild type (WT; BY4742). EST: estradiol; Hours: exposure to estradiol. Comparison of synIII + Cre between SC–Ura and YPD plates indicated a high frequency of loss of the URA3 plasmid in the induced synIII strain that prevents its growth on selective medium and not on rich medium. (B) Growth curves for wild type (BY4742) and synIII strains during Cre induction time course. Upon Cre induction, synIII shows much decreased fitness as compared to wild type. WT, wild-type (BY4742). The small decreased fitness effect visible on the uninduced synIII in this experiment compared to the WT might be explained by leakiness of the Cre plasmid.
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Figure S17: SUP61 essential tRNA complementation by a synthetic tRNA gene. First, a heterozygous diploid strain with SUP61S in the HIS3 plasmid was constructed that was heterozygous for synIII. The colonies were then screened for the loss of the HIS3 plasmid, which were then transformed with synthetic SUP61S cassette flanked by 500-bp homologous HO genomic sequence to integrate the donor at the HO locus. SUP61S, synthetic version of SUP61.
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Figure S18: SCRaMbLE leads to a gain of mating type a behavior in synIII heterozygous diploids. (A) Schematics of SCRaMbLE experiment behavior in synIII heterozygous diploids. Frequency of a-mater and α-mater colonies post-SCRaMbLE (induction with estradiol) in synIII/III and III/III strains. (B) Genotypic analysis of 12 gain of “a mater” synIII/III strains. Select synthetic PCRTags spanning synIII (~10 kb intervals) were used to assess the effects of SCRaMbLE. Red boxes indicate absence of PCRTags (presumed deletions); white boxes indicate presence of PCRTags. Clones 1 & 4, presumed deletion of right arm of synIII; Clones 2, 3, 5-10, & 12, presumed loss of entire synIII; and Clone 11, segment deletions within the right arm of synIII. Stars “*” indicate the position of loxPsym sites around these regions of the synIII chromosome.
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Table S1: SynIII minichunk plasmids
Minichunk Plasmids
SynIII minichunk synthetic coordinates
Start Stop pNA001 377 3360 pNA002 2604 5525 pNA003 4837 7822 pNA004 6991 9992 pNA005 9247 12190 pNA006 11448 14470 pNA007 13656 16704 #pNA008 15957 19975 #pNA009 19209 21034 pNA010 20272 23218 pNA011 22513 25491 pNA012 24688 27641 pNA013 26897 30022 pNA014 29219 32257 pNA015 31504 34450 pNA016 33703 36704 pNA017 35918 39144 pNA018 38130 41352 pNA019 40623 43599 pNA020 42851 45941 pNA021 45081 48168 pNA022 47407 50415 pNA023 49615 52303 #pNA024 51819 54436 pNA025 53759 56768 pNA026 56016 58624 pNA027 58251 60795 #pNA028 60142 63071 pNA029 62310 65291
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pNA030 64463 67422 pNA031 66774 69332 pNA032 68583 71593 #pNA033 70780 73348 pNA034 73072 75912 pNA035 75308 78094 pNA036 77338 80282 pNA037 79534 82556 pNA038 81802 84692 pNA039 84030 87021 pNA040 86271 88862 pNA041 88358 91081 pNA042 90335 93322 pNA043 92544 95550 #pNA044 94785 97386 pNA045 96965 100330 pNA046 99617 101869 pNA047 101114 103366 pNA048 102521 104851 pNA049 104099 106207 pNA051 105585 107676 pNA051 107077 109234 pNA052 108564 112198 pNA053 111451 115169 pNA054 114417 118160 pNA055 117337 121133 pNA056 120381 122627 pNA057 121873 124110 pNA058 123360 125432 pNA059 124847 126655 pNA060 126332 129580 pNA061 128849 132517 pNA062 131872 133987
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pNA063 133360 135600 pNA064 134713 136982 pNA065 136236 138468 pNA066 137710 141447 pNA067 140661 144345 pNA068 143666 147115 pNA069 146546 150079 pNA070 149331 151572 pNA071 150817 153206 pNA072 152457 156143 pNA073 155397 159091 pNA074 158343 162131 #pNA075 161901 165291 pNA076 164565 168240 pNA077 167496 171196 pNA078 170502 173840 pNA079 173106 176794 pNA080 176049 179720 #pNA081 178903 180524 *#pNA082 180019 182639 *pNA083 181876 184124 pNA084 183355 185579 pNA085 184832 187104 pNA086 186352 188602 pNA087 187846 190095 pNA088 189339 191568 pNA089 190781 194705 pNA090 193910 196318 pNA091 195480 197901 pNA092 197048 199408 pNA093 198616 201021 pNA094 200270 204163 pNA095 203398 207373
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pNA096 206516 208891 pNA097 208094 210551 pNA098 209670 212039 pNA099 211283 213535 pNA100 212769 215016 pNA101 214272 216498 pNA102 215767 219487 pNA103 218743 220974 *pNA104 220241 222455 #pNA105 221702 225023 pNA106 224652 228367 pNA107 227721 231390 pNA108 230579 234346 pNA109 233601 235849 pNA110 235097 237304 pNA111 236559 238842 pNA112 238077 240245 pNA113 239574 243307 pNA114 242549 246264 pNA115 245504 247802 pNA116 247013 249330 pNA117 248530 250868 pNA118 250052 252364 *pNA119 251576 255452 #pNA120 255142 258615 pNA121 257818 260126 pNA122 259337 261686 pNA123 260896 264829 pNA124 264031 267913 pNA125 267124 269429 pNA126 268674 270998 pNA127 270219 272499
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*Minichunks with pre-existing mutations: pNA082, 2324T deletion; pNA083, 467T deletion; pNA104, 1427T deletion; and pNA119, 825A deletion. The deletion numbers represent positions of mutations within the minichunks. #Minichunks that were digested with appropriate restriction enzymes to remove the mutated regions from overlapping BBs, but leaving sufficient overlap for HR.
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Table S2: Summary of eleven iterative one-step assemblies and replacements of native III with synthetic fragments needed to construct synIII
Replacement rounds
Starting strain Minichunks, Linker,
UTC fragment co-transformed
% of clones with right marker combination
PCR Tags Coordinate of last SYN
bp
^Clone kept
L1 BY4741
∆YCL069w [Ura–, Leu–]
1−4, Linker L1, Left end UTC
fragment 59% 1 [YCL064C1] − 2 [YCL063W1] 9246 clone 1: 9 kb-synIII
L2 9 kb-synIII [Ura–, Leu+]
4−13, Linker L2 10% 3 [YCL061C1] − 16 [YCL050C1] 30013 clone p1: 30 kb-synIII
L3 30 kb-synIII [Ura+, Leu–]
14−20, Linker L3 10.6% 17 [YCL049C1] − 28 [YCL039W1] 45934 clone p27: 46 kb-synIII
L4 46 kb-synIII [Ura–, Leu+]
21−26, Linker L4 1.3% 29 [YCL038C1] − 36 [YCL032W1] 58597 clone 3: 58 kb-synIII
*L5–A 58 kb-synIII [Ura+, Leu–]
27−42, Linker L5 1.2% 39 [YCL029C1] − 64 [YCL002C1] N/A clone L5-18
*L5–B clone L5-18 [Ura+, Leu+]
27−33, No Linker N/A 37 [YCL030C2] − 38 [YCL030C1] 92904 clone 16: 93 kb-synIII
L6 93 kb-synIII [Ura–, Leu+]
43−44, Linker L6 6.8% 65 [YCL001W1] 97411 clone 2: 97 kb-synIII
(Left arm)
R1 97 kb-synIII [Ura+, Leu–]
45−71, Linker R1 0.5% 66 [YCR002C1] − 67 [YCR003W1] 69 [YCR005C1] − 72 [YCR009C1] 76 [YCR011C3] − 98 [YCR030C1]
152497 clone 19: 152 kb-synIII
R2 152 kb-synIII [Ura–, Leu+]
71−78, 53−54, Linker R2
20% 99 [YCR032W4] − 113 [YCR037C2] 73 [YCR010C1] − 75 [YCR011C2]
172427 clone 26: 172 kb-synIII
R3 172 kb-synIII [Ura+, Leu–]
78−102, Linker R3 pRS413-SUP61
8.6% 114 [YCR037C1] − 148 [YCR072C2] 219791 clone 10: 219 kb-synIII
#R4 219 kb-synIII [Ura–, Leu+]
102−114, 46, 47, Linker R4
12% 149 [YCR073C4] − 165 [YCR088W2] 244011 clone 11: 244 kb-synIII
#R5 244 kb-synIII [Ura+, Leu–]
114−127, 46, 47, No Linker, Right end
UTC fragment N/A 166 [YCR088W1] −186 [YCR098C1] 272907 clone 28: 272 kb-synIII
L indicates synIII left arm; R indicates synIII right arm; *Round L5 was planned for replacement of PCRTags 37 to 64, using LEU2 as a selectable marker, but it was initially not possible to obtain a complete synthetic substitution. A clone in which PCRTags 37 and 38 amplified only the wild-type sequence and that was still [URA+] was selected for a second round of recombination with minichunks 27 to 33, using counter selection for URA3 by 5-FOA, allowing the next step to be carried out with a selection for [Ura+ Leu-] colonies. Transformations #R4 and #R5 also included minichunks 46 & 47 to encourage replacement of wild type YCP4 with synthetic YCP4S, but were not successful in this regard. ^For each clone, the amount of synthetic DNA (in kb) that was replaced in the native chromosome III is shown.
44
Table S3: Yeast strains used in this study
Strain name Description Replacement
round Genotype
BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
BY4741 ∆YCL069w MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 YCL069W::kanMX
HMSY001 9 kb-synIII L1 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 SYN3-8876bp-LEU2
HMSY002 30 kb-synIII L2 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 SYN3-29626bp-URA3
HMSY003 46 kb-synIII L3 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 SYN3-45560bp-LEU2
HMSY004 58 kb-synIII L4 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 SYN3-58222bp-URA3
HMSY005 93 kb-synIII L5A, B MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 SYN3-92904bp-LEU2
HMSY006 97 kb-synIII L6 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0a SYN3-97002bp-URA3
yLM043 97 kb-synIII * MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-97002bp-URA3
HMSY007 152 kb-synIII R1 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-152088bp-LEU2
HMSY008 172 kb-synIII R2 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-172018bp-URA3
HMSY009 219 kb-synIII R3 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-219111bp-LEU2 pRS413 (HIS3)-synSUP61(tRNA)
HMSY010 244 kb-synIII R4 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-243604bp-URA3 pRS413 (HIS3)-synSUP61(tRNA)
HMSY011 272 kb-synIII,
WT YCP4 tags R5 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-272123bp pRS413 (HIS3)-synSUP61(tRNA)
NAY001 272 kb-synIII,
Synthetic YCP4 tags MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-272123bp pRS413 (HIS3)syn-SUP61(tRNA)
yLM197
Strain NAY001
SUP61(tRNA)
integrated at HO
MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-272123bp HO::synSUP61-URA3**
yLM422 yLM197−URA3 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 SYN3-272123bp HO::synSUP61**
45
yLM108 to yLM117 synIIIxchr3 diploid MATa/α his3Δ1 leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0 (HIS3)-synSUP61(tRNA)
parents: HMSY011xBY4741
yLM098 to yLM107 synIIIxchr3 diploid MATa/α his3Δ1 leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0 parents: HMSY011xBY4741
NAY002 to NAY011 synIIIxchr3 diploid MATa/α his3Δ1 leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0 parents: HMSY011xBY4741
HO::synSUP61-URA3**
*Backcross to BY4742 was performed at this point.
**Essential tRNA cassette SUP61S integrated at the HO locus
46
Table S4: Sequence variants in the synIII chromosome
Position in
SynIIIv.3_41
Reference
SynIIIv.3_41
Variant
SynIIIv.3_42
ORF Amino acid
Substitution
Closest ORF
*68618 G T YCL025C Synonymous -
71593.1 - C YCL024W Frameshift -
92548 C A YCL002C Q117H -
*176111 A G YCR038C Synonymous -
#182368 T - - - YCR042C (277 bp)
195513 G C YCR054C P462A -
#221701 T - YCR073C Frameshift -
243006 T C YCR088W Synonymous -
#252434 A - YCR091W Frameshift -
For each variant (single base substitution or indel variant), we report position relative to the designed sequence, the reference base(s) and the experimentally determined variants. Variants occurring inside ORFs are annotated with gene name and corresponding amino acid substitution. For variants outside coding regions, we report the closest gene with the distance to nearest ORF boundary. Since these variants arose during assembly step in yeast, it suggests an error rate in the assembly process of about 3.3 X 10-5. As explained in the text, one expected loxPsym site at position 59583 was also absent from the sequence. We believe that this probably arose through a recombinational mechanism.
*This variant corresponds to the native sequence and therefore probably arose by recombination. #This variant corresponds to a pre-existed mutation in the corresponding minichunk.
47
Table S5: Wild type and synthetic PCRTags and the expected size of amplicons
PCR Tag #
ORF Pair WT or SYN
Forward Oligo Reverse Oligo ORF Size (bp)
Amplicon Size (bp)
Sequence identity (%)
1 YCL064C 1 WT GTCCTTTATAGTATTAGAGGAGCCGCCA TTCTCTAGGAACGGCCGTGATCTCTAAT
1082 271 50 SYN ATCTTTGATGGTGTTGCTGCTACCACCG CAGCTTGGGTACCGCTGTTATTAGCAAC
2 YCL063W 1 WT GTCTTCCTCGGATACGTATGAGTCTTTC ATTAGATGGCCTGAAGAATGAGGCGGAG
1271 280 59 SYN AAGCAGCAGCGACACCTACGAAAGCTTT GTTGCTAGGTCGAAAAAAGCTAGCGCTA
3
YCL061C
1 WT GGAGCTCAATAGTGGAACCCTTTTCTCA ACAGACTGCGCATGATGAGGATAAGACA
3290
421 47 SYN GCTAGATAGCAAAGGGACTCGCTTTTCG TCAAACCGCTCACGACGAAGACAAAACC
4 2 WT ATCATCGTCCTCATTTGAGGACGAATCG ACAGCGTATAGATAGCAGCGGTGCAACC
499 52 SYN GTCGTCATCTTCGTTGCTGCTGCTGTCA CCAACGAATCGACTCTTCTGGCGCTACT
5 3 WT TCTTTGCGTCTGCGTGAATGAAGAAGAG AGACAATCCGCCAGAGTTGACTGGGAAC
256 47 SYN TCGTTGGGTTTGGGTAAAGCTGCTGCTA CGATAACCCACCTGAACTAACCGGTAAT
6 YCL059C 1 WT ATCAACCTTTCTAGGCAATTGGGCAGGA ATTGGCGAGATCCGTTCCTTTCCCG
950 475 40 SYN GTCGACTTTTCGTGGTAGTTGAGCTGGT GCTAGCTCGAAGCGTCCCATTTCCA
7
YCL057W
1 WT TTGGTCGTTCACTCCCAGCGATATTAGT GGAGTTCACAGATGCGTCCCTAATTTCT
2138
223 48 SYN CTGGAGCTTTACCCCATCTGACATCTCT GCTATTAACGCTAGCATCTCGGATTTCC
8 2 WT ACTAGAGGCTACGATTACTGGTATGCTG AGATCCGGGGCCATTAAACCTCGATTCC
424 43 SYN TTTGGAAGCCACCATCACCGGCATGTTG GCTACCTGGACCGTTGAATCGGCTTTCT
9 YCL055W 1 WT TGCCACTACGCCATTTGGGTGTAAAATC TGGTACAGCACCATTAGTGGTGTCCTTC
1007 484 36 SYN CGCTACCACCCCTTTCGGTTGCAAGATT AGGAACGGCGCCGTTGGTAGTATCTTTG
10
YCL054W
1 WT TAGAAGGTCGGAAAGGGATGCCAAGTTT GGAGTCATCACTTGAACTCTCATCCGAA
2525
433 54 SYN CCGACGAAGCGAACGAGACGCTAAATTC GCTATCGTCAGAGCTAGATTCGTCGCTG
11 2 WT TGCATCCAAACTCTGTCCTGTCAACTCC TAATCTTGGGTCCAGCCTCTTTGGTGCC
448 43 SYN CGCTAGCAAGTTGTGCCCAGTTAATAGC CAATCGAGGATCCAATCGTTTAGGAGCT
12 3 WT GGCACATCAGTTAGCATTGGGTCAGAAA GTCTGAATCATCGTTAATCAAGCCCGCC
304 43 SYN AGCTCACCAATTGGCTCTAGGCCAAAAG ATCGCTGTCGTCATTGATTAGACCAGCT
13 YCL052C 1 WT CAGATATAGTGATGTCGTAGTCGTCGAG CTCAAATTCTTCGGATGTGCCGGAAAGG
1250 418 56 SYN CAAGTACAAGCTGGTGGTGGTGGTGCTA TAGCAACAGCAGCGACGTTCCAGAACGA
48
14
YCL051W
1 WT CTCTGTGGTCAAATCCTGTACGCCTGAT ATTGCTTGTTGCTGCGCTGTTGCTCGCA
1751
283 52 SYN TAGCGTTGTTAAGAGCTGCACCCCAGAC GTTAGAGGTAGCAGCAGAATTAGAAGCG
15 2 WT CGATGCGTTGAAGACAAGGCCTAGGTCA TTTAGAAGGGGTATAGGCCAGGGAAGAA
496 54 SYN TGACGCTCTAAAAACCCGACCACGAAGC CTTGCTTGGAGTGTAAGCCAAGCTGCTT
16 YCL050C 1 WT ATCAGAAAAGGCCTTCGAGCGTGGAACG AGCCAGTGGTTCTTCATTGGACCACAAA
965 355 54 SYN GTCGCTGAAAGCTTTGCTTCGAGGGACA TGCTTCTGGCAGCAGCCTAGATCATAAG
17 YCL049C 1 WT AACATGGTAGTACCTGGACCAAGCGCTA CATCTCGGTGCCATATAATTGGACGTCA
938 304 46 SYN GACGTGATAATATCGGCTCCAGGCAGAG AATTAGCGTTCCTTACAACTGGACCAGC
18 YCL048W 1 WT GGATTTGAACAGCTTGAGGGCCATTGGT TGGAAGGTAGACCAATGATGTGTCCCTA
1391 475 54 SYN AGACCTAAATTCTCTACGAGCTATCGGC AGGCAAATAAACTAGGCTGGTATCTCGG
19 YCL047C 1 WT TCTTAGTTGCGATTCAGCACTGAGATGG AGTATCTGTCGGGTTGACTGTTTTCTCG
776 250 50 SYN TCGCAATTGGCTTTCGGCAGACAAGTGA TGTTAGCGTTGGTCTAACCGTCTTTAGC
20
YCL045C
1 WT CAACTGCGAATCGGATCCTGGCAATAGA CCCAGATGAGCGTTTGTCTAACTCAAGC
2282
361 59 SYN TAGTTGGCTGTCGCTACCAGGTAGCAAG TCCTGACGAACGACTAAGCAATAGCTCT
21 2 WT CCTTTCAGATAGGGGGGATCTTGATTTG TGCTGTATTGGACGTCTTCGATTCTAGG
463 54 SYN TCGTTCGCTCAATGGGCTTCGGCTCTTA CGCCGTTCTAGATGTTTTTGACAGCCGA
22 YCL044C 1 WT AAGGCCAGTAGAAGAAGACTGCGGATTG TGGCTCTTTGCTACTTTCTCAGTCACTG
1253 439 65 SYN CAAACCGGTGCTGCTGCTTTGTGGGTTA CGGTAGCCTATTGTTGAGCCAAAGCTTA
23
YCL043C
1 WT AGAGTCCAAGGATCTTGAACCTTGGTAC CTTGAAAGGTGATGCCTCCCCAATCGTG
1568
358 49 SYN GCTATCTAGGCTTCGGCTGCCTTGATAA TCTAAAGGGCGACGCTAGCCCTATTGTT
24 2 WT AAACGCCTCTTCAGAGAGTTGAGGCAAA CGAAAGCGGTTTGCCTTTGGGTTACTTA
247 45 SYN GAAAGCTTCTTCGCTCAATTGTGGTAGG TGAATCTGGCCTACCACTAGGCTATTTG
25
YCL040W
1 WT ACTGTCAACGAACCCGGGATTTCACTTG AGATCTCCTAGAAATCGCGCGCACC
1502
319 51 SYN GTTGAGCACCAATCCAGGTTTCCATCTA GCTTCGTCGGCTGATAGCTCGAACT
26 2 WT CCAGTTGTCATCCGAAGTGCTGTCGCAT GGCACCCAAGGGTGACAAGGCT
325 55 SYN TCAACTAAGCAGCGAAGTTTTGAGCCAC AGCGCCTAGTGGGCTTAGAGCC
27
YCL039W
1 WT GGATGATGACGCCTCTTTCTCCTCTCCA GTTTGCCGGCCTCGAGGAAGATGAT
2237
496 61 SYN AGACGACGATGCTAGCTTTAGCAGCCCT ATTAGCTGGTCGGCTGCTGCTGCTA
28 2 WT TAGCTCTCCGCTAAGGTCCGAATGGCTT AGAGGCGGAATTTGTGACTGGATGGAAG
247 52 SYN CTCTAGCCCATTGCGAAGCGAATGGTTG GCTAGCGCTGTTGGTAACAGGGTGAAAA
29 YCL038C 1 WT CGCGGACAGGCCACCTAGTGAT TTCTACAGCGGTTTTGTTCTCCAAGGCA 1586 298 53
49
SYN AGCGCTCAAACCGCCCAAGCTC CAGCACCGCTGTCCTATTTAGCAAAGCT
30 2 WT GCTCCCCCAGAGATCTACTATCCCTGAA CGAACCATTTGTCGTTTCTGCGGTTTCA
268 48 SYN AGAACCCCACAAGTCAACGATACCGCTG TGAACCTTTCGTTGTCAGCGCTGTCAGC
31 YCL037C 1 WT GTTAGCATTACTCGACGATGTCGACGAT ATTGCCCACATCTTCCCCATGGAAACTT
1304 271 59 SYN ATTGGCGTTAGAGCTGCTGGTGCTGCTA TCTACCAACCAGCAGCCCTTGGAAGTTG
32
YCL036W
1 WT ACTAAGTAATGGGACGCTCTCTGTCAGA AGACAGCCGCTGTGATCTTTTGGACCTG
1700
439 56 SYN GTTGTCTAACGGTACCTTGAGCGTTCGA GCTCAATCGTTGGCTTCGCTTGCTTCGT
33 2 WT CATGGTGGAAGCACATTCACGCTCGAAT CTTAAGATCCCCGCTCACGTGATGACCA
421 45 SYN AATGGTTGAAGCTCACAGCCGAAGCAAC TTTCAAGTCACCAGAAACATGGTGGCCG
34 YCL034W 1 WT CAGCGATGCTCTCGCGTCTGCT TGAGGACTCGCTATCAATATACCTGCTG
1064 337 62 SYN TTCTGACGCCTTGGCTAGCGCC GCTGCTTTCAGAGTCGATGTATCGAGAA
35 YCL033C 1 WT ACACCTTGCACAACATATCTCCACCCTC TGAAAGGCCCAACACCGGTGCGTATTTA
506 214 40 SYN GCATCGAGCGCAGCAGATTTCAACTCGA CGAACGACCAAATACTGGCGCTTACTTG
36 YCL032W 1 WT ACCCAGTAGTGACGGTGTGTCTCTTTCA GTTCGCTGTTGAAACGGCAGATGGG
1040 208 57 SYN CCCATCTTCTGATGGCGTTAGCTTGAGC ATTAGCGGTGCTGACAGCGCTAGGA
37
YCL030C
1 WT AGAATACACCAACCCTAGACAACGCTCA CGGAGTTTCTTCTCTGTTCATTGCTAGC
2399
292 54 SYN GCTGTAAACTAGACCCAAGCATCGTTCG TGGTGTCAGCAGCTTGTTTATCGCCTCT
38 2 WT ATAAACGCCACGACCCAAATCGATGGCC GTCCTTGCCAAGTGGTAAATTCAGCGAT
394 47 SYN GTAGACACCTCGGCCTAGGTCAATAGCT AAGCCTACCTTCTGGCAAGTTTTCTGAC
39 YCL029C 1 WT AGCCTTTCTGTCTGATGACAGGATAGAG ATCGATATCGCAAACCTCGAGAAGAACG
1322 274 56 SYN GGCTTTTCGATCGCTGCTCAAAATGCTA TAGCATCAGCCAAACTAGCCGACGAACC
40 YCL028W 1 WT AGCACTGGCTTCTATGGCAAGCTCTTTT AGCCAAAGCTGAGAATGAACTGGAGTGG
1217 373 58 SYN TGCTTTGGCAAGCATGGCTTCTAGCTTC TCATAGCTCTAGCTTTAGCGCCCTAGCA
41
YCL027W
1 WT GGTGTCCATTTCAAATCCACCCATGTCA AGAACCCCTCAAGAACCATCGAGATAGT
1538
499 54 SYN CGTTAGCATCAGCAACCCTCCAATGAGC GCTGCCTCGTAGAAACCATCTGCTCAAA
42 2 WT CGATCTAATCACGGCAAGACCCCATTCA AGTAAGCGGCAATGGTTTAGAACGTGAC
226 52 SYN TGACTTGATTACCGCTCGACCACACAGC GGTCAATGGTAGAGGCTTGCTTCGGCTT
43 YCL026C-B 1 WT AGACTCAGGGATTTTGCTTGGCAAAGCA AAGGGATGAGGCCTTTGGTTCTGTAATC
581
232 50 SYN GCTTTCTGGAATCTTAGAAGGTAGGGCG TCGAGACGAAGCTTTCGGCAGCGTTATT
44 YCL026C-A 1 WT CAAATTAGCCCCCAATCCCAATAGTTCG TGCTGAAGCCAAGAAGAGACCAGAGTCT
208 43 SYN TAGGTTGGCACCTAGACCTAGCAATTCC CGCCGAAGCTAAAAAACGACCTGAAAGC
50
45
YCL025C
1 WT GAAAAGCTGAGACAAACCAGAAATGGCC CTCCTCCTTCTACTCCAGTGCTCGTTTA
1901
214 54 SYN AAACAATTGGCTTAGGCCGCTGATAGCT TAGCAGCTTTTATAGCTCTGCCCGATTG
46 2 WT ACCCAATTCTCCAAGAGATCTCCCTTGG CGCCGTTATTCTACTTTCCGTGCTGTCC
331 47 SYN GCCTAGTTCACCCAAGCTTCGACCTTGA TGCTGTCATCTTGTTGAGCGTTTTGAGC
47
YCL024W
1 WT TGACTCGAACAACCATACTAGCGCCTCT AAAGGACGATCTCTTCCTGCTAGAAGAG
3113
355 64 SYN CGATAGCAATAATCACACCTCTGCTAGC GAAGCTGCTTCGTTTTCGAGAGCTGCTA
48 2 WT CAAACTGGACTCTGTAAGGGGATCGAAT AGAACTATCGGTAGGATTCGAGGACAGT
229 56 SYN TAAGTTGGATAGCGTTCGAGGTAGCAAC GCTAGAGTCAGTTGGGTTGCTGCTCAAC
49 3 WT GCTACTAAATGTTAGAGGGGGACTATCG CGCATACAAAGATAAGACTGAGCGCCGA
208 54 SYN CTTGTTGAACGTCCGAGGTGGTTTGAGC AGCGTATAGGCTCAAAACGCTTCGTCGG
50 YCL017C 1 WT ACCCGAGGATAATGCGATATCCCTTAGT GGGAATAGGTGCCATCTATGTAAGAAGG
1493 343 47 SYN GCCGCTGCTCAAAGCAATGTCTCGCAAA AGGTATCGGCGCTATTTACGTTCGACGA
51 YCL016C 1 WT TAGCGCAGAACATGGTGAGTTCTCAAGT GTCTGAAGTTGTACTGTGTTCGCACGAC
1142 340 52 SYN CAAAGCGCTGCAAGGGCTATTTTCCAAC AAGCGAAGTCGTTTTGTGCAGCCATGAT
52
YCL014W
1 WT AATATCGCAGAGGCATCCTAAGTCTCCA TGATGACGAATTCGTCGCCTTCCCAATT
4910
466 56 SYN GATCAGCCAACGACACCCAAAAAGCCCT GCTGCTGCTGTTGGTAGCTTTACCGATG
53 2 WT CGAATCCGTCGATATATCTTCCAACTCG CAACTGGCAACTACTAGCCAAATCTCCA
223 52 SYN TGAAAGCGTTGACATCAGCAGCAATAGC TAGTTGACAAGAAGAGGCTAGGTCACCG
54 3 WT TAGCAAACTTTCCGGTGCATCCGATTTC CACTCTTTGAACTGGTGAAGGCTTGCTC
340 50 SYN CTCTAAGTTGAGCGGCGCTAGCGACTTT AACTCGTTGGACAGGGCTTGGTTTAGAT
55 4 WT ACTCTCTCCACAAGCGAGTAAAGTGCTG CGAGTCATCTAGATGCGCCTTAGGCAAA
496 48 SYN GTTGAGCCCTCAAGCTTCTAAGGTTTTG GCTATCGTCCAAGTGAGCTTTTGGTAGG
56 5 WT TCCGGCCATTGAGAATTTGAGTCCAAGT TCCTGATGTAAAGAAGCGCTTCCCCAGA
238 45 SYN CCCAGCTATCGAAAACCTATCTCCTTCT ACCGCTGGTGAAAAATCGTTTACCCAAG
57 YCL011C 1 WT GACAGAACCGAATCCTCTTGAAAATCCG TGCCATATCGAAGTTTGATGGTGCCCTC
1283 265 48 SYN AACGCTGCCAAAACCTCGGCTGAAACCA CGCTATCAGCAAATTCGACGGCGCTTTG
58 YCL010C 1 WT TGCTAGGTTTGCCAAAGCCGTTGGCGAA CTCAGAGGTTGCCTATAAGCCCAGAAGG
779 388 48 SYN AGCCAAATTAGCTAGGGCGGTAGGGCTT TAGCGAAGTCGCTTACAAACCACGACGA
59 YCL009C 1 WT GGCAGAGATACGTGTGGGTTTTGCAGAC GATGGCCAGAATCTCTCTATTGGGTACT
929 286 46 SYN AGCGCTAATTCGGGTTGGTTTAGCGCTT TATGGCTCGAATTAGCTTGCTAGGCACC
60 YCL008C 1 WT CACATGCGAAGTTGAGCTAAACTGTTGG GTCTCCACACCTAAAACCGCCATTGCCT 1157 373 52
51
SYN AACGTGGCTGGTGCTAGAGAATTGTTGA AAGCCCTCATTTGAAGCCACCTCTACCA
61 YCL005W 1 WT GTCGCTGAGCCGTTTAGTTGCACAAACA ACTAGTGGAAGCACGTTCAAGGACCGTA
770 232 45 SYN CAGCTTGTCTCGATTGGTCGCTCAAACC AGAGGTGCTGGCTCGTTCCAAAACGGTT
62
YCL004W
1 WT TCTACTTGATGGCCTTCGAGGAACAAGA GGCTCCCGAAAGAATGACCTCGTTATCA
1565
232 46 SYN CTTGTTGGACGGTTTGAGAGGTACCCGA AGCACCGCTCAAGATAACTTCATTGTCG
63 2 WT TGGGTCAGAGAGAGTGGATTGCCGATTG CACGGCAGTAATCGGGAGGCTTTGTTTA
430 45 SYN CGGTAGCGAACGAGTTGACTGTAGACTA AACAGCGGTGATTGGCAAAGATTGCTTG
64 YCL002C 1 WT TGCATTGGTCGGTATTACCAGTCTACCT TTCATCTCTGGTGAGGGAACAACTGTCT
755 481 45 SYN AGCGTTAGTTGGGATAACCAATCGGCCC CAGCAGCTTGGTTCGAGAACAATTGAGC
65 YCL001W 1 WT TCACGCTAAGGAGAGGTGGGCTGTATTG CGACAACAAGAGGGAAATGACAGTGGCT
566 295 47 SYN ACATGCCAAAGAACGATGGGCCGTTCTA GCTTAGTAGCAAGCTGATAACGGTAGCT
66 YCR002C 1 WT GGCGTTGCTAGACATATGAGCTGAGGAA CTTCAGGGGAAGAAAAACTCGTTGGAGC
968 217 50 SYN AGCATTAGAGCTCATGTGGGCGCTGCTT TTTTCGAGGTCGAAAGACCCGATGGTCT
67 YCR003W 1 WT ACAACTGGGATCTATCCACCGTTGGTTG ATGGCCGCATGATGGGCACTTATTCAAA
551 214 43 SYN GCAATTGGGTAGCATTCATCGATGGCTA GTGACCACAGCTAGGACATTTGTTTAGG
68 YCR004C 1 WT CAATTCAAGTGGAGACGCAGTTCTTGAG TGCGGGGATATTCGTTAGTACTTCCAGT
743 226 50 SYN TAGTTCCAAAGGGCTAGCGGTTCGGCTA CGCTGGTATCTTTGTCTCTACCAGCTCT
69 YCR005C 1 WT GCCAGGTGCTACCTCGTATATTGATGAA CTCAGCACTATCATCACCTTATCTGTCC
1382 322 57 SYN ACCTGGAGCAACTTCATAGATGCTGCTG TAGCGCTTTGAGCAGCCCATACTTGAGC
70
YCR008W
1 WT ATCATCTAGGCAGGGAAAGGCCTCATCG AGAGTGCGATGGTGATGCCGACTGGTAA
1811
214 64 SYN AAGCAGCCGACAAGGTAAAGCTAGCAGC GCTATGGCTAGGGCTAGCGCTTTGATAG
71 2 WT CACAAACGTTCCATCGGCGTCTAAATCA CCTCCCACAAATAGGTGCACTTGTATTG
418 50 SYN TACCAATGTCCCTAGCGCTAGCAAGAGC TCGACCGCAGATTGGAGCAGAGGTGTTA
72 YCR009C 1 WT GCTCAATTCTTTTTCAGCCCTTGGCAGT CTTGGACTCATTGAGAGCTGTGACAGCA
797 358 47 SYN AGATAGTTCCTTTTCGGCTCGAGGCAAC TCTAGATAGCCTACGAGCCGTTACCGCT
73 YCR010C 1 WT AAACGTAAAGATGGCCCACCCCAACAAA AGGTCTTTCAGCCTTCGCGTTGACG
851 319 46 SYN GAAGGTGAAAATAGCCCAACCTAGTAGG GGGCTTGAGCGCTTTTGCTCTAACC
74
YCR011C
1 WT TAGTCCGCTAAACAGTAGTGAGCCCAAA ATTCACAGGTCTCAGCTCGTTCGCTCTG
3149
340 58 SYN CAAACCAGAGAACAACAAGCTACCTAGC CTTTACCGGCTTGTCTAGCTTTGCCTTG
75 2 WT TGGCAAACGAATGGGAGACTTTGAGGAA AGGGCCAGGAGATTTCAGCTGTGATTTA
322 50 SYN AGGTAGTCGGATTGGGCTTTTGCTGCTG GGGTCCTGGTGACTTTTCTTGCGACTTG
52
76 3 WT GATATCTATAGACCCCTTAGCACCACAC GCCCCTCTGTGGCGGTCTATCA
472 47 SYN AATGTCGATGCTACCTTTGGCGCCGCAT GCCATTGTGCGGTGGCTTGAGC
77 YCR012W 1 WT CTTGGCTTCTCACTTGGGTAGACCAAAC ATCAGCCAAAGAGCTCAATTCGTGTCTG
1250 295 52 SYN TCTAGCCAGCCATCTAGGCCGACCTAAT GTCGGCTAGGCTAGATAGTTCATGTCGA
78
YCR014C
1 WT AATGTCACCACACTTGGAATAGCCCCTA CGGCATTGGGTCGGAAATTGCTAAACGC
1748
271 41 SYN GATATCGCCGCATTTGCTGTAACCTCGG TGGTATCGGTAGCGAAATCGCCAAGCGA
79 2 WT TGTCCAATCTGATACAAACTCCTCTGGG GCGACTCTTACAGGGGGATAAAGGAAGA
439 39 SYN GGTCCAGTCGCTAACGAATTCTTCAGGA AAGATTGTTGCAAGGTGACAAGGGTCGA
80 YCR015C 1 WT GACCTTATCAGAGCCTGTCAATAACCGA ATCACTGCCGTTGTTATCTTCAGGCGTA
953 442 52 SYN AACTTTGTCGCTACCGGTTAGCAATCGG AAGCTTGCCACTATTGAGCAGCGGTGTT
81 YCR016W 1 WT ATCCTCTACCAAAAAAGGGAAGCGTGTG AACACCAGACTCTGATGGCTCGTCTTTG
872 241 48 SYN GAGCAGCACTAAGAAGGGTAAACGAGTT GACGCCGCTTTCGCTAGGTTCATCCTTA
82
YCR017C
1 WT AAGTTCCCCAACTGGAGAGGGCAATAAA CCTGCTATCACTAACAGCTAGGTTCGTG
2861
382 50 SYN CAATTCACCGACAGGGCTTGGTAGCAAG TTTGTTGAGCTTGACCGCCCGATTTGTT
83 2 WT GCTAGTTAGATCCCTGTTCCCCATGGTA GATTGAGACGGTTCTTGCCTTTTCTTCC
373 48 SYN AGAGGTCAAGTCTCGATTACCCATAGTG AATCGAAACCGTCTTGGCTTTCAGCAGC
84 3 WT GAATAACAACGAGCCAAAACCAACAGCC GTTGATTTCTGTAGCTGTGGGAACTTCC
352 48 SYN AAACAATAGGCTACCGAAGCCGACGGCT ACTAATCAGCGTTGCCGTTGGTACCAGC
85 YCR018C 1 WT TTTTCCATAGGCGAGTCCACAGGGACTA AACTAGTCGGTCTGCCAGTATTACGAAG
665 202 47 SYN CTTACCGTAAGCCAAACCGCATGGAGAG TACCTCTCGAAGCGCTTCTATCACCAAA
86 YCR019W 1 WT GCTATGTTCTGGGTCTAGATGCTTAGAC AGAAACTGCAAAGCCGCCAAGGAACGAG
1091 454 54 SYN ATTGTGCAGCGGTAGCCGATGTTTGGAT GCTGACAGCGAAACCACCCAAAAAGCTA
87 YCR021C 1 WT GAATCCTCCTGTCAAAGATAGCCTTGGC TTCCGCCTTGGATATGGTACATTCGCTG
998 409 50 SYN AAAACCACCGGTTAGGCTCAATCGAGGT CAGCGCTCTAGACATGGTTCACAGCTTG
88
YCR023C
1 WT AGAAGACTGTGACTGCGAATTGTAGGCA TTCGAGTCCCTTGAGTTGTAGATCAGTC
1835
268 63 SYN GCTGCTTTGGCTTTGGCTGTTATAAGCG CAGCTCTCCACTATCTTGCCGAAGCGTT
89 2 WT ACTACCCTGTCCATGAAGCTCATTTTCG TGGCGCCACCATTAGTATTTCTGCCTCT
202 47 SYN AGAGCCTTGACCGTGCAATTCGTTTTCA CGGTGCTACTATCTCTATCAGCGCTAGC
90 YCR024C 1 WT GTACGAGGATGCAGTTGGCGACGTATTG ATCTAATGCTACCTGGCAAAGCACCCCT
1478 352 49 SYN ATAGCTGCTAGCGGTAGGGCTGGTGTTA CAGCAACGCCACTTGGCAATCTACTCCA
91 YCR026C 1 WT GCTGCTGCTGCTTCTTGATGATGGTGAT CCTTTTGTGTGATATTTGCGGTGTGGCA 2228 370 65
53
SYN AGAAGAAGAAGATCGGCTGCTAGGGCTG TTTGCTATGCGACATCTGTGGCGTTGCT
92 2 WT GAGTGAAGGATGGAAACCGTCTAGTGAA CGCGAAAGATAGTAGGGATGGTTCTAAC
355 54 SYN CAAGCTTGGGTGAAAGCCATCCAAGCTG TGCTAAGGACTCTCGAGACGGCAGCAAT
93 YCR027C 1 WT AGCTAGTTTCTCTCCTTCAGCTTTCGTG ATCGTTGACGGGCGTACGAGGCATAATA
629 211 41 SYN GGCCAACTTTTCACCTTCGGCCTTGGTA GAGCCTAACCGGTGTTAGAGGTATCATC
94
YCR028C
1 WT TATGACGACCGAGACAATTCCGCTTGAT CTTTTCTGCACAGTATCTTGGAGGCGTA
1538
253 52 SYN GATAACAACGCTAACGATACCAGAGCTG TTTCAGCGCTCAATACTTGGGTGGTGTT
95 2 WT AGATGCGAAACCCAAGTTCTCACCTCCC TAGCGGTTTGGTGGGATCTATGTTCAGT
373 49 SYN GCTAGCAAAGCCTAGATTTTCGCCACCC CTCTGGCCTAGTTGGTAGCATGTTTTCT
96
YCR030C
1 WT GGGTGGAGAACTTTGTTGTGGCGTTGAA TCTTGCCTCTGCATCTTCCAGTCTCACT
2612
460 55 SYN TGGAGGGCTAGATTGTTGAGGGGTGCTT ATTGGCTAGCGCTAGCAGCTCTTTGACC
97 2 WT AGAGGAGTGCTGAAACAACGATTGACCA TGCCTCTCCCAGTATTTCACTTCCTACT
331 56 SYN GCTGCTATGTTGGAATAGGCTTTGGCCG AGCTAGCCCATCTATCAGCTTGCCAACC
98 3 WT TGATTGTGGCTGAAGTGGAGGTTGAGAA GTCGTCCCACACCCTAAGATCTAAAGTG
448 52 SYN GCTTTGAGGTTGCAAAGGTGGTTGGCTG AAGCAGCCATACTTTGCGAAGCAAGGTT
99
YCR032W
1 WT AGCGGGAAGGTCATTCAACGATTTGACC AGAGCTCCAGGCCCTTTCAAGCGAACTA
6503
361 56 SYN GGCTGGTCGAAGCTTTAATGACCTAACT GCTAGACCAAGCTCGTTCCAAGCTAGAG
100 2 WT TCCAAATTCGTCCTGCAAGTGGTCATCG CAAATCTCCAACTGGCTCCGACTCAAGA
232 54 SYN CCCTAACAGCAGCTGTAAATGGAGCAGC TAGGTCACCGACAGGTTCGCTTTCCAAG
101 3 WT GCATTCGGTTTCAGGTAACCTTGAGAAC GCTCCCTGCATGTTGAGATATTGCGACC
211 50 SYN ACACAGCGTCAGCGGCAATTTGGAAAAT AGAACCAGCGTGTTGGCTGATAGCAACT
102 4 WT TGTGTCCATTGATTCTATACGGCTTGCG GCCTGACGCCAGAGAACTTTGATCCTCA
358 50 SYN CGTTAGCATCGACAGCATCCGATTGGCT ACCGCTAGCCAAGCTAGATTGGTCTTCG
103 5 WT TGATGGGATGAAACGGAGGCTTTTGCCT AACCGTCATATCCCGTGATTCTTGCGAT
403 41 SYN CGACGGTATGAAGCGACGATTGCTACCA GACGGTCATGTCTCGGCTTTCTTGGCTA
104 6 WT TACCAAACGACCCTTTCTACTTCGGGAT GCTTGCCTTTCCTATTCCATTTCTTGCC
229 43 SYN CACTAAGAGACCATTCTTGTTGCGAGAC AGAAGCTTTACCGATACCGTTTCGAGCA
105 7 WT TTACCGGGACCTATCGAAACCTATGGGC TAAACGATCTGCAGGGCCAAAACTTCCG
232 41 SYN CTATCGAGATTTGAGCAAGCCAATGGGT CAATCGGTCAGCTGGACCGAAAGAACCA
106 YCR033W 1 WT GAGCTCAGTCAGTAGGCCTTCGTCATCA TGATAATGCCGAGGGAAAGTGGGTAACG
3680 247 65 SYN ATCTAGCGTTTCTCGACCAAGCAGCAGC GCTCAAAGCGCTTGGGAAATGAGTGACA
54
107 2 WT GTCTGTAACTCCCAGTACTGCATCTGTA ATCCATTGATCGAGGTCTCCGAGAACTC
400 52 SYN AAGCGTTACCCCATCTACCGCTAGCGTT GTCCATGCTTCTTGGTCGTCGGCTAGAA
108 3 WT CCCAGATGTGGAAAGATCCTCTTCAACC CGTTCCAATGGATCTCCATTTGTTGCTC
352 47 SYN TCCTGACGTTGAACGAAGCAGCAGCACT GGTACCGATGCTTCGCCACTTATTAGAT
109 4 WT ACTTATACATCAGCAGGCTACCGGACTT CGAGGATGGATGCCGTAAGCTTTTTTCC
274 47 SYN GTTGATCCACCAACAAGCCACTGGTTTG GCTGCTAGGGTGTCGCAAAGACTTTTCT
110 YCR034W 1 WT ACCATTAAGCACTTTGCCCCCTGTGCTA GACCCAAGAAATAGATGTGGTGCCCATC
1043 418 47 SYN GCCTTTGTCTACCCTACCACCAGTTTTG AACCCAGCTGATGCTGGTAGTACCCATT
111 YCR035C 1 WT TGAAGGAGCAGCGAGGATGGAGATC GAGTCGCACTGGTCCAGTCTTCGATTTG
1184 403 48 SYN GCTTGGGGCGGCCAAAATGCTAATG ATCTCGAACCGGCCCTGTTTTTGACCTA
112
YCR037C
1 WT GCTACTCACAGATGAGCTGCGTGATTCA CAACATAGCTGCAGGAGAACCATCTTCA
2771
253 59 SYN AGAAGAAACGCTGCTAGATCGGCTTTCG TAATATCGCCGCTGGTGAACCTAGCAGC
113 2 WT ACCGCATGAGGCTAACAAGGCGCAA GGCAGTTTCATCTTCAGGCTTGTTGGTA
247 56 SYN GCCACAGCTAGCCAATAGAGCACAG AGCTGTCAGCAGCAGCGGTCTACTAGTT
114 3 WT TACGCAGACAAACGCTAACAACGAAGCA GGGCACTTTTGTCTCACATACTGTGTCA
268 48 SYN AACACAAACGAAAGCCAATAGGCTGGCT TGGTACCTTCGTTAGCCACACCGTTAGC
115
YCR038C
1 WT TGGATGTACGACGACTTTTAGGCGCTGA CGCTCTAAACGTATCTCCATGGTCACTT
1928
361 47 SYN AGGGTGAACAACAACCTTCAATCGTTGG TGCCTTGAATGTTAGCCCTTGGAGCTTG
116 2 WT GGAAATGGTGTGCGTTTGCTGCAAAGTT CGGTGCAACTGACCTCAGTGATTTACTT
268 43 SYN GCTGATAGTATGGGTTTGTTGTAGGGTG TGGCGCTACCGATTTGTCTGACTTGTTG
117
YCR042C
1 WT ATGGATCCTTATAGTCATCGAGCCCGTG GAGCACTTTTACCGGCAGTTCTAGGCCA
4223
487 50 SYN GTGAATTCGGATGGTCATGCTACCGGTA TTCTACCTTCACTGGTTCTAGCCGACCT
118 2 WT GTCCCCATCTTGTCTCAACTGAGAAGAA CGATGGTACTCCGTATGAGCATATTGTG
376 43 SYN ATCACCGTCTTGTCGTAGTTGGCTGCTG TGACGGCACCCCATACGAACACATCGTT
119 3 WT ACACACAATCACATCCCTCAAACTCGGA CAGTTCTGCATCATATTGGGTCCCATGT
391 48 SYN GCAAACGATAACGTCTCGTAGAGATGGG TTCTAGCGCTAGCTACTGGGTTCCTTGC
120 4 WT ATACGGGACGGATGACAAATGCACCATT ACCAAGGGAGAGAAGACTTGTGGCGAAG
331 45 SYN GTATGGAACGCTGCTTAGGTGAACCATA CCCTCGAGAACGACGATTGGTTGCTAAA
121 YCR044C 1 WT TGTGTACGACCAGTCAACATAGAGTCTC AAGCTCGGTCTTTCACTGTCGTGATTTG
1073 211 49 SYN GGTATAGCTCCAATCGACGTACAATCGT TTCTAGCGTTTTCCATTGCCGAGACCTA
122 YCR045C 1 WT CCTCTTCAATCGAATCTCACCAAGAGGT AGGCGTGGAAATTGAGTCGCTATCTCAT 1475 424 48
55
SYN TCGTTTTAGTCTGATTTCGCCCAATGGC TGGTGTTGAAATCGAAAGCTTGAGCCAC
123 YCR046C 1 WT GATCAGCGGCGAAAACAATGGCACTCTA CCTCTCAGAAATAGAGTCTCTGGATCCC
509 256 48 SYN AATCAATGGGCTGAATAGAGGAACTCGG TTTGAGCGAAATCGAAAGCTTGGACCCA
124 YCR047C 1 WT CCTGTGTCTTCTTTTCCTCCCGGTGAAC GGGCTCGTTTGACGCGGCTATTAGTATC
827 499 45 SYN TCGATGTCGTCGCTTTCGACCAGTAAAT TGGTAGCTTCGATGCTGCCATCTCTATT
125
YCR048W
1 WT AGGTCCGGTGCTGGAGAAACAGCTCAAA CGACGCATAGTAATCTGTGCAGCACCTG
1832
217 43 SYN GGGCCCAGTTTTGGAAAAGCAATTGAAG GCTAGCGTAATAGTCGGTACAACATCGA
126 2 WT GTCGCGCATCAGATGGAGGTATGTGTTG CGCCACGCAATTCAATAAAGCGTCCCAT
262 41 SYN CAGCCGAATTCGATGGCGATACGTTCTA AGCAACACAGTTTAGCAAGGCATCCCAG
127 YCR051W 1 WT CTCGTTCGTCGAGAACGGTGAAGATGGT TCTCTTGGAATCTGGTTCGTCCCCGGAA
668 340 39 SYN TAGCTTTGTTGAAAATGGCGAAGACGGC TCGTTTGCTGTCAGGTTCATCACCGCTG
128 YCR052W 1 WT AAGAAGGAGGCTGGATACGTCCATCAAT AAGGCTCAATGACGAATCGCGTTTCCTC
1451 310 57 SYN CCGACGACGATTGGACACCAGCATTAAC CAAAGATAGGCTGCTGTCTCGCTTTCGT
129
YCR053W
1 WT TTTCCGTTCTGATGAAGTCACCCCCTTG AGTACCGGTAACAGACAAAGTCTGGACG
1544
370 45 SYN CTTTCGAAGCGACGAAGTTACTCCACTA GGTGCCAGTGACGCTTAGGGTTTGAACA
130 2 WT CACTGGTTCTGCAGCCATCTACGGTTTA AACAGCACCGACGTTGTGTTTAGAGTTG
229 43 SYN TACCGGCAGCGCTGCTATTTATGGCTTG GACGGCGCCAACATTATGCTTGCTATTA
131 YCR054C 1 WT GTCTTTCTGGGGTGTATCATTATCCCTG GCCAGCGTCGTTAAATGAGAATAGCTCA
1691 427 48 SYN ATCCTTTTGTGGGGTGTCGTTGTCTCGA ACCTGCTAGCTTGAACGAAAACTCTAGC
132
YCR057C
1 WT CGATGATGAGAACATGACCTGACCCCTT TGGTTCCAGCAAACTGGGCCAATTACTA
2771
262 52 SYN GCTGCTGCTAAACATAACTTGGCCTCGC CGGCAGCTCTAAGTTGGGTCAATTGTTG
133 2 WT TATCGACCTCATAGCCGTCGAGAAGAGA TGTGGACGTCACCCCCCATTCTACTGTA
319 49 SYN GATGCTTCGCATGGCGGTGCTAAACAAG CGTTGATGTTACTCCACACAGCACCGTT
134 3 WT CACTACACGTGAACCATCCGGAGAGTAT GCTTTTAGCTGTCGGATTTACTAGTGGG
265 47 SYN AACAACTCGGCTGCCGTCTGGGCTATAA ATTGTTGGCCGTTGGTTTCACCTCTGGT
135 YCR059C 1 WT ATATGTTGCCGCAGAGCCATCCTGC CTCTGTTTTCCACCGCGGATCTGTCTGT
776 316 47 SYN GTAGGTAGCAGCGCTACCGTCTTGT TAGCGTCTTTCATCGAGGTAGCGTTTGC
136
YCR061W
1 WT TGGCACCACTTCTGTGCTACTTCTCGTT TGAAGAGAGCGAGAACAACGTGTCTCTG
1895
214 59 SYN CGGTACTACCAGCGTTTTGTTGTTGGTC GCTGCTCAAGCTAAATAGGGTATCTCGA
137 2 WT CCTTCCTATACTGCAATCGCCCTCGCTT CGATGCTAATGAGACGGGGACAGAAAGC
388 58 SYN TTTGCCAATCTTGCAAAGCCCAAGCTTG GCTAGCCAAGCTAACTGGAACGCTCAAA
56
138
YCR065W
1 WT CTCACCGGATCACTCGTCCCCA ACCCTTACCATCGCAGGACTTTTCAGTC
1694
355 48 SYN GAGCCCAGACCATAGCAGCCCT GCCTTTGCCGTCACAGCTTTTTTCGGTT
139 2 WT TTCTCTACTCGACCCACTTCCGTATTCC GCTTATCAACCGCGATGGGGTTTTCTCT
205 56 SYN CAGCTTGTTGGATCCTTTGCCATACAGC AGAGATTAGTCGGCTAGGAGTCTTTTCC
140 YCR066W 1 WT ACCACTGAGTTCCAAACCATCCAAAAGG TGAGGTCTCACCACATGAGCTTTTGTCG
1463 295 54 SYN GCCTTTGTCTAGCAAGCCTAGCAAGCGA GCTAGTTTCGCCGCAGCTAGACTTATCA
141
YCR067C
1 WT AATAGAGTCCTGGGATGCTGATGTTGAC TTCGCTATCACACATGCCGTCTTCATCT
3197
490 66 SYN GATGCTATCTTGGCTAGCGCTGGTGCTT AAGCTTGAGCCATATGCCAAGCAGCAGC
142 2 WT TGTGCTTGACGCGCTCTCCACGGATAAA GGAGACGGCAACTAGCTCATTCTCAAAA
388 59 SYN GGTAGAGCTAGCAGATTCAACGCTCAAG TGAAACCGCTACCTCTAGCTTTAGCAAG
143 3 WT CGTGGATGCCGGTTTTGAGTTGTCTAGA ACCATCGCTTCCACTGAATTCAGAGTCG
367 56 SYN GGTGCTAGCTGGCTTGCTATTATCCAAG GCCTAGCTTGCCTTTGAACAGCGAAAGC
144
YCR068W
1 WT ATGCAACGGAGCTAGTTCAAGTTGCTCA CCTCGATGAGGATTCCCAGTCTCTGCTT
1562
259 57 SYN CTGTAATGGTGCCTCTAGCTCTTGTAGC TCGGCTGCTGCTTTCCCAATCTCGAGAA
145 2 WT CCCTGGAGAGCTACTACCTTCAAAAAGA ATTGCGGCCTACACAGGTAGATGAAGAG
469 52 SYN CCCAGGTGAATTGTTGCCAAGCAAGCGA GTTTCGACCAACGCAAGTGCTGCTGCTA
146 YCR069W 1 WT TCCCGTTTCGAAGTCGATGAAAGAGGCG GAATTCCGAGGTGTTAGAATCAGGTCCA
956 340 46 SYN CCCAGTCAGCAAAAGCATGAAGGAAGCT AAATTCGCTAGTATTGCTGTCTGGACCG
147
YCR072C
1 WT ACATGACACCAGTAGTCGGCAGTCCGAT TTTGAAGTCGCATGCGCACTGGGTTAAT
1547
442 49 SYN GCAGCTAACCAACAATCTACAATCGCTG CCTAAAAAGCCACGCTCATTGGGTCAAC
148 2 WT TGAGACAATATACCTACCATCAGGGCTG CCAAGGTCTATTGTATAGTGGCTCTCAC
394 52 SYN GCTAACGATGTATCGGCCGTCTGGAGAA TCAAGGCTTGCTATACTCTGGTAGCCAT
149
YCR073C
1 WT CACGCCAGATTGGTGCAAATATGCCAAA CCTACTATCGCTAGCATCCTCATTATCG
3995
406 59 SYN AACACCGCTTTGATGTAGGTAAGCTAGG TTTGTTGAGCTTGGCTAGCAGCTTGAGC
150 2 WT TAGTACAACTAGTGCACCCAATGAGCTC CATTCTAGTATCGAAAGGGGAGGATTCC
208 56 SYN CAAAACGACCAAAGCGCCTAGGCTAGAT TATCTTGGTTAGCAAGGGTGAAGACAGC
151 3 WT ATAGACGGGGAACGCCAGTAGTGATCTT CCGTTTCTCACCGATAAGTAGTTCGGAT
268 52 SYN GTAAACTGGAAAAGCCAACAAGCTTCGC TCGATTTAGCCCAATCTCTTCTAGCGAC
152 4 WT CAAACACCTTTCCAAGAAGGCTCTTCCC TTCAGGCTCGGCAGTTAAGGGAAAACTT
211 49 SYN TAGGCATCGTTCTAGAAAAGCTCGACCA CAGCGGTAGCGCTGTCAAAGGTAAGTTG
153 YCR073W-A 1 WT AGAGGCCTCTTGCAAAAGCACAGCATCT GAAGAGTGATGCGATATGACCATCGGGG 947 466 50
57
SYN AGAAGCTAGCTGTAAGTCTACCGCTAGC AAACAAGCTAGCAATGTGGCCGTCTGGA
154 YCR075C 1 WT TGACGCCAGATCATGGCCTCGAGAT CGCATCACTGGCCATTTTTTCACTGCTA
782 397 51 SYN GCTAGCCAAGTCGTGACCTCTGCTT GGCTAGCTTGGCTATCTTCAGCTTGTTG
155 YCR076C 1 WT GCTACTTGGATTCCCCAGAGGATTGGGA GGGTTCAACATCGTTTCTCTCTGAGCTA
752 445 55 SYN AGAAGAAGGGTTACCCAATGGGTTTGGC AGGCAGCACCAGCTTCTTGAGCGAATTG
156
YCR077C
1 WT AGAGGAGGACCCTGAAGAGGCC TAGGGCGTATTTGGAACATTCTGGACAC
2390
337 60 SYN GCTGCTGCTACCGCTGCTAGCT CCGAGCTTACCTAGAACACAGCGGTCAT
157 2 WT ACCCGACGCAGGCTGCATTGGA TTTTGGAAATCCTCACAGCAGCGGCAGC
400 49 SYN GCCGCTAGCTGGTTGCATAGGG CTTCGGTAACCCACATTCTTCTGGTTCT
158 YCR079W 1 WT CTCGTGGATCATATCGCACTCTGGATTG CGACCTTTCAGATCGCGGCAAATGCAAG
1328 328 57 SYN TAGCTGGATTATCAGCCATAGCGGTCTA GCTTCGTTCGCTTCTTGGTAGGTGTAGA
159
YCR081W
1 WT GCATGAATTGAGTTCGTCTCACACTTCG TAGCAGGTGCATGGAAATGGCATCATCT
4283
226 50 SYN ACACGAACTATCTAGCAGCCATACCAGC CAACAAATGCATGCTGATAGCGTCGTCA
160 2 WT TTTTTCGGCCCAAAAGAGGGTGGTATCA GCTCAGAGGAGTTTTCGACAGTTGCAAA
364 50 SYN CTTCAGCGCTCAAAAACGAGTTGTTAGC AGACAATGGGGTCTTGCTCAATTGTAGG
161 3 WT GACATTACACGGGCCTGGTTTTCAGTTG CCCAGCAGCATTTGACCTCTTCTTTTTG
268 43 SYN CACCTTGCATGGTCCAGGCTTCCAACTA ACCGGCGGCGTTGCTTCGTTTTTTCTTA
162 4 WT CCAACGCATCATCGCTGATTTATCAGCT AACGAAAGAGGACACTTGGAATGGTGGT
250 41 SYN TCAACGAATTATTGCCGACTTGAGCGCC GACAAAGCTGCTAACTTGAAAAGGAGGC
163
YCR084C
1 WT GGATGATGGGGAAGACGAAGTGTTCAGG TTCTCTGGTGGCCCGTCTATCTGACGAT
2141
202 59 SYN GCTGCTAGGGCTGCTGCTGGTATTCAAA CAGCTTGGTTGCTCGATTGAGCGATGAC
164 2 WT GGAATCTAGATCCAAAAGGAAAGGTGGG TGGTTCGCCATCGGCCTTCCCA
478 53 SYN GCTGTCCAAGTCTAGCAAAAATGGAGGA CGGCAGCCCTAGCGCTTTTCCT
165
YCR088W
1 WT CGCCCCAAGCTTACCTTCTAGAAACTCG ATTGCTGGGGAAGAGACCTTTTGAGCCG
1778
343 56 SYN TGCTCCTTCTTTGCCAAGCCGAAATAGC GTTAGATGGAAACAAGCCCTTGCTACCA
166 2 WT TTCTGCGCCATTGAAGACAAGGGCC CTTAGAAACTGGAGCAGGGCTCTTGGAA
259 53 SYN CAGCGCTCCTCTAAAAACCCGAGCT TTTGCTGACAGGGGCTGGAGATTTGCTT
167
YCR089W
1 WT GTCAAGTTCCTCTTTAGCCTCCACCAAA AGAAGAAGGTAAAGAGTACCCCGAAGAG
4829
265 66 SYN CAGCTCTAGCAGCTTGGCTAGCACTAAG GCTGCTTGGCAAGCTATAACCGCTGCTA
168 2 WT CTCTGTCCCGTTATTACCATCATCTAGC AATAGACGACGAGACTGGCGATAACGAC
208 68 SYN TAGCGTTCCATTGTTGCCTAGCAGCTCT GATGCTGCTGCTAACAGGGCTCAAGCTT
58
169 3 WT CTCTACCGTTACATTTGGATCGACCAGC CGTAGAGCTAGAGGTTGATAAAAGGGGG
439 63 SYN TAGCACTGTCACCTTCGGTAGCACTTCT GGTGCTAGAGCTAGTGCTCAACAATGGA
170 4 WT CATAGCGTCTTCTCTACCATTGTCCTCT CGAGTAACCATCACAGCTGCTAGATGTG
364 66 SYN TATCGCTAGCAGCTTGCCTCTAAGCAGC GCTATAGCCGTCGCAAGAAGAGCTGGTA
171 5 WT TTCATCCTCTTATTCGTATGTGCAGCCC AGAAAGGGTGGACGAGGCTTTCTCCGAT
226 63 SYN CAGCAGCAGCTACAGCTACGTTCAACCA GCTCAAAGTGCTGCTAGCCTTTTCGCTG
172
YCR091W
1 WT TTCGTTGTCCGCAAGCCCAAGCTCA CGGAGAAGAGCTACCTGAGATCTCATTA
2162
355 70 SYN CAGCCTAAGCGCTTCTCCTTCTAGC TGGGCTGCTAGAGCCGCTAATTTCGTTT
173 2 WT CGAGGCAAAAAGGCTTGGTTCCAAATCA ACTTCTATGTGAGCTGTTATGCCTTGGC
388 56 SYN TGAAGCTAAGCGATTGGGCAGCAAGAGC AGATCGGTGGCTAGAATTGTGTCGAGGT
174
YCR092C
1 WT GTTGCATGATGTGGCTGCTAACGACAAA TCAAAGGGAAAGCGAATCAGTACGGTCA
3143
379 52 SYN ATTACAGCTGGTAGCAGCCAAGCTTAGG CCAACGAGAATCTGAAAGCGTTCGAAGC
175 2 WT CTTAGAAACTAAGCGTATGAGAGACGGC GAAGCATGATCCAGGTGCCAGCAAATCA
469 52 SYN TTTGCTGACCAATCGGATCAAGCTTGGG AAAACACGACCCTGGCGCTTCTAAGAGC
176 3 WT CGGGAAAGAACAGTACGCAAACTGCCTA TCCACTCAAAGGCAGTGTTTCTTCCAAG
427 50 SYN TGGAAAGCTGCAATAAGCGAATTGTCGG CCCTTTGAAGGGTTCTGTCAGCAGCAAA
177
YCR093W
1 WT TCTTTCTTCTTCACCTGCAGAGGAGCTT CGACTGGGATGGCTGATTCAAACGGCTT
6326
484 61 SYN CTTGAGCAGCAGCCCAGCTGAAGAATTG GCTTTGGCTAGGTTGGTTTAGTCGAGAC
178 2 WT CATTATAGAGTCACTTGACCGCTCCTCT AGACCCAGGTGAAAGTATTTCGGACAGT
208 56 SYN TATCATCGAAAGCTTGGATCGAAGCAGC GCTACCTGGGCTCAAGATTTCGCTCAAC
179 3 WT CATCGCTATTGATGCTCTAGGATCGCTA CAGCTCCTGCCTAGTGCTGGAATCAGAT
259 54 SYN TATTGCCATCGACGCCTTGGGTAGCTTG CAATTCTTGTCGGGTAGAGCTGTCGCTG
180 4 WT TAGCCTTTTTGCCACTAGTGAGAGTCCT ATCTTCCGAAGAGGCCAAGTGTTCTAAG
349 50 SYN CTCTTTGTTCGCTACCTCTGAATCTCCA GTCTTCGCTGCTAGCTAGATGTTCCAAA
181 5 WT AAGGTTAAGAGATAGCGACTTGCCAAGG CAAAGCCGGAACATCTCTCAAGAGGTCC
325 48 SYN ACGATTGCGAGACTCTGATCTACCTCGA TAGGGCTGGGACGTCTCGTAGCAAATCT
182 6 WT ATTTGGCGGCGAGGGTTCAATTTCACAC TGCAGCCGTCTTCAACTTAGACTCATCA
241 48 SYN TTTCGGTGGTGAAGGCAGCATCAGCCAT AGCGGCGGTTTTTAGTTTGCTTTCGTCG
183 YCR094W 1 WT ACTGTCTCCTCAAAGTGTGCTTCCGTTG GGAATTAGCGATCAACCCACAGGGATAT
1175 472 47 SYN CTTGAGCCCACAATCTGTTTTGCCACTA GCTGTTGGCAATTAGACCGCATGGGTAG
184 YCR095C 1 WT CGAATGCGTCCTATTATTCTCAAGGGGC GGCAGTTCCGGTAAAAAGCTCTTCTGTA 1088 481 54
59
SYN GCTGTGGGTTCGGTTGTTTTCCAATGGT TGCTGTCCCAGTTAAGTCTAGCAGCGTT
185
YCR098C
1 WT ACCACCGAAGGCTAGTGGCAAATTTGTC GTCGACTAGAGTTTCCAACGCAGCCCTA
1556
334 47 SYN GCCGCCAAAAGCCAAAGGTAGGTTGGTA TAGCACCCGAGTCAGCAATGCTGCTTTG
186 2 WT CTTACCAGTCCAGCCATTGGATACCAAA TATTAGTAGTGAAGCGTCAGCAACCGCT
274 43 SYN TTTGCCGGTCCAACCGTTGCTAACTAGG CATCTCTTCTGAAGCTAGCGCTACTGCC
The wild-type (WT) and synthetic (SYN) PCRTags that did not yield any product under the conditions that were employed for the PCR reaction, are shaded.
60
Table S6: SynIII restriction sites removed
ORF RestrictionEnzyme
Wildtype Synthetic Name Start Stop
YCL069W 5062 5064 AlwNI CAGAAGCTGCTCAGAAGCCGCT
YCL061C 10803 10805 AlwNI CAGAGTCTGATCAGAGTCACTT
YCL057W 15720 15722 AlwNI CAGATTCTGTGCGGATTCTGTG
YCL054W 22544 22546 MmeI TTATAATAAATTGATTTGGGTTTTCCTTATAATAAATTGATTTGGGTTTTTC
YCL054W 23486 23488 BlpI GCTAAGCAAGCTAAACAA
YCL054W 24245 24247 Bpu10I GCTAAGGCAGCTAAAGCA
YCL051W 27038 27040 MmeI AAAATGAATATTATGAGCAAAAGCTTAAAATGAATATTATGAGCAAAAGCTG
YCL051W 27173 27175 MmeI CAAAACGCCAATGCACGGAATTCCAACAAAACGCCAATGCACGGAATTCTAA
YCL050C 29038 29040 MmeI AAAGACCATGTGTCTCTTGTCGGATTAAAGACCATGTGTCTCTTATCGGATT
YCL049C 30613 30615 MmeI GACACTGAACCTCATTTTCCAACATTGACACTGAACCTCATTTTCTAACATT
YCL048W 33283 33285 MmeI TAACCTCTTTGGTTTCTGTTGAAATTTAACCTCTTTGGTTTCTGTTGAAATC
YCL048W 33535 33537 Bpu10I GCTAAGGAGGCTAAAGAG
YCL048W 33559 33561 BpuEI CACTTGAGCATTTGAG
YCL048W 33934 33936 MmeI TAAGAGTTCATCAAACGTGTTGGATTTAAGAGTTCATCAAACGTATTGGATT
YCL047C 34543 34545 BpuEI CTTGAGGACCTC
YCL047C 34876 34878 MmeI CACATCATTTCCAGACGAGTTGGAAACACATCATTTCCAGACGAGTTGGGAA
YCL047C 34891 34893 Bpu10I GCTTAGGCAGCTTGGGCA
YCL045C 36726 36728 Bpu10I ATCCTTAGCATCTTTAGC
YCL045C 37077 37079 BpuEI CTTGAGGCTTGGGG
YCL044C 37943 37945 BlpI GCTTAGCGAGCTTAGACT
YCL044C 38582 38584 MmeI TCCAACAGTGACTGTCTAACAGTGACTG
YCL044C 38723 38725 MmeI GTGGAGAAGCGGTTCAAAGTTGGAAAGTGGAGAAGCGGTTCAAAGTTGGGAA
61
YCL043C 39428 39430 Bpu10I TTCCTCAGCTTCTTCAGC
YCL043C 39650 39652 MmeI TAGTTTAGCAATCAAAACGTCGGATGTAGTTTAGCAATCAAAACATCGGATG
YCL043C 39908 39910 MmeI TAGCCTTAGACTCCAACACGATCTTGTAGCCTTAGACTCCAATACGATCTTG
YCL040W 42087 42089 MmeI ACTCCGGGACATTGATCCGTTGGACCACTCCGGGACATTGATCAGGTGGACC
YCL040W 42123 42125 MmeI TTCCGCATCGCGGACACCGTCGGAAATTCCGCATCGCGGACACCGTCGGGAA
YCL040W 42165 42167 Bpu10I GCTCAGGGTGCTCAAGGT
YCL040W 42213 42215 MmeI GCATTAACCAACGACACCGTCGGAACGCATTAACCAACGACACCGTCGGGAC
YCL040W 42441 42443 BpuEI CTCAAGCTGAAG
YCL040W 42750 42752 MmeI TTACAGAGTCTCAGACTGCCCACCACTTACAGAGTCTCAGACTGCCCACCAC
YCL039W 44054 44056 BpuEI ACCTTGAGACTTTGAG
YCL039W 44822 44824 MmeI CGTGCGGGCTGGATGGTGGTTGGATCCGTGCGGGCTGGATGGTGGTTGGGTC
YCL038C 45767 45769 MmeI TTCACTTTCAGGTAAAACTTGACTGATTCACTTTCAGGTAAAACTTGACTGA
YCL038C 45923 45925 MmeI ATGCGTTTTATCGGTAAGCAGTCCAAATGCGTTTTATCGGTAAGCAGCCCAA
YCL036W 50309 50311 BglI GCCAAACTGGCGCGCCAAACTTGCGC
YCL034W 53041 53043 BglI GCCGTAGCGGCGGGGCGTAGCGGCGG
YCL030C 58304 58306 BlpI GCTTAGCATGTTTAGCAT
YCL028W 61758 61760 BglI GCCTCCATGGCGCATCCATGGC
YCL028W 61947 61949 BglI GCCTCCATGGCTCGCATCCATGGCTC
YCL027W 63010 63012 BlpI GCTCAGCCAGCTCAACCA
YCL026C-B 64637 64639 BglI GCCTCCAAGGCAAGCCTCCAATGCAA
YCL024W 73283 73285 BsmBI AAATGGAGACGGTAAATGGGGACGGT
YCL017C 74786 74788 BsmBI TCACAGAGACGAGTCACAGAGACCAG
YCL014W 77565 77567 BsmBI CTTTACTCTGAAACTTTACTCTGAAA
YCL011C 84516 84518 BsmBI GCCTCGGTCGCGTGCCTCGGTCCCTT
YCL010C 84972 84974 BsmBI CTTCCGTCTCTTT
62
CTTCTGTCTCTTT
YCL005W 89993 89995 BlpI GCTAAGCTTGCTAAGTTA
YCL004W 90642 90644 BsmBI ATATTCATTGCGTATATTCATTGCAT
YCL004W 91503 91505 BsmBI CGTCTCATCAAATCGTCAGTTCAAAT
YCR030C 151701 151703 AarI GTGGTAAAGCAGGTGCGGTGGTAAAGCAGGCGCG
YCR030C 151956 151958 AarI AAGTTTGCGCAGGTGGTAAGTTTGCGCGGGTGGT
YCR045C 185380 185382 AarI CTGCAGACGCAGGTGAGCTGCAGACGCAGGACTG
YCR046C 187019 187021 AarI CACCTGCTACCTGC
YCR047C 187674 187676 AarI ACCGTCCAAATTCACCTACCGTCCAAATTTACCT
YCR048W 190046 190048 AarI TGGTTCCACCTGCTCTTTGGTTCCACCTTCTCTT
YCR057C 198374 198376 AarI GTCAATGTTGCCCACCTGTCAATGTTGCCTACCT
YCR057C 199931 199933 SgrAI CACCGGTGAGCACCGGACTG
Unique restriction sites were generated in synIII either by introduction of synonymous mutations in ORFs, or by removal of redundant sites by the same method to leave only one pre-existing instance of a given site in the synIII sequence.
63
Table S7: SynIII restriction site “landmarks” added
ORF Restriction Enzyme Name Start Stop Pre-existing/ introduced (P/I) Sequence
(WT/Syn) 9247 9255 AlwNI P 19537 19543 BlpI P 26359 26364 MmeI P 30017 30022 BpuEI P 39138 39144 Bpu10I P
YCL038C 45935 45943 RsrII I GGGTCCCAG CGGTCCGAG
52293 52303 BglI P YCL030C 58619 58624 BsaI P YCL025C 68581 68595 SfiI I TAGTCCAGCTGGACC
TAGGCCAGCTGGGCCYCL016C 76594 76600 SanDI P
88857 88862 BsmBI P 97388 97393 SacII P
YCR008W 109229 109234 AvrII P YCR017C 126643 126655 SfiI I AGCAGATTTTGCTTG
GGCCGATTTGGCCTG 136238 136246 DraIII P 147109 147115 AarI P
YCR030C 152457 152469 SfiI I GGCACCAGTGGCAGAGGCCCCAGTGGCCGA
YCR033W 163089 163094 EciI P YCR037C 172389 172396 SgrAI P YCR042C 180399 180405 BstEII P
190789 190795 AarI P YCR059C 201009 201021 SfiI P YCR067C 211291 211296 BspMI P YCR072C 218743 218749 SanDI P
231384 231390 PasI P YCR088W 243974 243985 AscI I AGACGAGCAACT
CGGCGCGCCACT
64
251576 251587 PpiI P YCR093W 259335 259349 SfiI I GAAGCTATTTTAGCC
GAGGCCATTTTGGCC 265584 265590 PasI P
65
Table S8: PCRTags for non-essential segments analysis
Segments Primer Name Forward Primer Reverse Primer PCR Amplification
Region in synIII
1 Custom Primer 01 CAACAAACTGTAGAATGGAGATGCAG GACATCACAGAAGTGGCACAATTC 1431…1743
2 Custom Primer 02 GGAGGAAACTAAGTACTCTTCGCAG CTACCGACAATGAGCATATTCATTG 3678…4070
3 Custom Primer 03 TACAGACTTCAACACAATCAGAATC TACTAAGTACATTAATTAGTTAATTG 5502…5729
4 YCL063W1 AAGCAGCAGCGACACCTACGAAAGCTTT GTTGCTAGGTCGAAAAAAGCTAGCGCTA 8347…8626
5 YCL055W1 CGCTACCACCCCTTTCGGTTGCAAGATT AGGAACGGCGCCGTTGGTAGTATCTTTG 18746…19229
6 YCL050C1 GTCGCTGAAAGCTTTGCTTCGAGGGACA TGCTTCTGGCAGCAGCCTAGATCATAAG 28651…29005
7 YCL047C1 TCGCAATTGGCTTTCGGCAGACAAGTGA TGTTAGCGTTGGTCTAACCGTCTTTAGC 34561…34810
8 YCL039W2 CTCTAGCCCATTGCGAAGCGAATGGTTG GCTAGCGCTGTTGGTAACAGGGTGAAAA 43672…43918
9 YCL038C1 AGCGCTCAAACCGCCCAAGCTC CAGCACCGCTGTCCTATTTAGCAAAGCT 46037…46334
10 YCL037C1 ATTGGCGTTAGAGCTGCTGGTGCTGCTA TCTACCAACCAGCAGCCCTTGGAAGTTG 49155…49425
11 YCL034W1 TTCTGACGCCTTGGCTAGCGCC GCTGCTTTCAGAGTCGATGTATCGAGAA 53199…53535
12 YCL033C1 GCATCGAGCGCAGCAGATTTCAACTCGA CGAACGACCAAATACTGGCGCTTACTTG 53809…54022
13 YCL030C2 GTAGACACCTCGGCCTAGGTCAATAGCT AAGCCTACCTTCTGGCAAGTTTTCTGAC 58766…59159
14 YCL027W1 CGTTAGCATCAGCAACCCTCCAATGAGC GCTGCCTCGTAGAAACCATCTGCTCAAA 63123…63621
15 YCL026C-B1 GCTTTCTGGAATCTTAGAAGGTAGGGCG TCGAGACGAAGCTTTCGGCAGCGTTATT 64553…64784
16 YCL026C-A1 TAGGTTGGCACCTAGACCTAGCAATTCC CGCCGAAGCTAAAAAACGACCTGAAAGC 65940…66147
17 YCL025C2 GCCTAGTTCACCCAAGCTTCGACCTTGA TGCTGTCATCTTGTTGAGCGTTTTGAGC 67486…67816
18 Custom Primer 04 CAAAGGTGAATGCTTTTGGATCAAT CTCAAATTGCTCCTCTGGGTTGTGC 82665…83040
19 YCL011C1 AACGCTGCCAAAACCTCGGCTGAAACCA CGCTATCAGCAAATTCGACGGCGCTTTG 83871…84135
20 YCL010C1 AGCCAAATTAGCTAGGGCGGTAGGGCTT TAGCGAAGTCGCTTACAAACCACGACGA 84918…85305
21 YCL009C1 AGCGCTAATTCGGGTTGGTTTAGCGCTT TATGGCTCGAATTAGCTTGCTAGGCACC 86150…86435
22 Custom Primer 05 CTGTAGCAAAAACGACAGCGAAG CTGGCACATTTGTTAAAGTGGCG 94238…94541
23 Custom Primer 06 GAGGACTCGTTGACGTAGAATTA GATAGAACGAGTACAACACCCGATC 96071…96449
24 YCR004C1 TAGTTCCAAAGGGCTAGCGGTTCGGCTA CGCTGGTATCTTTGTCTCTACCAGCTCT 101171…101396
25 YCR005C1 ACCTGGAGCAACTTCATAGATGCTGCTG TAGCGCTTTGAGCAGCCCATACTTGAGC 102630…102951
26 Custom Primer 07 GTGTGGGTTTATTAAAATAGTTAG GTAACAGTGAAGAGCAGCATCTATATG 105829…106166
27 YCR008W1 AAGCAGCCGACAAGGTAAAGCTAGCAGC GCTATGGCTAGGGCTAGCGCTTTGATAG 109111…109324
28 Custom Primer 08 GGCATTCGTATCTTATTCCTCGCTTC ACTATCTGCTCTATCTGACCAACCG 110946…111236
66
29 YCR014C1 GATATCGCCGCATTTGCTGTAACCTCGG TGGTATCGGTAGCGAAATCGCCAAGCGA 120506…120776
30 YCR016W1 GAGCAGCACTAAGAAGGGTAAACGAGTT GACGCCGCTTTCGCTAGGTTCATCCTTA 124155…124395
31 YCR017C1 CAATTCACCGACAGGGCTTGGTAGCAAG TTTGTTGAGCTTGACCGCCCGATTTGTT 125659…126040
32 YCR018C1 CTTACCGTAAGCCAAACCGCATGGAGAG TACCTCTCGAAGCGCTTCTATCACCAAA 128705…128906
33 YCR019W1 ATTGTGCAGCGGTAGCCGATGTTTGGAT GCTGACAGCGAAACCACCCAAAAAGCTA 131330…131783
34 Custom Primer 09 CCTGGGGCTCTAGGAGTATCACG GTTTGTACATCTATTTATCACAAG 132317…132680
35 Custom Primer 10 GCTACAAAATATTGAATGTGAATC GTCTAATCGATAGTAGGCCATTTC 133780…134088
36 YCR021C1 AAAACCACCGGTTAGGCTCAATCGAGGT CAGCGCTCTAGACATGGTTCACAGCTTG 134483…134891
37 Custom Primer 11 GATAATGGTAGCAATAATGGCAATAC AGCCTTCCGTGCTTGCCAGTATAT 141288…141686
38 YCR026C1 AGAAGAAGAAGATCGGCTGCTAGGGCTG TTTGCTATGCGACATCTGTGGCGTTGCT 142572…142941
39 YCR027C1 GGCCAACTTTTCACCTTCGGCCTTGGTA GAGCCTAACCGGTGTTAGAGGTATCATC 145907…146117
40 Custom Primer 12 GAACTGCGTCAGGTCATCTCGTCCTC CCTGAGCAGGTGACTTAGGTGGAGGAGC 146800…147120
41 YCR030C2 GCTGCTATGTTGGAATAGGCTTTGGCCG AGCTAGCCCATCTATCAGCTTGCCAACC 151458…151788
42 YCR032W4 CGTTAGCATCGACAGCATCCGATTGGCT ACCGCTAGCCAAGCTAGATTGGTCTTCG 157487…157844
43 YCR033W2 AAGCGTTACCCCATCTACCGCTAGCGTT GTCCATGCTTCTTGGTCGTCGGCTAGAA 163248…163647
44 YCR034W1 GCCTTTGTCTACCCTACCACCAGTTTTG AACCCAGCTGATGCTGGTAGTACCCATT 167406…167823
45 YCR059C1 GTAGGTAGCAGCGCTACCGTCTTGT TAGCGTCTTTCATCGAGGTAGCGTTTGC 200475…200790
46 YCR061W2 TTTGCCAATCTTGCAAAGCCCAAGCTTG GCTAGCCAAGCTAACTGGAACGCTCAAA 202623…203010
47 YCR066W1 GCCTTTGTCTAGCAAGCCTAGCAAGCGA GCTAGTTTCGCCGCAGCTAGACTTATCA 208837…209131
48 YCR067C2 GGTAGAGCTAGCAGATTCAACGCTCAAG TGAAACCGCTACCTCTAGCTTTAGCAAG 210492…210879
49 YCR069W1 CCCAGTCAGCAAAAGCATGAAGGAAGCT AAATTCGCTAGTATTGCTGTCTGGACCG 216191…216530
50 YCR073C4 TAGGCATCGTTCTAGAAAAGCTCGACCA CAGCGGTAGCGCTGTCAAAGGTAAGTTG 219780…219990
51 YCR075C1 GCTAGCCAAGTCGTGACCTCTGCTT GGCTAGCTTGGCTATCTTCAGCTTGTTG 225184…225580
52 Custom Primer 13 CGTGTACCAGTCTATTTAAACTGG GAATGTCGTCGTTGCTAATCC 236096…236412
53 YCR088W2 CAGCGCTCCTCTAAAAACCCGAGCT TTTGCTGACAGGGGCTGGAGATTTGCTT 242674…242932
54 YCR089W5 CAGCAGCAGCTACAGCTACGTTCAACCA GCTCAAAGTGCTGCTAGCCTTTTCGCTG 244981…245206
55 YCR091W1 CAGCCTAAGCGCTTCTCCTTCTAGC TGGGCTGCTAGAGCCGCTAATTTCGTTT 251967…252321
56 YCR095C1 GCTGTGGGTTCGGTTGTTTTCCAATGGT TGCTGTCCCAGTTAAGTCTAGCAGCGTT 266075…266555
57 Custom Primer 14 CTTCTAAACACAGATATTATGAAAATG GTTTCACAATTCTAACATAGGATGG 268941…269249
58 YCR098C2 TTTGCCGGTCCAACCGTTGCTAACTAGG CATCTCTTCTGAAGCTAGCGCTACTGCC 269931…270204
67
Table S9: Replication origins in native chromosome III (from SGD) and synIII.
ARS Name Location on Chromosome III Modification on synIII ARS300 838 − 1551 Deleted ARS301 11146 − 11401 ARS302 14575 − 14849 Deleted ARS303 14871 − 15213 Deleted ARS320 15214 − 16274 Partially deleted ARS304 30200 − 30657 ARS305 39159 − 39706 ARS306 74458 − 74677 ARS307 108780 − 109295 ARS308 114321 − 114939 Insertion of loxPsym site ARS309 131985 − 132328 ARS310 166503 − 167348 ARS313 194265 − 194513 Modification (PCRTag) ARS314 197378 − 197609 ARS315 224816 − 225061 ARS316 272852 − 273095 ARS317 292388 − 292921 Deleted ARS318 294404 − 295034 Deleted ARS319 315354 − 316239 Deleted
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