Supplementary Information

For the submission: A tool for multiple targeted genome deletions that is precise, scar-free, and suitable for automation, Aubrey et al.

• Supplementary information relating to the initial 11 ORFs
• Supplementary information relating to the 96 ORFs

• 

  Figure S1. This gel demonstrates PCR1 products for the initial 11 ORFs in lanes 1-11 when a 10:1 ratio of primers (forward:reverse) was employed. Other lanes on this gel show PCR2 results, and are discussed later. Although amplification in PCR1 was good when equimolar amounts of the forward and reverse primers were used, the amplification product was sometimes weak or not visible when a 10:1 ratio of primers was employed. Alternative primer ratios might have proven effective, but this was not explored further. For example, when a 10:1 ratio of primers (forward:reverse) was employed in PCR1, only eight of the initial 11 ORFS amplified well. This can be seen in the lanes numbered 1-11. Lanes 2, 3 and 9 show poor amplification.

• 

  Figure S2. This gel shows the contrasting situation where equimolar amounts of primers are used for PCR1. The expected product was still obtained in PCR3 but sometimes with further amplification of the PCR1 and PCR2 products. Occasionally, even when PCR1 was successful at a 10:1 primer ratio, the PCR3 amplification produced further amplification of PCR1 and PCR2 products, presumably caused by failure of the SOEing reaction during the first 10 cycles of PCR3.

• 

  Figure S3. When testing the primers selected on the basis of these initial criteria, PCR2 failed for the ORF YGR125W and electrophoretic analysis indicated primer dimer formation that had not been predicted. This gel shows the problem for YGR125W and demonstrates that the reverse primer is at fault. The reverse primer is used in lanes 26, 27, 29 and all show far more dimer than the neighbouring lanes that did not have the reverse primer.

• 

  Figure S4. Replacement PCR2 primers were designed for YGR125W and YGL202W using the new improved method, and they gave the expected PCR2 product without apparent dimer production.

• 

  Figures S5a and S5b. In addition to products of the expected size (approx 1.25 kb), PCR2 gave an additional product approximately double the expected size (approx 2.5 kb). This additional product was seen on several gels, including the two given here.

• 

  Figure S6. Tests on PCR2 for YMR272C to vary the extension time showed that the larger product was still evident despite reduced extension time.

• 

  Figure S7. Tests on PCR2 for YMR272C excluding the primers (including just the forward primer (F), including just the reverse primer (R), including both primers (F+R), including no primers (NP) showed that both primers were needed to produce any product.

• ORF ID	PCR1 product size	PCR2 product size	PCR3 product size
  YMR272C	774	1266	1890
  YDR093W	680	1281	1796
  YGL202W	736	1457	1852
  YHR163W	592	1266	1708
  YPR073C	667	1266	1783
  YGR125W	595	1281	1711
  YNL169C	976	1266	2092
  YJL134W	567	1281	1683
  YPL262W	855	1281	1970
  YDR074W	981	1266	2097
  YMR205C	707	1266	1823

  Table S1. Table of expected sizes of the products produced for the 11 ORFS.

• ORF ID	PCR1 forward (primer 1)	PCR1 reverse (primer 2)	PCR2 forward (primer 3)	PCR2 reverse (primer 4)
  YMR272C	GCTTACATTTGCAGACGTCAAAAAACCTGACAAACC	CTTCGTACTAGTTTTAGCTTACCACCGCTTTTAGTG	GTGGTAAGCTAAAACTAGTACGAAGTTATTGGTCAGCCTCGTCCAAAATTGTCAACCTAGGAAAACACACATTACTTGCCTC	TTATTCATATTTCATTTTGGACAATGGGGCATCGGGGCCTAAGTAGATCCCAATACAACAGAT
  YDR093W	GTGATCATTTCTGTATAAGTTTTCTCAACTGAATATATAAATATATATCATTACTATGCT	ATATGTGTTGAGTTTATGCTATGATTCTTTTTAAAGAATTCTATTATTAAAAGGATTAGG	AATTCTTTAAAAAGAATCATAGCATAAACTCAACACATATTGATTACAATTTTTTTCTTCCTTTGTACTATGTATACCAACACACATTACTTGCCTC	TTAACGGTCCCTGCTGCGCTGGCTGAGCAGCGTCTCTGCATGATTGATCCCAATACAACAGAT
  YGL202W	AAGACATTAACATCATTTTTCGTGCTGTTAGGGTGGTG	GATAGTAACGATCGGTTGTCCTATTCAATGTCTGTATC	AATAGGACAACCGATCGTTACTATCAGAGGTAAATACGTTGGAAGAATATATATAAGGTGAAAAACACACATTACTTGCCTC	CTATTTGGAAATACCAAATTCTTCGTATAAAGTATCACCTAATCTGATCCCAATACAACAGAT
  YHR163W	CAAGACGGTGCATTTCTGGGCTCCTACTTTGAAATGGGGTCTGG	TTGTCGCGGACCAGGGGTAAAGCAATTGATGGTGCATTGCCTTC	TTGCTTTACCCCTGGTCCGCGACAAAAAAGACACACATGCGAGCTTTCGAACCTCAGATGCTAATCACACATTACTTGCCTC	CTAAAAAGTTTTCGTTTGAACTTTTCCAAAAGCTTCCTCATTAACGATCCCAATACAACAGAT
  YPR073C	TTGTTTCTCTACCAGATTTCTTCACTTTTGACAAACAACCCCTAGAATACAGACTATC	TTTCGTGTTAACTTTCCCTTCTCAGTTTTCTATCGCTTATCAAAAATCACAGGGTTTC	CTGAGAAGGGAAAGTTAACACGAAAGCTAGCAAGTAATGATGGTGCGGTCTACATCGAGTAAAAACACACATTACTTGCCTC	TTATAACTCTTTTTTTAAAAACTGTTTGGAGAAATAGGTGATTTGGATCCCAATACAACAGAT
  YGR125W	AATCTATATAGATATATGTATATAATAAAAGTTTTCAATCTGCTGACC	ATTTCTAAATTAAATTTTTGAGATTTTAAACTGTCTTGCTTATAGACC	AGCAAGACAGTTTAAAATCTCAAAAATTTAATTTAGAAATGAGCGTACACGTATATTTATTTAATTTCTTTTATTGCCGGCACACATTACTTGCCTC	TCATGCGCTAACAAGTGTGTAACCTAGTAATTCTTTGAATCTGGTGATCCCAATACAACAGAT
  YNL169C	ACCACTAGACCTGCAAACTCGACTTTCAATGGTTTAAATGAAG	GCTGGCTTTGCTTTTCCTTCTTCTTCAAAAAATAACGACCAAG	AAGAAGAAGGAAAAGCAAAGCCAGCAAGCAATCATATGTAAAGTTAGCATTTATTTTGCTGTATACACACATTACTTGCCTC	TCATTTTAAATCATTCTTTCCAATTATGCCTAATTTCTGTCCCATGATCCCAATACAACAGAT
  YJL134W	CACATTGTCGTCAAAAGGTAATCTCCCCAGAAGTTTTCACTC	TTTTTATCTATACAAATGGCTTGTGTCCGCTGGTCTGGTAGG	TACCAGACCAGCGGACACAAGCCATTTGTATAGATAAAAAAACAAAAGGAACAAAAAAAAAAAAATAGACAAAATGTAGTCACACATTACTTGCCTC	TTATGCTATATTTAAGAGGGAAAATAGGACGGGGCTGCACATTACGATCCCAATACAACAGAT
  YPL262W	TTATCCTTCTTATTCTTCTTTGCCATTAATCACTTCTAC	GATTGTATCTCTTATCCGTTTAATAGTTAGACTTTATGG	TCCATAAAGTCTAACTATTAAACGGATAAGAGATACAATCACGAGCTAAATACCTAATAATATACAAGTTTTTTATGTCTCACACATTACTTGCCTC	TTATTTAGGACCTAGCATGTGTTCAGGAACAACCCATTCATCAAAGATCCCAATACAACAGAT
  YDR074W	CTATCTTCTCTTGGCAAGAGTGTTCGAGATATGGTAG	TTCGGCACAGAAATAGTGACAGGCAGTGTTATTTTGG	TGCCTGTCACTATTTCTGTGCCGAAAACACCCTTTTTAACGAAATGGTTATGACTAGACAGACATCACACATTACTTGCCTC	TCAAACCTTTGCGCCGGTGTAAGAAGCCGATCTTTTCATAACATAGATCCCAATACAACAGAT
  YMR205C	CCATCTCCACGCTAAAAGGAAAACGAAGATTAAGATAAAGTTGGG	TGCGTATGGTTAGTTCTTGGCCAATGCTAAACTAGTCTCTAAATC	ATTGGCCAAGAACTAACCATACGCAAAGAAAATGACCTTTTATTACACTTTCTATTATTAATGTCCACACATTACTTGCCTC	TTAATCAACTCTCTTTCTTCCAACCAAATGGTCAGCAATGAGTCTGATCCCAATACAACAGAT

  Table S2. Table of primer sequences for the 11 ORFs

• ORF ID	PCR2 forward (primer 3)
  YGL202W	AATAGGACAACCGATCGTTACTATCAGAGGTAAATACGTTGGAAGAATATATATAAGGTGAAAAAACACTCCTCAGAAGCTC
  YGR125W	TCATGCGCTAACAAGTGTGTAACCTAGTAATTCTTTGAATCTGGTCTAGAGATCCCAATACAAC

  Table S3. Further forward primers were then chosen for PCR2 for YGL202W and YGR125W

• ORF ID	CPS A to CPS D with gene	CPS A to CPS D gene deleted	CPS A to RinklURA3	CPS D to FinklURA3
  YMR272C	1801	646	709	1198
  YDR093W	5460	621	659	1223
  YGL202W	2240	737	744	1254
  YHR163W	1410	660	769	1152
  YPR073C	1041	555	651	1165
  YGR125W	3725	614	651	1224
  YNL169C	2051	548	650	1159
  YJL134W	1785	555	660	1156
  YPL262W	2167	700	766	1195
  YDR074W	3393	702	667	1296
  YMR205C	3196	316	552	1025

  Table S4. Expected product sizes using the confirmation primers. The four resultant strains were verified using confirmation primers from the EuroFAN project.

• 

  Figure S8. Confirmation gel for strains YHR163W, YPR073C, YGR125W, YJL134W after selection on FOA, with expected product sizes shown at bottom of lanes.

• 

  Figure S9. Confirmation gel for strains YGR125W and YNL169C after selection on FOA, with expected product sizes shown at top of lanes.

• ORF	Raw sequencing data files
  YHR163W	4F, 4R
  YPR073C	5_1F, 5_1R, 5_2F, 5_2R
  YNL169C	7_2F, 7_2R, 7_4F, 7_4R
  YJL134W	8F, 8R

  Table S5. Sequence results for the four strains with confirmed deletions.

• ORF	Alignment files
  YHR163W	YHR163Wdeletant.txt
  YPR073C	YPR073Cdeletant.txt
  YNL169C	YNL169Cdeletant.txt
  YJL134W	YJL134Wdeletant.txt

  Table S6. Alignments of sequencing results with expected sequence after the deletion.

• Well	Strain	Reason chosen	PCR1 notes	PCR2 notes	PCR3 notes	Colonies on URA-
  A1	YAL023C	32 good				273
  A2	YAL056W	32 good				168
  A3	YBL069W	32 good		weak		30
  A4	YBR052C	32 good				200
  A5	YCL063W	32 good		weak		9
  A6	YCR095C	32 good	wrong size		weak	84
  A7	YDL174C	32 good				100+
  A8	YDL191W	32 good				22
  A9	YDR222W	32 good				100+
  A10	YER122C	32 good			no visible product, some dimer	12
  A11	YER158C	32 good	wrong size		no visible product, lots of the PCR1 wrong size product	0
  A12	YFL001W	32 good				18
  B1	YGL254W	32 good		weak		20
  B2	YGR163W	32 good		weak		140
  B3	YGR241C	32 good				13
  B4	YIR013C	32 good				170
  B5	YJL065C	32 good				120
  B6	YJR153W	32 good				210
  B7	YKL132C	32 good				172
  B8	YKL191W	32 good				132
  B9	YLR065C	32 good		weak?		72
  B10	YLR079W	32 good			no visible product, some PCR1	2
  B11	YLR091W	32 good	no visible product	no visible product	no visible product, some PCR1	0
  B12	YML097C	32 good	no visible product, some dimer		no visible product, some dimer	24
  C1	YMR035W	32 good				240
  C2	YMR103C	32 good				106
  C3	YNL129W	32 good				200+
  C4	YNR057C	32 good			no visible product, made PCR1	4
  C5	YOR076C	32 good		weak		72
  C6	YOR106W	32 good				68
  C7	YPL202C	32 good				208
  C8	YPR054W	32 good			no visible product, produced a small product	13
  C9	YAL058W	GC content, 3prime 0,1, overall 37.29	no visible product			44
  C10	YAR028W	GC content, 3prime 2,3, overall 35.42			no visible product, some small product	12
  C11	YBL033C	GC content, 3prime 4,4, overall 40.00		weak	no visible product, some PCR2	21
  C12	YBL055C	GC content, 3prime 2,4, overall 43.14	weak, dimer		no visible product, some PCR2	52
  D1	YBL091C	GC content, 3prime 0,2, overall 35.71				432
  D2	YBR077C	GC content, 3prime 5,5, overall 38.24		weak	no visible product, dimer	12
  D3	YCL050C	GC content, 3prime 3,4, overall 21.43	no visible product			500+
  D4	YDR071C	GC content, 3prime 3,5, overall 24.49	no visible product		no visible product	1
  D5	YDR330W	GC content, 3prime 3,3, overall 36.73		weak		105
  D6	YER115C	GC content, 3prime 2,6, overall 33.33		no visible product	no visible product	0
  D7	YHL044W	GC content, 3prime 2,2, overall 42.59				440
  D8	YHR207C	GC content, 3prime 7,5, overall 39.02	no visible product		weak	29
  D9	YIL079C	GC content, 3prime 3,3, overall 44.00	weak plus dimer	weak	weak?, produced PCR2	50
  D10	YIL111W	GC content, 3prime 4,4, overall 37.84			no visible product, some PCR2, some small product	112
  D11	YJL058C	GC content, 3prime 4,5, overall 37.25				154
  D12	YJL128C	GC content, 3prime 1,4, overall 30.00	no visible product		weak product, some PCR2	32
  E1	YKL023W	GC content, 3prime 2,5, overall 57.14				32
  E2	YKL166C	GC content, 3prime 2,2, overall 43.75	weak		no visible product, made PCR1	9
  E3	YKR039W	GC content, 3prime 4,5, overall 21.95	weak			114
  E4	YLR218C	GC content, 3prime 6,6, overall 48.39		weak		153
  E5	YLR255C	GC content, 3prime 2,4, overall 60.00			weak	81
  E6	YLR350W	GC content, 3prime 7,5, overall 33.33				239
  E7	YML048W	GC content, 3prime 4,6, overall 53.85				213
  E8	YML088W	GC content, 3prime 3,4, overall 45.10	no visible product		no visible product, lots PCR2	249
  E9	YMR018W	GC content, 3prime 5,5, overall 38.64	no visible product, lots dimer		weak, lots PCR1 and some small product about 600	44
  E10	YMR278W	GC content, 3prime 1,2, overall 39.22	no visible product, dimer			53
  E11	YNR058W	GC content, 3prime 6,6, overall 44.44				332
  E12	YOL158C	GC content, 3prime 4,5, overall 40.00	no visible product		no visible product, some PCR2	12
  F1	YOR008C	GC content, 3prime 2,5, overall 27.27				42
  F2	YPR037C	GC content, 3prime 3,4, overall 51.35		weak		201
  F3	YPR040W	GC content, 3prime 2,4, overall 47.73				152
  F4	YPR051W	GC content, 3prime 1,2, overall 45.24				36
  F5	YBR273C	self dimer tests				492
  F6	YAL030W	self dimer tests				736
  F7	YAL065C	self dimer tests	wrong size?			512
  F8	YAR002W	self dimer tests	no visible product		no visible product, some PCR2	9
  F9	YBL049W	self dimer tests			weak, some small product	69
  F10	YBR144C	self dimer tests	two weak bands		no visible product, lots PCR2	320
  F11	YBR185C	self dimer tests		weak		135
  F12	YBR255W	self dimer tests				113
  G1	YCL044C	self dimer tests		ok, but dimer present	no visible product, dimer	29
  G2	YCL047C	self dimer tests				294
  G3	YBR268W	self dimer tests				445
  G4	YAL002W	dimer tests				77
  G5	YBL082C	dimer tests			no visible product	160
  G6	YCR045C	dimer tests				500+
  G7	YDL106C	dimer tests				500+
  G8	YIL148W	dimer tests	no visible product		no visible product, lots PCR2	42
  G9	YJR073C	dimer tests	no visible product, lots dimer		no visible product, some PCR2, some small product	100
  G10	YLR011W	dimer tests		weak		14
  G11	YCL044C	dimer tests		weak		101
  G12	YDL022W	dimer tests	weak			4
  H1	YDL175C	dimer tests			no visible product	121
  H2	YGL211W	dimer tests		weak	no visible product, made PCR1	72
  H3	YAL009W	pcr2 dimer tests				259
  H4	YBR185C	pcr2 dimer tests				824
  H5	YBR270C	pcr2 dimer tests	weak		no visible product	117
  H6	YCR025C	pcr2 dimer tests				1004
  H7	YDL012C	pcr2 dimer tests			weak	72
  H8	YDR375C	pcr2 dimer tests			no visible product, also lots small product	70
  H9	YLR035C	pcr2 dimer tests		weak?		67
  H10	YLR412W	pcr2 dimer tests			multiple product	500+
  H11	YML089C	pcr2 dimer tests				140
  H12	YPL033C	pcr2 dimer tests	no visible product, dimer		weak	18

  Table S7. Table of properties of the PCR results for the 96 ORFs.

• Choices of ORFs were determined by the following: 32 ORFS that were predicted to have good primers under every condition 32 ORFS to test the new dimerism scoring method. These should include a good range of predicted outcomes, including primers that were predicted to fail because of dimer. These 32 ORFs shouls have primers that are good in all other respects 32 ORFS to test the effect GC content (16 to test the effect of overall average GC content, and 16 to test the effect of GC content in the 8 bases at the 3' end) The 32 ORFS chosen to have good primers: have primers are non-essential as given by the MIPS viability catalog or by the collection notes that came with our Eurofan collection of single gene deletants. have primers with: no unwanted non-specific binding and low hairpin, dimer and self dimer scores (<= 10) between 40% to 50% GC content overall at least 37% GC content in 8 bases at 3' end for both primers (at least 3/8 at 3' end) have confirmation primers and these make products of less than 3000 bases The 32 ORFS chosen to test dimerism scoring: have primers are non-essential as given by the MIPS viability catalog or by the collection notes that came with our Eurofan collection of single gene deletants. have primers with: between 40% to 50% GC content overall at least 37% GC content in 8 bases at 3' end for both primers (at least 3/8 at 3' end) have confirmation primers and these make products of less than 3000 bases have no unwanted non-specific binding have a range of dimer and self dimer scores The 32 ORFS chosen to test effect of GC content: have primers are non-essential as given by the MIPS viability catalog or by the collection notes that came with our Eurofan collection of single gene deletants. have primers with: no unwanted non-specific binding and low hairpin, dimer and self dimer scores (<= 10) have confirmation primers and these make products of less than 3000 bases demonstrate a range of GC content from lowest to highest with half (16 ORFs) to test overall GC content range and half (16 ORFs) to test 3' end GC content The resulting choices, showing the overall GC percentage, the number of GCs in the last 8 bases and the result of an 8 base sliding window average across the length of the primer are given in GCExptsByOverall.txt and GCExptsByEnd.txt.

  Algorithm S1. Summary of reasons/methods for choosing the 96 ORFS.

• ORF ID	Sequences of forward and reverse primers	Percent G+C	G+C at 3' end	Commenta
  YCL050C	CTTTTTTTTATTGTGTTGGTTTTTATATGTTTTGTTATGTATTGTTTATTTTCCCT TTTTATTTTAATTTTTACTTTTCTGTTTGTTCTAAAATCTATCTAAACTGGCTTTC	21.4 (12/56)	3/8 4/8	Required annealing at 55 ° C
  YKR039W	TTATTTATTTTTTTTTATACCATTTCCTTTTGATAAGGGGT TTTTTATTTCTTTTTTTTGTTTCTTATAAATGTTGCTGTCC	22 (9/41)	4/8 5/8	Weak product
  YDR071C	TTTGTTTTACAACCAAAAGTTTTAAAAAAGTGGATTCATATAAGGTTAG TTTAACTTTTTATTTAATCGTAATGTATTGGTGTTATTTTTTCTCCGAG	24.5 (12/49)	3/8 5/8	Required annealing at 55 ° C
  YOR008C	AAATAATGTGGAGATCTTATCTTGTTTTTCTCTTCTTTATGACA TATTTAAATAGAATTTTTTATCCTGAGTACTAAATCAGCCAACG	27.3 (12/44)	2/8 5/8
  YJL128C	GTAGTATAGTATTGCAACAGGAAAAAAAAATCTTATTGCGTAGCACTCTGTATCTATTTT CTTTGAGAAATTAACGTCTCGATCTGCTTAATATAATAATGTATCTATAGAAATGTACGG	28.8 (17/59)	1/8 4/8	Required annealing at 55 ° C
  YER115C	AATAGTAAGGAACAAACTGTTGATTTAACTGCAGATGCCATAGACTTA ATACAAGTTTGAACAAAATACAACTCAAAGTAAAATGGTAGCTGCACG	33.3 (16/48)	2/8 6/8
  YAR028W	GTTGTAAACCGCTAATGATTATGTTCCTAATACCTGTGTGTTTGGTTT GGTCAATTGATATCTTGCCTTAATATGGCACTTATAGATGTTATCCTG	35.4 (17/48)	2/8 3/8
  YJL058C	CGGAAGTGAATGAGAATACCAACAGTTTTACTGATGATTATATTGACGGAG AATGAAAGAACTTTATTTCTACAGAGGTCAATTGGCGTTTCTGTCACCTAG	37.3 (19/51)	5/8 4/8
  YOL158C	AGAATCATCTCTACTACACACTGCATTATGAACTGCAGCCACATTTATCTAAGGG TTTCAGAGAGACATTCACGAGCACTTTTTTGCTATATTAGAGTTCTGCTAGGACC	40 (22/55)	4/8 5/8	Required annealing at 55 ° C
  YBL055C	GAGAAAGTGAGTGGGAATGGACAAGATTATTTGGGTGAGGGTGTTTGCTTA ATAACTATAACAGTACTGGCGCCTCAAAAAAATGCCTTGGCAGCGAAAGTG	43.1 (22/51)	2/8 4/8
  YML088W	CTCTGAAGAGTTTAGCGGGCTCTCAGACGGTTTTTTCGCTTTTGGCATTTT GCTGTCTTATTCCCTAGTGTCGGCCACCTGATAAAGGTTTTTAACTCGTAC	45.1 (23/51)	3/8 4/8	Required annealing at 55 ° Cb
  YPR040W	GCCGTCCTGCTAGATGTCTGTACGGGATACTTCTCAATCAACTT TTTTTCTGGGGAGCTGTTGTGTCTAAAGCTGCCCTTAGGTCATG	47.7 (21/44)	2/8 4/8
  YPR037C	TGCCCTTATACCGCGTAGTGTAGGTCCAGTCGTACTT CCCTTCGTTCCTTGCACAATCTGTCCAGACTTTCCTC	51.4 (19/37)	3/8 4/8
  YML048W	GCTTTTGCCCTTGACCCTGAATGCGGCAGATCCAGCGACTTCATCGAGGTTA GCTTGCAATCGCCCTCTATCAAAACAAAGTCCACCGATCTCTGCTGCCCCTC	53.8 (28/52)	4/8 6/8
  YKL023W	TTCCCCATATTGGGAACCTGCTCTGCCCTGCTCAGCACGTAG GCTTGCACGTTATACCTGGCCGGGATTGGGGCGCTATTCTTG	57.1 (24/42)	5/8 2/8
  YLR255C	AGGGTGGAGAGTTAGAGCTGGTGTTGGCACCGGGCGTAGTTGCTG TCACCCGTCGGGCGAAATCCACCCTCGCGGCCTCGTTTAGAATAC	60 (27/45)	4/8 2/8

  Table S8. Results of investigation into overall GC content. ^a Where there is no comment, the expected amplicon was obtained using the standard annealing temperature of 58 ° C (See Methods). ^b Only 6 of the 21 bases at the 3' end of the forward primer are G or C. In general, reactions involving primers with an overall G+C content below 30% either gave a weak product or no visible product at all. However, this was not always the case. Furthermore, some primer pairs with higher G+C contents also failed to yield a visible product. In spite of the apparent failure of some of these reactions under standard conditions, all primer pairs chosen to test overall G+C content yielded the expected product when the reduced temperature touchdown PCR was employed (touchdown PCR with annealing from 60-55 ° C for 5 cycles and then 55 ° C for a further 25 cycles). We do not advocate the use of a lower annealing temperature for all ORFs since stringency is reduced. We also examined the efficacy of amplification in relation to the frequency of G+C residues at the 3' extremity of primers with an overall G+C content above the 30% threshold (Supplementary information Table S9). We expected reactions to fail if primers lacked a GC clamp and contained two or less G+C residues among the last 8 at the 3' end. In fact, some primers of this nature did generate amplicons under standard annealing conditions (58 ° C) whereas others did not.

• ORF ID	Sequences of forward and reverse primers	Percent G+C	G+C at 3' end	Commenta
  YLR350W	ACGATTATGTATATCCATATATATATATACACTACAAGCCGACCG GATGAAACTGTTTCTTATGCGTTTTTATAATGGTATAGTCTTGCG	33.3 (15/45)	7/8 5/8
  YHR207C	AAGGATAATAACAGAATTTAATGGCTTGATATGGACGGGCG TTTTGTGCCACTTTTCGTTATTTTGTATCAGTGCACGCTTG	39.0 (16/41)	7/8 5/8	Required annealing at 55 ° Cb
  YNR058W	TAGTACATTGTGCAACCTGTGCCATAATATTGACACCGAACTTCTTCAGGGCCT TAGTTGTTATCCTAGCAATAGAAAACGAATTGCGCTGTTGACATCCTCCCGACC	44.4 (24/54)	6/8 6/8
  YLR218C	GTAGTCTTTACGGTAATGGAGTTGGCCCTTG AAATGAAGCGAATGGATGAGAAGAGGGGAGG	48.4 (15/31)	6/8 6/8
  YBR077C	CGTAGGTGTTTGACATTGAATATTTATACCGGCT ATTGCTTCAATATTTTGGATACCGTTTGCTGCTG	38.2 (13/34)	5/8 5/8
  YMR018W	AATTATATTCTGCGCTTGCCTTTTATATTAAGCGTCACAACGCC CGTGCTTACAAAAGGTACCCTTTATTCTTAATGATTCTCCACTC	38.6 (17/44)	5/8 5/8	Required annealing at 55 ° C
  YBL033C	GGTTCAAAAAGTGGAAGTAGTAGTGCTCTT CCTGTAGGTTTGTTGTAGTTTTATGTACGC	35.4 (12/30)	4/8 4/8
  YIL111W	ATGATATTGTTGAAGGATATCGACTTGCTCTCAAAGG GCTCTTTACAATGAATATGTGGCGTTATTCCATTGTG	37.8 (14/37)	4/8 4/8
  YDR330W	CACGATGTAACCCAGTTATAGAACCTATTCTGTAGTTTGTTGCAGTTTT ATAAAAGGTATGTTATTATTGCCACACGTCGTAGTCTATGCGTTTGTAG	36.7 (18/49)	3/8 3/8
  YIL079C	CTTTCCTCGATGAAAGTTGGTTCAGGATCCAAGTACAAGGACTGTTGCTT AATATCTTAGCTCCGTGGTCCGCTTCCTTTGTATTCCTCATGTGAGAATG	44.0 (22/50)	3/8 3/8
  YHL044W	GCAGTACTACAACTCTGTGCGGTGGGAAAAATTTATTCTTCGAGGCACAGTATT AGCCTTAGCGGGCCCTCACTTGCTTTTGAATTTGTCTTTATACCACAAGTTAAC	42.6 (23/54)	2/8 2/8
  YKL166C	CCAGGTACGAGTGATTTAGGAAGGTCTGAAGAAAAGCGACTATGCATT TTTGTGCAGGCTCGCTCTTTCCTTGTACAACCACCGATATACAATATG	43.8 (21/48)	2/8 2/8
  YPR051W	CAACCAAGGTTCGTCGTCAGTAAGCTAGTTACGGAATGGTAA GTCCTGACCCAACGCCTTTTCTGTAGTAGTGGCTTTTTTATC	45.2 (19/42)	1/8 2/8
  YMR278W	TTTAATCACCAAGAACCCTAGCACTTATAAAGCGCGTCTAGCGAAAAAGTT TTTTTGAAATTGCAGCAGCTAATGGCTCGAGAGTACTGTTGAGTATTTAGG	39.2 (20/51)	1/8 2/8	Required annealing at 55 ° C
  YBL091C	TGCTTACTTTTCGTTTCCACTTTATCTTGCTGTTCGTTCGAGCAACAGTATTTATT TTTTCAATACGGTAGAGCTTCTACAGTACTTGTTGATGTAAAAGCCCACTTATTAG	35.7 (20/56)	0/8 2/8
  YAL058W	CAAGTACATTCAAAATACAAGAAAGGCTGGCTGGGATAGCACCAATGAGACTATAAAAA AGTACTACTTTGTGTGGCCGTTATCTATATTAGAAATGCGTTGCCGTTCACTTTTTTAC	37.3 (22/59)	0/8 1/8	Required annealing at 55 ° C

  Table S9. Results of investigation into G+C content of the 8 bases at the 3' end. ^a Where there is no comment, the expected amplicon was obtained using the standard annealing temperature of 58 ° C (See Methods). ^b This reaction gave a product of unexpected size. See also Table S8.

• ORF ID	PCR1 forward (primer 1)	PCR1 reverse (primer 2)	PCR2 forward (primer 3)	PCR2 reverse (primer 4)
  YAL023C	TTCCTGTATTTGCGTCTGCCACCTACGTGA	GATTGCTGGACCACGGTTCGAAACAGAATG	TGTTTCGAACCGTGGTCCAGCAATCATTAAGATCATCGTAGTAAGTACATAAGTAAAAAACAAGACACACATTACTTGCCTC	TCATGCTTCTTGCTTGTCGGCAATGTCCCAAGTGGAAAACCAGTTGATCCCAATACAACAGAT
  YAL056W	AGGCGCAAATTATTGGGAATAAGACTTGGCTG	GCCTATGATTTAGTAATGCGACAATGGACCTG	TTCATTGGCAGGTCCATTGTCGCATTACTAAATCATAGGCAAAGGCAGTTTTCAATGCTTTCACTTTGTAAACTTTGTTTCACACATTACTTGCCTC	TTATGCACTAGGATTTACGCTAGGAAAAATACTTTTCGTTATGGTGATCCCAATACAACAGAT
  YBL069W	CCTTGTCCCTGTGGAGATAGGTTTCAAATATATCTGGATGACATGTTTGAGGGCGA	TGTTCAACGTTTGCCTCTTGGTATGCAATGTCGTATTTGTAGCTTCCGGAATGCTC	CATACCAAGAGGCAAACGTTGAACAATACCCACTTACATTTACAATATGTTTTATTTTTAAACTTAGCGACAAGAAGAGATAG	CTAGAATTTTTCGACGTTCATGATTAACTTTCCTTGGGCACGTTGGATCCCAATACAACAGAT
  YBR052C	TGTTTGCTTCGTGTTGACCTATTAGCTCACAAGATAGAGCTTGTTTGGAA	TATCTATGTGGCGTAGTATGTGCTTATAATAGTGAGTAGTATGCTGGAGG	AAGCACATACTACGCCACATAGATAAACCGTATATATCTTTACATAAGTACGATATGGTATAACTCACACATTACTTGCCTC	TTACAAATCTTTGATACGGTCGTAAAAGGCCTTCCCTTGAAGTTGGATCCCAATACAACAGAT
  YCL063W	TAGCGAAAACCTGAGGAGATCTTTTACCGTCCTT	TAATCGATGTGTCCTTGTTTCCTTATGCGAGCTG	AAGAAACAGCTCGCATAAGGAAACAAGGACACATCGATTATTAATGCCTCCTTTACCTAACCTACTCTTCTTTTGCTCCAAGCGACAAGAAGAGATAG	TTAAAACAGCAGTTCTGTATTCAAAGCATCTTGCAATTCTTCTATGATCCCAATACAACAGAT
  YCR095C	ATCCTTTGTTCACTTTCTCTTTCTGCACCTACATCACCGTC	TATGAATTCAGGTTATGGATCGCACCAGTGCTGTAAGTGTC	GTGCGATCCATAACCTGAATTCATATTTACATTTTTACGTATATGTATAGATGATTTCATGATAACACACATTACTTGCCTC	CTATGTAAAAATATGTTCCCTATAAAAGTGATGCTCTTCCGGAACGATCCCAATACAACAGAT
  YDL174C	ATAGTTGATATTGTCGTTAGAGTCCAGTTGTTGGGCG	TGTTCTTGCTTTTGTTTCCAGCTGGATCGGAATGATG	CCAGCTGGAAACAAAAGCAAGAACAGTATCTGATTTTCCTTTTTCACCCTTCACGTAAACCTGAACACACATTACTTGCCTC	TCACCTGTAATCATTAGCGGGCTCGTTTGGATCAGTTTTAAAGATGATCCCAATACAACAGAT
  YDL191W	TACAACGCATTTTCATTTGTTTTACAGCACCCCTG	TTCGAATATACTGTCTCACTGTACCTATTCTACGG	GGTACAGTGAGACAGTATATTCGAAACAAAATAATAAAGACTGACAACTGCAGAACAGAGAGGATCACACATTACTTGCCTC	TCATCTCAAACATACCATTTCGAATATACTGTCTCACTGTACCTAGATCCCAATACAACAGAT
  YDR222W	CAAGACTTTCGAATACGAGCCCCCTAAGTTCAATTTAAGTGAATGACTGGAA	CACTAAATGAGTCTCTATGTATGTACGATGTATCTTATACGCCCTCACTGTC	GTATAAGATACATCGTACATACATAGAGACTCATTTAGTGACAAAAGCGTGAACACTTCCACATGAAATTCGTTTTTTGTCACACATTACTTGCCTC	TCAACTAATGAAGGTGCTCTCAACGATAGAAATACCTTTCTCGTGGATCCCAATACAACAGAT
  YER122C	TGCGCTTGACAAGAATGAAGAATGTTCTCTCCCTCAAA	TGTCGCTATGTGTATACTAAATTGCTCAAGAAGACCGG	ATCCGGTCTTCTTGAGCAATTTAGTATACACATAGCGACAGTTTAATTTTCTCTTTAATTGAATTTTTTTTTTTTCATTGCACACATTACTTGCCTC	TTATTTTCTTAAGTAGTCCCTTAAATAACTACCGAGTTTTTCAGCGATCCCAATACAACAGAT
  YER158C	ATGCGTAGAAGAAGGCCAAACACAACCGGAGGAA	GCTGATTAAGAGTGAGTGGAAGTAGTAGCGAGCG	CTACTTCCACTCACTCTTAATCAGCCGGGTTCATCAAGCTTTTTTCCTTCTTGAGACTCTGTTGTCACACATTACTTGCCTC	TTATAAGTAGTAATGTGTATACTTAGCGCTATCCTGGTGAACGCGGATCCCAATACAACAGAT
  YFL001W	TACGACACAGTAATTTAATTTCAAAGAGGAGGGCGCCTGG	TCAAGGGAGGTTATAGTAGGGCAGTAAAGATTGCATGTGG	ACTGCCCTACTATAACCTCCCTTGAAATAATATATAAACCTGTATAATATAACCTTGAAGACTATCACACATTACTTGCCTC	TTACTTATTTTTGTTGTTCTTTTTCTTGGAGTATTTAGCATTAACGATCCCAATACAACAGAT
  YGL254W	AGTGTTGGTGATGAGTTCTCCTCCCTATACCCT	TGTTTTGGTGGCCACGTATTCTGGTACCACTTG	ACCAGAATACGTGGCCACCAAAACAACGCCATAGAAGAGCAATTTCCGTCCTCTACTCATGTGATAGCGACAAGAAGAGATAG	TCAGTATTCGAATAAATCCCAGACGCTAGGCTTATATAAATGAATGATCCCAATACAACAGAT
  YGR163W	AACAGATTCATGAGGAAGAGGAGAGACAACGTCAATT	GTTGTATGTGTATTAGTACCGTTGTCCTGGAGTTTTG	TAACAAAACTCCAGGACAACGGTACTAATACACATACAACAAGACGTAAGGCATGAAAATATTAGGGTATATAGATACATACAATCATATGGGAGAAG	TCACACATCGTCCTCAATACTATTTTTAGCTTGACTGGCTCTTGCGATCCCAATACAACAGAT
  YGR241C	TTCTCCAATACTCCAATTGCAACGCACCATATTATCCGGGAGAATACACG	TGTTTCACAAACACGCCTCTATATCTATTATAGGGGTAGCCGAGACAGTC	GATATAGAGGCGTGTTTGTGAAACATGAGCGATAGATACTACAGTTTTTTGTTCTCAACAGGCCGAGCGACAAGAAGAGATAG	CTATATATCTATTAAGTTAGGATTTTCTTGCTGATGCTGCTGTTGGATCCCAATACAACAGAT
  YIR013C	CTGTATTAGCTTGTCATCTAAGCCTTCGTCATTGAAGTATCCC	AGCGACCCTGTAATGTTATGTTTCTAGCTAGGAACAGAAAGTG	AGAAACATAACATTACAGGGTCGCTCCCATTATACTTTTTCAGCTTTCAACCTATCGGGACAAAACACACATTACTTGCCTC	TCAGAATCTGACAGTTCCGGTAAGTTCTTTGGGGATCCTTCTCTTGATCCCAATACAACAGAT
  YJL065C	TGACAGTGGGTATTGCTAGGAAACTCTCTAGGGACAAGGCGCAT	TGTGTTAGCCTGATCACATCGAGCGAGCTATCCTGACGCTATTC	CGCTCGATGTGATCAGGCTAACACACTACACAAGCACATTGCATAGAAGGATTTTTCTTCTCTCTCACACATTACTTGCCTC	TTATTCTTGATCAGTAACGTCTTCGTCGTCGTCTTCGTCATTATCGATCCCAATACAACAGAT
  YJR153W	AGGGGTTAAAGGGCATTGAGTTTTTGTGAGTGGAAATGCCCCCGTTATA	TGCGTTTGTCCATCAATGTGGGTAGATAGTCTTCCCGTTTGAAGTTCTG	CTACCCACATTGATGGACAAACGCATCCTCTTTTAAAGTACTCATATGACTATACATACCTTCTTCACACATTACTTGCCTC	TTAACAGCTTGCACCAGATCCAGATGGGATTCCAGAACATCCAGAGATCCCAATACAACAGAT
  YKL132C	GAACGAGGACCTAACGCTAACAGCAGAGTCCATAT	CTGCTTCTTATGTAATCTGTTGCCGGCCAAACGAG	CGGCAACAGATTACATAAGAAGCAGTTTTTAGATATATGATCATATCCTACAATGCAATTACCGACACACATTACTTGCCTC	CTATACTGGCAAGTGACAGTTGTGTAAGCGCAATAGCTCTTTGCAGATCCCAATACAACAGAT
  YKL191W	ATATAATCGACCGTTTCACCAACACTTCTCAACTCATCAGCCACTTTGTC	TTTTCAACCTTTGCACGATCTTTCAACGCTGCCACTTAGAAATCCATCAC	TCTAAGTGGCAGCGTTGAAAGATCGTGCAAAGGTTGAAAACTCTTATAATTTGTTTCCCTCGACTTCTCTATTTAAATCCCACACATTACTTGCCTC	TCATTTGTTTTCCTTTTTCATAGCGTCTTCGCGATCAAATCCATAGATCCCAATACAACAGAT
  YLR065C	CCACGGTCAGTGTATAGTTGCCTACAGTTTCACCGTAAGTTAGCAAACCTACCCA	TCTACCAACACAAACTTAGACCACTGCCTTTGGTGTCTTCTTGAAGCCACACGTG	AGTGGTCTAAGTTTGTGTTGGTAGAACAAAGCTTTTCAAACGTTTTTGAAATTTTTTGCTTTCTACACACATTACTTGCCTC	TTAACGATACTTGTACTTAATTTTAGAGTCAGTTTCACCCCTCCTGATCCCAATACAACAGAT
  YLR079W	TCATGATGGTCTCTGATGTACTAAAGGGGTTATCGGAGTCCATAACATGCGAT	TTTCGTGTAATAGTCCCTGCTTCAAGGGTCAAAATTCCGTAGAGGTTTGGTTG	TTGAAGCAGGGACTATTACACGAAAAGAAATGAAAAAAAAAAGTTTCTGGAACGATTATATTTTAAGCGACAAGAAGAGATAG	TCAATGCTCTTGATCCCTAGATTGAAACAATGCCTTTGGCTTGAAGATCCCAATACAACAGAT
  YLR091W	AGCTTCGCAATGGGTGGTCTGTGAGAAATG	TGTTGCTCCAAGTTCGCAATCGCTGTTCTG	CAGCGATTGCGAACTTGGAGCAACATTGTTGTTGGCTGTTATACTGGAGTAAATGTCACGTGCCACACACATTACTTGCCTC	CTAATCAACTTCCCACCGGGTGCAATGAAACATCAAAGGCTCCGTGATCCCAATACAACAGAT
  YML097C	CCTGGGATGTATGGACTCTACCCTTTTTTTTACAGAATCACGTAGCACAGCAT	GGCTGTTGATTCCTGTTCTTTGTTGTTTGATGAAGAGAAACTCAAGCCCGTTG	CAACAAAGAACAGGAATCAACAGCCTGATCTCATGCACATATTTCATGTATAGAGTAGCCTAGCACACACATTACTTGCCTC	TTATTCTGACAGAGAAAGTAGAGCATCAACACAGGGCCCAATACGGATCCCAATACAACAGAT
  YMR035W	TAGCAGCCTTTACCGCGCAACAAAATCCATATACTATGGACGAG	TACTGTACAGTACAATGCAACAGCTTCATGAATAGCAGCACGCC	AGCTGTTGCATTGTACTGTACAGTAAAATCAGCTTATTTTTATGAATTTAGAAGTGACCCCGGTACACACATTACTTGCCTC	TCATTCTAAAATGGCTCTTTTGCTAATGCAGTCTCTACCGGTACTGATCCCAATACAACAGAT
  YMR103C	GATAATGACAAAACAGACACTTTCTGTGGTACTCC	TCTATTTTCTGTAGTTCTTCTTGTCCCCGTTAACC	ATTGTGGTTAACGGGGACAAGAAGAACTACAGAAAATAGATCCCGTAAAGGGAAAAAGCTAACCTCATTCGCCTCACAGGAGCGACAAGAAGAGATAG	CTAGGTAAGGCTCCCCATGGCTGAGCCCGCGCAAGGAAATCCAGGGATCCCAATACAACAGAT
  YNL129W	ACTTTCTCCAAAAGGGCCTCAATACATGGTT	ATTTTGAGGTTCTACTCTAGCTCACACTTCG	GTGAGCTAGAGTAGAACCTCAAAATGAAAGCGCCACAAAATCGATGAGAAGTATAAAAAAAAAAACACACATTACTTGCCTC	CTAATCCTTACAAAGCTTTAGAATCTCTTGGCACACCCAGCTTAAGATCCCAATACAACAGAT
  YNR057C	TTGAGATCGGTAACTTCTTCTGCATTCCAATGGTCCTCGAAACCGTT	GACCTGTGCGCTAGTGATTTTTTGAGTTCTTTTCACTGGCCTACTTG	TCAAAAAATCACTAGCGCACAGGTCTTTAAATAAAAAAGTTGCAAGGCTTTCATCAGCTATATATAGCGACAAGAAGAGATAG	CTATTCTACGTCTTGTTGGGTCATTAATGACTCCACCGATGGCAGGATCCCAATACAACAGAT
  YOR076C	TTCAAAAGCTCGGCGGCTATGACTTCTTGCTCCTCTTGTTGACTTCGCA	GTCAGGTGGTACTGTAAGTTTCGAAGACCACCTCCTCGTACGTGTATTG	TTCGAAACTTACAGTACCACCTGACTCCTACAACTAAGAAATTATACTAGGCATTCATACTTATTCACACATTACTTGCCTC	TTACTGGCATGCAATTCTGCCAACTGCGATAACCTTATGATCTATGATCCCAATACAACAGAT
  YOR106W	GTAGCAACAACGCTCATGTTGCTTCACAATCTCCAATTTTTGACATCAGCGATTA	AATCTCAACTTTCTGCAGTGGATATTAGTTGGCCAATTTGTTAATGCTGATCCCG	CAAATTGGCCAACTAATATCCACTGCAGAAAGTTGAGATTTAATCTCCTTAAACGCGCATTGAGCACAGACTTTCTGGTACACACATTACTTGCCTC	CTAACTTAATACAGCAAGCAATACCACCATGCACACAACTATTATGATCCCAATACAACAGAT
  YPL202C	CCCATAGAGAATTCAAATTCTTCTCCACCCGTCTC	TTCTTGGGGGTCGCTTTCTCTTGATTAATTTGCGC	ATCAAGAGAAAGCGACCCCCAAGAATTATTTAGTTTTCAACTCATTAAAACGGTATCACGTTTAACACACATTACTTGCCTC	TTAATATTTTGATATTAAGGCTGGGTCAATAGAATTTAATTCATTGATCCCAATACAACAGAT
  YPR054W	AGAGGAAAAGAAATTAAATCACACAGACAAAAACGCGGGGAGGAAG	TCTTGGAGCGTTGAGCACGTCTACATTTCTATCGATATGATGTATC	TGTAGACGTGCTCAACGCTCCAAGATAACCACTACACAAATAGGTATATCATGAAGTTATATTCTCACACATTACTTGCCTC	CTATAAAGACGAGGAGGACAAATCGGTTTTGAAATTTTTCACTTCGATCCCAATACAACAGAT
  YAL058W	CAAGTACATTCAAAATACAAGAAAGGCTGGCTGGGATAGCACCAATGAGACTATAAAAA	AGTACTACTTTGTGTGGCCGTTATCTATATTAGAAATGCGTTGCCGTTCACTTTTTTAC	GATAACGGCCACACAAAGTAGTACTTGGACAAGTGTTAGTTTATAACATGGTCTCAATAATTGCACACACATTACTTGCCTC	CTATGTAAATACTACACAACAAAGAACCGACGTCGTCAAGAGCACGATCCCAATACAACAGAT
  YAR028W	GTTGTAAACCGCTAATGATTATGTTCCTAATACCTGTGTGTTTGGTTT	GGTCAATTGATATCTTGCCTTAATATGGCACTTATAGATGTTATCCTG	CATCTATAAGTGCCATATTAAGGCAAGATATCAATTGACCAGCGGAAGCATTTTATTCACCAAGTATACTTACTTTTCTTACACTCCTCAGAAGCTC	TCAAGGTAAATCAGCGTCAGGATATTGCTTTCTCCAGTATTCACTGATCCCAATACAACAGAT
  YBL033C	GGTTCAAAAAGTGGAAGTAGTAGTGCTCTT	CCTGTAGGTTTGTTGTAGTTTTATGTACGC	ACAAAAAGTGGCGTACATAAAACTACAACAAACCTACAGGACACAAGTGTAGCATAATTATTAGTACATATAGTGCTTTTAGCGACAAGAAGAGATAG	TTATATTGCCAGCGTCGATGTTGATGACAACGCAGAGTTCATACGGATCCCAATACAACAGAT
  YBL055C	GAGAAAGTGAGTGGGAATGGACAAGATTATTTGGGTGAGGGTGTTTGCTTA	ATAACTATAACAGTACTGGCGCCTCAAAAAAATGCCTTGGCAGCGAAAGTG	GAGGCGCCAGTACTGTTATAGTTATCATCTATTGGTGCAAAGAGCAAAGTTACATTATTTTTAGTAGCGACAAGAAGAGATAG	TTACTCTCCAAATATCTTACAGGTCGTCTTCCACGTGGTATCTATGATCCCAATACAACAGAT
  YBL091C	TGCTTACTTTTCGTTTCCACTTTATCTTGCTGTTCGTTCGAGCAACAGTATTTATT	TTTTCAATACGGTAGAGCTTCTACAGTACTTGTTGATGTAAAAGCCCACTTATTAG	TACATCAACAAGTACTGTAGAAGCTCTACCGTATTGAAAAGGTAAAATGCGCTTTCAAATGGCCTCCTCACTAGGTATATCACACATTACTTGCCTC	TCAGTAGTCATCACCTTTCGAAACGACTTCCTTTTTGTGAGCATGGATCCCAATACAACAGAT
  YBR077C	CGTAGGTGTTTGACATTGAATATTTATACCGGCT	ATTGCTTCAATATTTTGGATACCGTTTGCTGCTG	AGTTATCAGCAGCAAACGGTATCCAAAATATTGAAGCAATTGGGTTTAACAATATGTATCGAGGCCACACCTTTTCATGCACAATCATATGGGAGAAG	TCAACCTAGCTTGTAGCCAAACAAATCAGTCAACTCTCTCATAGCGATCCCAATACAACAGAT
  YCL050C	CTTTTTTTTATTGTGTTGGTTTTTATATGTTTTGTTATGTATTGTTTATTTTCCCT	TTTTATTTTAATTTTTACTTTTCTGTTTGTTCTAAAATCTATCTAAACTGGCTTTC	AGATTTTAGAACAAACAGAAAAGTAAAAATTAAAATAAAAGTCCTTCACAAGTACAAGTATACACGTATACATGTATGTACACACATTACTTGCCTC	TCAATAGTTGTATTCGTTTGGTTTTTGACCAGAAGTGTTTGGGAAGATCCCAATACAACAGAT
  YDR071C	TTTGTTTTACAACCAAAAGTTTTAAAAAAGTGGATTCATATAAGGTTAG	TTTAACTTTTTATTTAATCGTAATGTATTGGTGTTATTTTTTCTCCGAG	AAAATAACACCAATACATTACGATTAAATAAAAAGTTAAAATCATTAGTTTAACGCACTTTACAAATGTCTAATTATAAACACACATTACTTGCCTC	CTAGTTGTCGTATTCTTCCTTAATTAGTTCTCTCTCCATATCGATGATCCCAATACAACAGAT
  YDR330W	CACGATGTAACCCAGTTATAGAACCTATTCTGTAGTTTGTTGCAGTTTT	ATAAAAGGTATGTTATTATTGCCACACGTCGTAGTCTATGCGTTTGTAG	CATAGACTACGACGTGTGGCAATAATAACATACCTTTTATACAAATTGTATGGTGGCAGGTACCTAGATGTAATTAGGTAAGCGACAAGAAGAGATAG	TTATTCTATTTCAGGGTCAAGCTTCTCAAGTAGTAAAGAACTATTGATCCCAATACAACAGAT
  YER115C	AATAGTAAGGAACAAACTGTTGATTTAACTGCAGATGCCATAGACTTA	ATACAAGTTTGAACAAAATACAACTCAAAGTAAAATGGTAGCTGCACG	TACCATTTTACTTTGAGTTGTATTTTGTTCAAACTTGTATACACAGGAGAAACAATTCCATAAATAAACTTTGTAAGATACACACATTACTTGCCTC	TTAAAAATATAGGGATGAAAACTCATCGGCTATACTGTCATAGACGATCCCAATACAACAGAT
  YHL044W	GCAGTACTACAACTCTGTGCGGTGGGAAAAATTTATTCTTCGAGGCACAGTATT	AGCCTTAGCGGGCCCTCACTTGCTTTTGAATTTGTCTTTATACCACAAGTTAAC	AAGCAAGTGAGGGCCCGCTAAGGCTTTGGAAGGTATTTCTAGGTTCTACTTTAGTTTTTTATAACCACACATTACTTGCCTC	TCAAACTGTGACCCCCATCTCTGGATACTTATCCTCCCAGTACTTGATCCCAATACAACAGAT
  YHR207C	AAGGATAATAACAGAATTTAATGGCTTGATATGGACGGGCG	TTTTGTGCCACTTTTCGTTATTTTGTATCAGTGCACGCTTG	AAGCGTGCACTGATACAAAATAACGAAAAGTGGCACAAAAGAGATAAACGCATACTAAATTACTGTGTCTGTTATTGTTTCACACATTACTTGCCTC	TTATCTTTCATCCACTGCGACCGAAACGTTTGAATCGAATCTGACGATCCCAATACAACAGAT
  YIL079C	CTTTCCTCGATGAAAGTTGGTTCAGGATCCAAGTACAAGGACTGTTGCTT	AATATCTTAGCTCCGTGGTCCGCTTCCTTTGTATTCCTCATGTGAGAATG	AAGCGGACCACGGAGCTAAGATATTGATTATATGTGAGTTTTTTTAATTTTTTTTCCATTCTCATAGCGACAAGAAGAGATAG	TCAATAACGGCCATTTCTTTGGCTTTTGTTAGAAGATGATCGGCCGATCCCAATACAACAGAT
  YIL111W	ATGATATTGTTGAAGGATATCGACTTGCTCTCAAAGG	GCTCTTTACAATGAATATGTGGCGTTATTCCATTGTG	ACGCCACATATTCATTGTAAAGAGCTTTTTGTTTTGAGTTTTAAAAGATATCCATTTACTAACATCACACATTACTTGCCTC	TCAGTGTTTGTTATACATGCTCTTTACAATGAATATGTGGCGTTAGATCCCAATACAACAGAT
  YJL058C	CGGAAGTGAATGAGAATACCAACAGTTTTACTGATGATTATATTGACGGAG	AATGAAAGAACTTTATTTCTACAGAGGTCAATTGGCGTTTCTGTCACCTAG	AAACGCCAATTGACCTCTGTAGAAATAAAGTTCTTTCATTAAAGCATCTAACACTTTATTTCAATCTCCTATTTAATATCCACACATTACTTGCCTC	CTATATACTCATTCTAATGCCTGCTAATAACTCTTCGATAATTCGGATCCCAATACAACAGAT
  YJL128C	GTAGTATAGTATTGCAACAGGAAAAAAAAATCTTATTGCGTAGCACTCTGTATCTATTTT	CTTTGAGAAATTAACGTCTCGATCTGCTTAATATAATAATGTATCTATAGAAATGTACGG	ATTATTATATTAAGCAGATCGAGACGTTAATTTCTCAAAGCGTTAATATCCAAATAAAAGCAAACAGGCACGTGAATATACACACATTACTTGCCTC	CTATAAACCACCCATATGTAATGCCGGTACATTTTTAGATAAACCGATCCCAATACAACAGAT
  YKL023W	TTCCCCATATTGGGAACCTGCTCTGCCCTGCTCAGCACGTAG	GCTTGCACGTTATACCTGGCCGGGATTGGGGCGCTATTCTTG	TCCCGGCCAGGTATAACGTGCAAGCATGCATTCTAATGACATAACTATTTGACGACGTGAATTAGAGCGACAAGAAGAGATAG	TCAAAATTCTATAGGCTGTCCATTCATGTCAGTTGAGGAAGGAAAGATCCCAATACAACAGAT
  YKL166C	CCAGGTACGAGTGATTTAGGAAGGTCTGAAGAAAAGCGACTATGCATT	TTTGTGCAGGCTCGCTCTTTCCTTGTACAACCACCGATATACAATATG	CAAGGAAAGAGCGAGCCTGCACAAAGGAGGTTCAGTGGGATAATTGTAATTGAAAAAAAAAAAAACACACATTACTTGCCTC	TTAAAATTCTTTCATTAAATCCATATATGGATCCTCCCCTTGAATGATCCCAATACAACAGAT
  YKR039W	TTATTTATTTTTTTTTATACCATTTCCTTTTGATAAGGGGT	TTTTTATTTCTTTTTTTTGTTTCTTATAAATGTTGCTGTCC	GACAGCAACATTTATAAGAAACAAAAAAAAGAAATAAAAATGAATCGAACAACGACAAAAAAAGACTTTATTTTTTAGATCACACATTACTTGCCTC	TTAACACCAGAAATTCCAGATTCTATACCATCTTGGCTTTGTGGCGATCCCAATACAACAGAT
  YLR218C	GTAGTCTTTACGGTAATGGAGTTGGCCCTTG	AAATGAAGCGAATGGATGAGAAGAGGGGAGG	CTCTTCTCATCCATTCGCTTCATTTAAATCTAAATGTCGTGATGTATAACTTGTATATAATAGACCACACATTACTTGCCTC	TCATTTCTTCTTTTCCGAATCCTTACTGGTCGTCCCATCCACGTCGATCCCAATACAACAGAT
  YLR255C	AGGGTGGAGAGTTAGAGCTGGTGTTGGCACCGGGCGTAGTTGCTG	TCACCCGTCGGGCGAAATCCACCCTCGCGGCCTCGTTTAGAATAC	AGGGTGGATTTCGCCCGACGGGTGATCAATATTTCTTGAAGTACGATTTCTGTGTGTATAGATTCCACACATTACTTGCCTC	TTAGCGAGCCTTTAAGCACAGAACAATCTCTATAGCCAACATCTTGATCCCAATACAACAGAT
  YLR350W	ACGATTATGTATATCCATATATATATATACACTACAAGCCGACCG	GATGAAACTGTTTCTTATGCGTTTTTATAATGGTATAGTCTTGCG	GACTATACCATTATAAAAACGCATAAGAAACAGTTTCATCTTGGCTCTGCCTATACGCATATATGTATATATATATATATCACACATTACTTGCCTC	CTAACTAATTTGAGCACGGCCCGTAATACCAGGGATGGATATTCTGATCCCAATACAACAGAT
  YML048W	GCTTTTGCCCTTGACCCTGAATGCGGCAGATCCAGCGACTTCATCGAGGTTA	GCTTGCAATCGCCCTCTATCAAAACAAAGTCCACCGATCTCTGCTGCCCCTC	GTTTTGATAGAGGGCGATTGCAAGCTTGTAAAACCAAAACACTACCATCCAGACTTTTTTTTTATCACACATTACTTGCCTC	TTAATTAGATTTTTTCAAATCATTCTTTTTTATACCAAAAGTAGCGATCCCAATACAACAGAT
  YML088W	CTCTGAAGAGTTTAGCGGGCTCTCAGACGGTTTTTTCGCTTTTGGCATTTT	GCTGTCTTATTCCCTAGTGTCGGCCACCTGATAAAGGTTTTTAACTCGTAC	GGCCGACACTAGGGAATAAGACAGCAAGTATGTACATTTGGAAGAGCATATGTTAAATATTTTATCACACATTACTTGCCTC	TCAATTGATTTCACTCAATGACAACGCAATGGCCAATTGTAAGTCGATCCCAATACAACAGAT
  YMR018W	AATTATATTCTGCGCTTGCCTTTTATATTAAGCGTCACAACGCC	CGTGCTTACAAAAGGTACCCTTTATTCTTAATGATTCTCCACTC	ATAAAGGGTACCTTTTGTAAGCACGGTTTTAGTTAATTTTTTTGCTATATAGTGCATTCAAAATACACACATTACTTGCCTC	CTAATCCATTTTGTTATAAATAGCTTTTAAGGTTGATATTAACGAGATCCCAATACAACAGAT
  YMR278W	TTTAATCACCAAGAACCCTAGCACTTATAAAGCGCGTCTAGCGAAAAAGTT	TTTTTGAAATTGCAGCAGCTAATGGCTCGAGAGTACTGTTGAGTATTTAGG	AACAGTACTCTCGAGCCATTAGCTGCTGCAATTTCAAAAATACTATTTTAACCTATACATTAAGACCAAGCGACTTAAAGCACACATTACTTGCCTC	TCAAAATTTTGTAACTATATTCATTTCATCTGGTCTAAACCATTCGATCCCAATACAACAGAT
  YNR058W	TAGTACATTGTGCAACCTGTGCCATAATATTGACACCGAACTTCTTCAGGGCCT	TAGTTGTTATCCTAGCAATAGAAAACGAATTGCGCTGTTGACATCCTCCCGACC	CAACAGCGCAATTCGTTTTCTATTGCTAGGATAACAACTATATACGATCTCATCAAGTCATTTGCCAAGGAATTCTTATTCACACATTACTTGCCTC	TCATTGGTTTATGTGGGATTTCCACTCATGTAATACTTCAATCAAGATCCCAATACAACAGAT
  YOL158C	AGAATCATCTCTACTACACACTGCATTATGAACTGCAGCCACATTTATCTAAGGG	TTTCAGAGAGACATTCACGAGCACTTTTTTGCTATATTAGAGTTCTGCTAGGACC	AGTGCTCGTGAATGTCTCTCTGAAAGCTGGAGAGTACTAGCGATACAAATATAGTGTTAAGCTCTAGCGACAAGAAGAGATAG	TCAATATCCAATTACACGACGGAGCCATGATTGTTGTTTGATTTTGATCCCAATACAACAGAT
  YOR008C	AAATAATGTGGAGATCTTATCTTGTTTTTCTCTTCTTTATGACA	TATTTAAATAGAATTTTTTATCCTGAGTACTAAATCAGCCAACG	GGCTGATTTAGTACTCAGGATAAAAAATTCTATTTAAATAAGAAACCCAAAAAAAATTATTATATTCTTAAACTATTGCCCACACATTACTTGCCTC	TCAATCAGCTTCGTCTGGATTGACCACTGTTAAAACGTTGTTTTTGATCCCAATACAACAGAT
  YPR037C	TGCCCTTATACCGCGTAGTGTAGGTCCAGTCGTACTT	CCCTTCGTTCCTTGCACAATCTGTCCAGACTTTCCTC	GACAGATTGTGCAAGGAACGAAGGGACAAAGAAAGATTACATTAATCAATTATAGTAATAGTAAAAGCGACAAGAAGAGATAG	TCAACCGTGCTGTTTAGCCTCCTTCTCGAGAGACACGCGTTTACCGATCCCAATACAACAGAT
  YPR040W	GCCGTCCTGCTAGATGTCTGTACGGGATACTTCTCAATCAACTT	TTTTTCTGGGGAGCTGTTGTGTCTAAAGCTGCCCTTAGGTCATG	TAGACACAACAGCTCCCCAGAAAAACAATCAAATATACAAAACAATACGTACAAATACATACACGCACACATTACTTGCCTC	TTAGAACTGAATTATTTCACATTGTCTTTTGATCATCGGCGTGTTGATCCCAATACAACAGAT
  YPR051W	CAACCAAGGTTCGTCGTCAGTAAGCTAGTTACGGAATGGTAA	GTCCTGACCCAACGCCTTTTCTGTAGTAGTGGCTTTTTTATC	TACAGAAAAGGCGTTGGGTCAGGACAAGGAAAAACACTCGATGATGATAAAATATATATTAATAAAGCGACAAGAAGAGATAG	TTATGTGGCCAGCCGGCCATGCATTAGGAACGTAGACCGAGTGCAGATCCCAATACAACAGAT
  YBR273C	TAGGTTCAAGTGAGTAAATTCTATCTTTGACCAGGGTCCCCAGTTT	TTGGTTACGGCTGTTTGGTTTAGCTATCCTTGCTTCTTTGTAAGTG	AGCTAAACCAAACAGCCGTAACCAAGTAGTTAATGGCTTCAACTCCATTTATGACTAGTATTAGACACACATTACTTGCCTC	TTATTTTTTATCTTCGTCCTTTTTCTTTTCTAAATGGACATTATTGATCCCAATACAACAGAT
  YAL030W	ATGCACGATTTATAGTGATCTAGGGGTACCTCCTCGG	GGACAATGAATACGTTGCGCTTGCTATGTCTTTGGCG	AGCAAGCGCAACGTATTCATTGTCCCTTTCTACATCGTTGATACTTATGCGTATACATTCATATACACACATTACTTGCCTC	TTAGTATAATGTTAATTTCCTAGATGGTACTCTGTGGCTTTCTGTGATCCCAATACAACAGAT
  YAL065C	CATTCCAACCAGTAGTTCCACCTCTGGTTCTTCTGAGAGCGAAACTA	TTTAGAGCTGACAGACTCTTCATTCGTAGTCTGTGGCCTCCATGTTG	GAATGAAGAGTCTGTCAGCTCTAAAACGCACGGCATCATCTTATATTAATAATTCTATCATCACGCACACATTACTTGCCTC	TTACCATACGATTGCCAGCAATACGGTGGAAATAAAAACACTTATGATCCCAATACAACAGAT
  YAR002W	CTCGATTTGCATAAGTGTGCCTTAGTATGC	TGGATGTCGATTTTAGGATATCTTGCGGTG	ATCAAATAAGCACCGCAAGATATCCTAAAATCGACATCCATAATGAAAATTTTAGCCGTGACATAATTACCGTATAGCCCCACACATTACTTGCCTC	TCAAAAGGTATATAGGGACTTGAAAGCCTCAACTTTATTTTCATCGATCCCAATACAACAGAT
  YBL049W	CGTGCACCTTTTCCTCGAGATAAACATATAGCGATGACTTAGAGAACAA	TTTCTTCTACAGTAAGATAAGCTTCTCCCTTTTCCTTCAGTGCTGCTAC	CTGAAGGAAAAGGGAGAAGCTTATCTTACTGTAGAAGAAATGTCTTCCTTTGTCTGCTATCTAGCACCTCTCGTCTTTTACACACATTACTTGCCTC	TCAAGTACATTTACAAATGTTTTTCAGCTCTAAGATAAACTTTCCGATCCCAATACAACAGAT
  YBR144C	TGCTTCAGAAACAGACACATTAATAACTCCATGAGAACCGCC	GTGACAATGAAATAATCAAATTGTGACATCTGCTGACGCGGG	CGCGTCAGCAGATGTCACAATTTGATTATTTCATTGTCACTAAGAATTAGCAACTACTTTCATTCATCTTCGACGCAACTAGCGACAAGAAGAGATAG	CTAGCACATGAAAAAAAAGGATATATATAATTTTACAAGTAGTGTGATCCCAATACAACAGAT
  YBR185C	ACATTTGATAACCCTCAGAGATGATCCTCG	CTCATAAGCTTAGCTGAATGGATAGGCTTG	CTATCCATTCAGCTAAGCTTATGAGGGCAATATGTATATAAATGCGAAAAGTTTTGGAACATCTGAGCGACAAGAAGAGATAG	TTAGCTTGGAGGTAAACGATATATATCACCGTTAACTTTCATTCTGATCCCAATACAACAGAT
  YBR255W	GCTTACGTGCTAATGTCCTTACCCTCGAGT	GTTTCATCCGTTTAAAGTACCTCCCCCTCC	GGGAGGTACTTTAAACGGATGAAACTTTGCGGGTCTATGTATTTATAGAAATAAACCATCTGCACCACACATTACTTGCCTC	TTACCACAGCAATGCCTTAATGATTTTTAGAACTAAAAAAATTGTGATCCCAATACAACAGAT
  YCL044C	AAAACGATGTCAGAGGCGTCGTAATTGAAGGTTACCCAACAA	CTTTTATTACGGTAATTGGAAAAGGAGAGGGAATGGAGGAGG	TCCTCCATTCCCTCTCCTTTTCCAATTACCGTAATAAAAGGTCTTTGCGGATTGCTTAGTTGTACCGTGCGTATATTAAAAGCGACAAGAAGAGATAG	CTAATGTGTCTTTTCATTAGTGAGAGCCTTGGGGGTAGGCCCTGGGATCCCAATACAACAGAT
  YCL047C	GAGAAATCCTCATTACCCAAGAGCACAAGGAT	ATTAGTCTGCTAGTGCACGTACTGCTATCAAC	GCAGTACGTGCACTAGCAGACTAATAAGAGAAAGATAGAAATTCAAATGCCTTTCATGGAGATATCACACATTACTTGCCTC	TCAATCAAATATTGTCTTATTATTGATCAAATAATCGTATATGCAGATCCCAATACAACAGAT
  YBR268W	CACAATAAGCACTTCAAAGATGTTGAAGAAAACCTGGAACACATGTTTAGGACCC	CTTCAGTCTTCAGCAGCTATTTCCTTTGCTGTAGATATAACTGCTTTAAGAAGCC	AGGAAATAGCTGCTGAAGACTGAAGGCCTCCGTTAATAAGATCAAGTTTGGTATCCTTGTAAATACACACATTACTTGCCTC	TTAAAGTTGGCTCAAGAAATTGTTTTGCTTGATTCGCTGCCTGTTGATCCCAATACAACAGAT
  YAL002W	AAGCCAGCTGCTAAGTCCATTGTCACTCTA	TCTAGGTGTAATGAGTAATGGTCTCGGCAG	GAGACCATTACTCATTACACCTAGACATTTCTAAATATTTAATACAACTTTGGTTACATAAAAGTCACACATTACTTGCCTC	TTATACTATTTTTGGGTTAGATTCCGTCTGGCAAATTAAACAAGAGATCCCAATACAACAGAT
  YBL082C	AGTGTTGTTGTATTTCATGTGATTGTAATGGGGGTGGTAG	ACTACTAGTATCTAACTCTTTCTCTCTCCCATACACACTC	GAGTGTGTATGGGAGAGAGAAAGAGTTAGATACTAGTAGTAACAGATGTATAAATATCATATAATCGTATTGTTTTAAATCACACATTACTTGCCTC	TCAGTTGAGCTTTTTTTCCATAGAGCTGGTGGTACGAAGATGGCTGATCCCAATACAACAGAT
  YCR045C	AAGAAGAAGAAGAAGAATCGAGTCTTCCCTCTGAG	GACCAGTGTTTTTTTGCTGTTTTTCTTGGCGGATG	TCTAACATCCGCCAAGAAAAACAGCAAAAAAACACTGGTCTGTTTTTCTCTTTCTCTCATAAATAAATTATTGCATAATGCACACATTACTTGCCTC	CTACAAATTTTTCATAAAATCACGCCTCTTCAATCGAATCTCACCGATCCCAATACAACAGAT
  YDL106C	CTTCTCGACTTCTTTTGTTTTCTTGTTTCTCCGCT	AGCGTATTGTATACGTTATTTAGCAGTAAGCCGTG	CAAGTCACGGCTTACTGCTAAATAACGTATACAATACGCTTGAAAATAATTCTGTTTTTTTTTTTTTGCGATTGCATTTTCACACATTACTTGCCTC	TCATATCCATCTATGCTCGTCAGTTAGTTCGTTAGTGTTCTTCAAGATCCCAATACAACAGAT
  YIL148W	GTGCATCTATTTCCCGCCTATATCTGGAACGTTCTTCATT	GTTTGAATCTTGGTCGTCTCAACTATGGGATAATAAGCGG	TAGTTGAGACGACCAAGATTCAAACACATAGTTTTTCGAACGTTCAGAGCTTAAAGGGACAATTACACACATTACTTGCCTC	TTAGAATGGATATGGTGCATACATTTGCATGTTTGAATCTTGGTCGATCCCAATACAACAGAT
  YJR073C	CGTGAAACTTCCGTACCATTTCTTGCAACAATTGGTAAACAGCA	CGCTTGACTTGCGCTATTCTTGTTGTCTTCAATTGCTGTTTATG	CAACAAGAATAGCGCAAGTCAAGCGGTGCTCTATTTCTATATATTCTATAATGTTAGAAGCCTATCACACATTACTTGCCTC	TTACATATTCTTTTTGGCCTTATCACGGTTAGCGTAGATCATGGCGATCCCAATACAACAGAT
  YLR011W	CGCCGAGCTTGGCCATTACAATATAGAGCACATGCTATGA	GTTTAATGCGGTTGTCGACTAAGCACCTCAGCTGGTAATG	TGCTTAGTCGACAACCGCATTAAACACCATAAAATTTAACTGTAGTCGCAATAAAAGATGAAAAGAGCGACAAGAAGAGATAG	TTATTTATTCCTCGTTGTTTCGATGCACGATGCGAGTAATTGCAGGATCCCAATACAACAGAT
  YCL044C_2	TGTTTTGTACTATGCCCCATGGTGTGGTCACTGTAAGAGATT	CTTTTATTACGGTAATTGGAAAAGGAGAGGGAATGGAGGAGG	TCCTCCATTCCCTCTCCTTTTCCAATTACCGTAATAAAAGGTCTTTGCGGATTGCTTAGTTGTACCGTGCGTATATTAAAAGCGACAAGAAGAGATAG	CTAATGTGTCTTTTCATTAGTGAGAGCCTTGGGGGTAGGCCCTGGGATCCCAATACAACAGAT
  YDL022W	TATTTTGTACAGCTGATGGGACCTTGCCGTCTTCATTTT	CTTTATATTATCAATATTTGTGTTTGTGGAGGGGGGGGG	ACCCCCCCCCTCCACAAACACAAATATTGATAATATAAAGATTTATTGGAGAAAGATAACATATCATACTTTCCCCCACTCACACATTACTTGCCTC	CTAATCTTCATGTAGATCTAATTCTTCAATCATGTCCGGCAGGTTGATCCCAATACAACAGAT
  YDL175C	CGTCTACAGAACCCCTGAAGAACACGAAACCTGTAGCCAA	GACTGCGAGGAAGTGATGTAAGCTGCTATCGTTGTACAGG	CAGCTTACATCACTTCCTCGCAGTCCTTTAAATAGGAAGCCCTTTAACAAGCTGTTCTTGGTTACCACACATTACTTGCCTC	TTATCTTCTTTTCCCCAAGGTACCACTCCTATATGGTTGATAAGAGATCCCAATACAACAGAT
  YGL211W	AAACTGCTGACGAAGTGGGATGGGATGAGATGATAA	GACGGTTCCTTTGCTTTACTCTTACCATATCGTCTC	GTAAGAGTAAAGCAAAGGAACCGTCCATATATATAGCTCGCAGTAGAAGAATGCAGCGTAACATAAGCGACAAGAAGAGATAG	TCAAAAGCTGAGTTTCTCAAGAGCTCTCAGCTTCAAGGCTGCACCGATCCCAATACAACAGAT
  YAL009W	TTGTCACTACTCAACGTATTCGCACTACTAACACTG	GACCTCCTTTATTTAGCTTTCCACTACCTTTCTTCC	AGTGGAAAGCTAAATAAAGGAGGTCAAGAGTTGGAGGGCTTCTTCCTTCGAATAAGAGGTCATATCACACATTACTTGCCTC	TCATTCTGATTTAGGTCGGAGTTCGTGCGCTTGTTTGCGTCTTCTGATCCCAATACAACAGAT
  YBR185C_2	AATACTTCCTTGCCCCTCTCTCATTAATTCTTGGCCAGTTATAATCGA	CTCATAAGCTTAGCTGAATGGATAGGCTTGCTTTCTGATGGAAATTTG	CTATCCATTCAGCTAAGCTTATGAGGGCAATATGTATATAAATGCGAAAAGTTTTGGAACATCTGCACACATTACTTGCCTC	TTAGCTTGGAGGTAAACGATATATATCACCGTTAACTTTCATTCTGATCCCAATACAACAGAT
  YBR270C	AATATTTCGTCACGTTGCGGTGAGCAAGGATCTTTTGGCT	TGCTTGACTCTGTAGTTTAAGACTCGCCTAGCCACTTATG	GAGTCTTAAACTACAGAGTCAAGCATTAAATATTTTATTTAGTAATATACAATAGTGGCATATAGCACACATTACTTGCCTC	TTATATACTCATGCGTATGCCCTCAAGAAGTTCACCAATAATTCTGATCCCAATACAACAGAT
  YCR025C	TTATTGGTATGGGGCCATATTTTCCACAGGGATACATTGAGC	AAACCTCCTCGTTTAGGAAAGAGTTAACACGCCTAGTTTCAG	AACTCTTTCCTAAACGAGGAGGTTTTAGAAGATAGCTTCAATTTTTTCCGATAATAATTTCCCATCACACATTACTTGCCTC	TTACGCAGTGACAAGTCTATATTGACATCTTTCTGCTTTCTGCTTGATCCCAATACAACAGAT
  YDL012C	CGCTAGGTTGCTAAGGTTTCGTCTTTTCTATCTTACGAT	GAGATGTGTATTTGCTTGGTTTATGGGCTATTGTATGGG	ACCCATACAATAGCCCATAAACCAAGCAAATACACATCTCTTTTACAAGTTTTGCCTTTAGAAGATCACATCGTACTAACCACACATTACTTGCCTC	TTAACGTACGCTTTGTTTGGAGGCAGAGTTTCCGTAATAATCTTGGATCCCAATACAACAGAT
  YDR375C	AATTGACAAATTCTCTACTGCTAAACACCCCTTTTGTTCCGAGTTCGCCAGGGATA	GGTAGATTGAGGGCACACTTCATTTTTTTTCAAATCGTCCCTTGGTACGTCTACTG	AAATGAAGTGTGCCCTCAATCTACCGTGCTAAGTGGCGCCTTACTACATAACTTGTATATAATAACACACATTACTTGCCTC	CTAGAAAATATGATTAGCGTTCCGTAAGCTGGAGACCATTTTCAAGATCCCAATACAACAGAT
  YLR035C	CACTCATCTTAGCCACAGTGGAGGGTTTACCTATGAA	CCAAAACTATGAAAAGGCGTCTGTTCCTCAAACCGAG	AACAGACGCCTTTTCATAGTTTTGGTGATCGGGTGCACTATCACCACTCAGCACAATAGATTACTCACACATTACTTGCCTC	TCAGGGGCTGTCTGGCGTGAAGAGGCACCATTCATTTGTCGTGGCGATCCCAATACAACAGAT
  YLR412W	TTTTGCGCTTGTGGTGGTAGCTCAATGGCTTACTCGTT	ACAGCAGAAGACCGGTCAAGGTCATTAACTTCGGTATC	ATGACCTTGACCGGTCTTCTGCTGTCATATCATTCACTACATAGTACTTTATAACTAAACTGTAACACACATTACTTGCCTC	TTAGTACTCTATAGCATGTAACGTCAAGTCAGAGAAGGAATTGATGATCCCAATACAACAGAT
  YML089C	CATCGTGAATTAAGTTCCGGAAATGCGTAGAAAG	AGGGAATAACAACAAATTGGAAATGTGCGGCTAG	ATTACGCTAGCCGCACATTTCCAATTTGTTGTTATTCCCTAAAGTTTTCCACATAATAAACCAAGACAACAGGATTGTTTCACACATTACTTGCCTC	TCACGGATCAATTTTATAAAGCATACAACTAAAGAGAAAAGTGTAGATCCCAATACAACAGAT
  YPL033C	CCAACCTTGTCCTCATCAAGTCAAATGGCGAATATGGCTT	GTCAGATTTTTGGGCTCTATTACTAGTGGACCTGTGCTTC	GAAGCACAGGTCCACTAGTAATAGAGCCCAAAAATCTGACGGGTACTACAAATCGAAGTAACAGTGATAAAATTGCTAAGCACACATTACTTGCCTC	TTAAGTAAAAGAGCTTTTGAAACCAGTTATCAAAAGAATATCACCGATCCCAATACAACAGAT

  Table S10. Table of primer sequences for the 96 ORFs

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  Figures S10a, S10b, S10c. Gel images showing PCR1 products for the 96 ORFs.

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  Figures S11a, S11b. Gel images showing PCR2 products for all except 8 of the 96 ORFs. The remaining 8 are shown in the gel in Figure S12.

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  Figure S12. The remaining eight of the PCR2 results are shown in the first 8 non-marker lanes of this gel, which then also contains the first 24 results for PCR3.

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  Figures S13a, S13b, S13c. Gel images showing the remaining PCR3 products for the 96 ORFs.

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  Figure S14. Temperature gradients to investigate PCR1 failures for YLR091W, YML097C, YAL058W and YCL050C.

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  Figure S15. Temperature gradients to investigate PCR1 failures for YDR071C and YHR207C.

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  Figure S16. Temperature gradients to investigate PCR1 failures for YJL128C, YML088W, YMR018W and YMR278W.

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  Figure S17. Temperature gradients to investigate PCR1 failures for YBR255W, YAR002W, YIL148W and YJR073C.

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  Figure S18. Temperature gradients to investigate PCR1 failures for YPL033C.