chemical synthesis of the mouse mitochondrial genome · mitochondrial genome sequence as it exists...

Nature Methods

Chemical synthesis of the mouse mitochondrial genome Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman

Supplementary Figure 1 Timeline to synthesize the mouse mitochondrial genome. Supplementary Figure 2 Detailed strategy to synthesize the synthetic mouse mitochondrial genome.

Supplementary Figure 3 Assembly of the mouse mitochondrial genome from 75 sequence-verified segments following three rounds of assembly and 45 cycles of PCR amplification.

Supplementary Figure 4 Producing a circular mouse mitochondrial genome following its release from the vector. Supplementary Figure 5 Assembly of the mouse mitochondrial genome from 75 sequence-verified segments following

three rounds of assembly and 80 cycles of PCR amplification. Supplementary Figure 6 Overlapping DNA molecules containing 16-bp non-complementary sequence can be

assembled and the products can be PCR-amplified.

Supplementary Table 1 Optimization of the one-step oligonucleotide assembly method.

Supplementary Table 2 Oligos used to construct nucleotides 1375-1534 from pCC1BAC.






Supplementary Table 8 Oligos used to construct mtDNA segment 29 from 16 40-mer oligos

Supplementary Table 9 Oligos used to construct Mmus mtDNA Segment 29; Reverse complement of standard oligo design.

Supplementary Table 10 The design of the mouse mitochondrial genome at the nucleotide level.

Supplementary Table 11 The number of colonies (CFU) produced following individual transformation of each of the 75 first-stage assemblies.

Supplementary Table 12 Cloning efficiencies and synthesis error rates for each of the 75 mouse mitochondrial genome segments.

Supplementary Table 13 DNA sequence characterization of four E. coli clones containing the complete mitochondrial genome following four rounds of assembly and 45 cycles of PCR amplification.

Supplementary Table 14 Additional oligos used in this study.

Supplementary Table 15 DNA sequence characterization of four E. coli clones containing the complete mitochondrial genome following four rounds of assembly and 80 cycles of PCR amplification.

Supplementary Note 1

Nature Methods: doi: 10.1038/nmeth.1515

Supplementary Figure 1. Timeline to synthesize the mouse mitochondrial genome.

A 16.5-kb DNA molecule can be synthesized in five days upon delivery of oligonucleotides. This time frame includes four stages of DNA assembly, two PCR amplification steps, and 2-3 rounds of cloning and sequencing.


Supplementary Figure 2. Detailed strategy to synthesize the synthetic mouse mitochondrial genome.

(a) The 75 first-stage assemblies (340 bp), produced from eight overlapping oligos, are shown. At the termini of each segment are 20 bp of pUC19 sequence (red) and 8-bp NotI restriction sites (N). Following NotI digestion, the pUC19 sequence is released. This exposes 40 bp of overlapping sequence. (b) The 15 second-stage assemblies (1,260 bp), produced following the assembly of five first-stage assemblies, are shown. At the termini of each assembled product are 40 bp of pBR322 sequence (purple) and 8-bp AscI restriction sites (A). Following AscI digestion, the pBR322 sequence is released. This exposes 40 bp of overlapping sequence. (c) The three third-stage assemblies (5,660 bp), produced following the assembly of five second-stage assemblies, are shown. At the termini of each assembled product are 40 bp of pSMART sequence (blue) and 8-bp SbfI restriction sites (S). Following SbfI digestion, the pSMART sequence is released. This exposes 40 bp of overlapping sequence. (d) The complete 16,299-bp mouse mitochondrial genome with two 6-bp PmlI restriction sites (P) and 221 nucleotides of repeated sequence (16,532 bp total synthesized sequence), produced following the assembly of the three third-stage assemblies into the pCC1BAC vector (orange), is shown. (a-d) Mouse mitochondrial genome sequence is shown in green.


Supplementary Figure 3. Assembly of the mouse mitochondrial genome from 75 sequence-verified segments following three rounds of assembly and 45 cycles of PCR amplification.

(a) The 75 sequence-verified segments were assembled in sets of five and then analyzed on 0.8% E-gels. (b) Assembly products from (a) were PCR-amplified and analyzed on 0.8% E-gels. (c) PCR products from (b) were assembled in sets of five and analyzed on a 0.8% E-gel. (d) Assembly products from (c) were PCR-amplified and analyzed on a 0.8% E-gel. (e) PCR products from (d) were assembled in the presence of the pCC1BAC vector and analyzed by field-inversion gel electrophoresis on a 0.8% E-gel using the U-5 program (parameters are forward 72 V, initial switch 0.1 sec, final switch 0.6 sec, with linear ramp and reverse 48 V, initial switch 0.1 sec, final switch 0.6 sec, with linear ramp). (a-e) L indicates the 100-bp DNA ladder (NEB), M indicates the 1-kb DNA ladder (NEB), and XT indicates the 1-kb extension DNA ladder (Invitrogen).


Supplementary Figure 4. Producing a circular mouse mitochondrial genome following its release from the vector.

(a) The 16.5-kb mouse mitochondrial genome that was synthesized has 7.5 kb intervening vector (V) sequence once it is cloned into pCC1BAC. To release the vector, PmlI sites (P) were designed at the termini of the linear mitochondrial genome sequence. To produce a circular mitochondrial genome sequence as it exists naturally, nucleotides 1-221 (red) were repeated following the last nucleotide in the sequence (16,299). This creates 221 bp of repeated sequence at the ends of the linear genome following PmlI digestion, which can then be intramolecularly assembled into a 16.3-kb circular mitochondrial genome without intervening vector sequence. (b) The mitochondrial genome plus vector (24 kb total) was digested with PmlI and then assembled at 50 °C as described in the Online Methods section but with 4 x more T5 exo (due to larger overlap). Samples were analyzed every 2 min by electrophoresis on a 1% agarose gel. Electrophoresis was carried out at 4.5 V/cm for 3 h in 1X TAE buffer in the absence of ethidium bromide. The gel was stained with SYBR Gold and scanned with a GE Typhoon 9410 imager. XT indicates the 1-kb extension DNA ladder and BT indicates the BAC-Tracker Supercoiled DNA ladder (Epicentre).


Supplementary Figure 5. Assembly of the mouse mitochondrial genome from 75 sequence-verified segments following three rounds of assembly and 80 cycles of PCR amplification.

(a) The 75 sequence-verified segments were assembled in sets of five and then analyzed on 0.8% E-gels. (b) Assembly products from (a) were PCR-amplified and analyzed on 0.8% E-gels. (c) PCR products from (b) were assembled in sets of five and analyzed on a 0.8% E-gel. (d) Assembly products from (c) were PCR-amplified and analyzed on a 0.8% E-gel. (e) PCR products from (d) were assembled in the presence of the pCC1BAC vector and analyzed by field-inversion gel electrophoresis on a 0.8% E-gel using the U-5 program. (a-e) M indicates the 1-kb DNA ladder (NEB), and XT indicates the 1-kb extension DNA ladder (Invitrogen).


Supplementary Figure 6. Overlapping DNA molecules containing 16-bp non-complementary sequence can be assembled and the products can be PCR-amplified.

(a) Mitochondrial genome segments 46-50 were PCR-amplified using Hot-start Phusion polymerase with primers “Not1 +8 For” and “Not I + 8 Rev” (Supplementary Table 14). One hundred microliter reactions were setup using 2 µl template (25 ng/µl), 500 nM each primer, 200 µM each dNTP, 1X HF Buffer, and 20 U/ml enzyme. Cycling parameters were 98 °C for 30 s, then 30 cycles of 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s, followed by a single 72 °C incubation for 5 min. Products were analyzed on a 0.8% E-gel (Invitrogen) and estimated to be 25 ng/µl each. (b) Without purification, equal volumes of the products were pooled and 1 µl (+ 4 µl water), 2.5 µl (+ 2.5 µl water) samples were added to 15 µl enzyme-reagent mix and assembled at 50 °C for 1 h. Assembly products were analyzed on a 0.8% E-gel. (c) One microliter samples of the assembly reactions were PCR-amplified in 100-µl reactions using Phusion polymerase with 500 nM each of primers “pBR322 CPCR 20bp-Clone FOR” and “pBR322 CPCR 20bp-Clone REV” (Supplementary Table 14), 200 µM each dNTP, 1X HF Buffer, and 20 U/ml enzyme. Cycling parameters were 98 °C for 30 s, then 15 cycles of 98 °C for 5 s, 50 °C for 30 s, and 72 °C for 30 s, followed by a single 72 °C incubation for 5 min. Products were analyzed on a 0.8% E-gel. L indicates the 100-bp DNA ladder (NEB) and M indicates the 1-kb DNA ladder (NEB).


Supplementary Table 1. Optimization of the one-step oligonucleotide assembly method.

oligo length

(nt)

# oligos

bp overlap

insert size (bp)

variation from method

sequence madenM each

oligoCFU

correct insert size

fraction

correct sequence fraction

Oligo sequence Table

90 4 40 160 None 1.3 2500 N/A N/A90 4 40 160 None 7.8 40000 10/10 4/690 4 40 160 None 46.8 40000 N/A N/A90 8 40 360 None 1175 bp to 1534 bp pCC1BAC 7.5 7300 4/4 1/4 Supplementary Table 390 12 40 560 None 975 bp to 1534 bp pCC1BAC 7.5 2700 1/4 N/A Supplementary Table 460 8 20 284 None 0 50 N/A N/A60 8 20 284 None 1.25 50 N/A N/A60 8 20 284 None 7.5 710 N/A N/A60 8 20 284 None 45 3970 10/10 8/1060 8 20 284 None 270 50 N/A N/A60 12 20 444 50 °C 60min 0 60 N/A N/A60 12 20 444 50 °C 60min 7.5 200 N/A N/A60 12 20 444 50 °C 60min 18.75 2440 9/10 52/8860 12 20 444 50 °C 60min 45 1140 N/A N/A60 12 20 444 50 °C 60min 112.5 170 N/A N/A60 12 20 444 50 °C 60min 270 140 N/A N/A60 12 20 444 50 °C 60min 0 90 N/A N/A60 12 20 444 50 °C 60min 7.5 280 N/A N/A60 12 20 444 50 °C 60min 18.75 1710 10/10 41/7460 12 20 444 50 °C 60min 45 570 N/A N/A60 12 20 444 50 °C 60min 112.5 110 N/A N/A60 12 20 444 50 °C 60min 270 160 N/A N/A40 16 20 284 None 15 1550 N/A N/A40 16 20 284 None 30 2400 9/10 38/9340 16 20 284 None 45 970 N/A N/A40 16 20 284 None 60 650 N/A N/A40 16 20 284 None 75 470 N/A N/A40 16 20 284 None 270 160 N/A N/A60 8 20 284 50 °C 30min 45 9880 N/A N/A60 8 20 284 50 °C 30min, - Pol 45 120 N/A N/A60 8 20 284 50 °C 30min, - Lig 45 250 N/A N/A60 8 20 284 50 °C 0min 45 180 N/A N/A60 8 20 284 50 °C 15min 45 110 N/A N/A60 8 20 284 50 °C 30min 45 18880 N/A N/A60 8 20 284 50 °C 60min 45 36000 N/A N/A60 8 20 284 25°C 60min 45 100 N/A N/A60 8 20 284 30°C 60min 45 110 N/A N/A60 8 20 284 35°C 60min 45 120 N/A N/A60 8 20 284 40°C 60min 45 8060 N/A N/A60 8 20 284 45°C 60min 45 33660 10/10 N/A60 8 20 284 50°C 60min 45 8260 10/10 N/A60 8 20 284 55°C 60min 45 80 N/A N/A60 8 20 284 60°C 60min 45 0 N/A N/A60 8 20 284 65°C 60min 45 30 N/A N/A60 8 20 284 50°C 2 h 0 330 N/A N/A60 8 20 284 50°C 2 h 15 3930 N/A N/A60 8 20 284 50°C 2 h 30 9190 N/A N/A60 8 20 284 50°C 2 h 45 10520 N/A N/A60 8 20 284 50°C 2 h 60 11030 N/A N/A60 8 20 284 50°C 2 h 75 9870 N/A N/A60 8 20 284 50°C 2 h 90 7850 N/A N/A60 8 20 284 50°C 2 h, RC 0 370 N/A N/A60 8 20 284 50°C 2 h, RC 15 1740 N/A N/A60 8 20 284 50°C 2 h, RC 30 3160 N/A N/A60 8 20 284 50°C 2 h, RC 45 5860 N/A N/A60 8 20 284 50°C 2 h, RC 60 6790 N/A N/A60 8 20 284 50°C 2 h, RC 75 7700 8/10 N/A60 8 20 284 50°C 2 h, RC 90 7310 N/A N/A

1375 bp to 1534 bp pCC1BAC Supplementary Table 2

Supplementary Table 8Segment 29: Mouse mtDNA

cooridinates 6115-6398

663 bp to 1106 bp pCC1BAC



Supplementary Table 5






Segment 29: Mouse mtDNA cooridinates 6115-6398

Segment 29: Mouse mtDNA cooridinates 6115-6398 produced by Reverse complement of oligos

Supplementary Table 51243 bp to 1526 bp pCC1BAC


Several overlapping oligonucleotide sets (see Supplementary Tables 2-9) were assembled into the pUC19 cloning vector. We tested the number of oligos in a single reaction, their length, the amount of overlap, orientation, oligo concentration, reaction temperature, reaction time, and the absence of Phusion pol (- Pol) or Taq Ligase (- Lig). Success was measured first by colony forming units (CFU) following E. coli transformation. CFUs 5-10 x above background (vector only) generally indicated that the oligos were appropriately assembled into the full-length product. The correct insert size fraction was measured by colony PCR or NotI restriction


digestion of isolated plasmid DNA. The clones were sequenced with the M13F and M13R primers (Supplementary Table 14). Unless otherwise indicated, assembly reactions were carried out as described in the Online Methods section but at 50 °C for 30 min. The indicated insert size does not include the 20-bp pUC19 sequence and NotI sites found at each termini. A sequence from pCC1BAC (Epicentre) was arbitrarily selected to synthesize for optimization experiments. We also made use of mouse mitochondrial genome segment 29. As many as 16 oligos can be assembled at once, can vary in size from 40-90 nucleotides, and can overlap by 20-40 bp. Longer incubation times and incubation at 45 °C (and not 50 °C as typically used) improves the assembly efficiency. The optimum oligo concentration can vary depending on the number of oligos being assembled. The reverse complements (RC) of the oligos assemble with similar efficiencies as oligos designed in the opposite direction. The assembly reactions are dependent on Phusion polymerase and Taq ligase.

Supplementary Table 2. Oligos used to construct nucleotides 1375-1534 from pCC1BAC.

Oligo name Oligo sequence

UAF 1-50 D cttgcatgcctgcaggtcgactctagaggatcgcggccgcgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaata

11-100 RC ttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccga

61-150 D cgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatat

UAR 111-160 RC acgacggccagtgaattcgagctcggtacccggcggccgctgccattgggatatatcaacggtggtatatccagtgatttttttctccat

Supplementary Table 3. Oligos used to construct nucleotides 1175-1534 from pCC1BAC.

Oligo name Oligo sequence




111-200 RC ttgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccat

161-250 D tcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagac

211-300 RC caagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggt

261-350 D aataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacggt

UAR 311-360 RC acgacggccagtgaattcgagctcggtacccggcggccgctcaccagctcaccgtctttcattgccatacgaaattccggatgagcattc


Supplementary Table 4. Oligos used to construct nucleotides 975-1534 from pCC1BAC. Oligo name Oligo sequence




111-200 RC ttgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccat

161-250 D tcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagac

211-300 RC caagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggt

261-350 D aataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacggt

311-400 RC tcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattc

361-450 D tatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttcc

411-500 RC gttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgt

461-550 D acacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagc

UAR 511-560 RC acgacggccagtgaattcgagctcggtacccggcggccgcccagggattggctgagacgaaaaacatattctcaataaaccctttaggga


20bpCmR-p1-1 caggtcgactctagaggatcgcggccgcgatcggcacgtaagaggttccaactttcacca

CmR-p1-2 taactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctct

CmR-p1-3 cgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaa

CmR-p1-4 tgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttcc

CmR-p1-5 gttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaa

CmR-p1-6 aggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatg

CmR-p1-7 tcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatcc

8X20bpCmR-p1-8 gaattcgagctcggtacccggcggccgctgaataaaggccggataaaacttgtgcttatt



20bpCmR-p1-1 caggtcgactctagaggatcgcggccgcgatcggcacgtaagaggttccaactttcacca

CmR-p1-2 taactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctct

CmR-p1-3 cgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaa

CmR-p1-4 tgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttcc

CmR-p1-5 gttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaa

CmR-p1-6 aggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatg

CmR-p1-7 tcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatcc

CmR-p1-8 atgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatt

CmR-p1-9 cccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacggtgagctggtga

CmR-p1-10 tcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttc

CmR-p1-11 tgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccac

20bpCmR-p1-12 gaattcgagctcggtacccggcggccgcccggaaatcgtcgtggtattcactccagagcg Supplementary Table 7. Oligos used to construct nucleotides 663-1106 from pCC1BAC. Oligo name Oligo sequence

20bpCmR-p2-1 caggtcgactctagaggatcgcggccgcagtgaataccacgacgatttccggcagtttct

CmR-p2-2 gttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtc

CmR-p2-3 tggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttt

CmR-p2-4 tcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaac

CmR-p2-5 gtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgtt

CmR-p2-6 gcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgt

CmR-p2-7 tacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtga

CmR-p2-8 ctgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaac

CmR-p2-9 tgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaag

CmR-p2-10 caggcgtagcaaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccg

CmR-p2-11 acgcctggttgctacgcctgaataagtgataataagcggatgaatggcagaaattcgatg

20bpCmR-p2-12 gaattcgagctcggtacccggcggccgcttgacagcttatcatcgaatttctgccattca


Supplementary Table 8. Oligos used to construct mtDNA segment 29 from 16 40-mer oligos. Oligo name Oligo sequence

40mer Seg 29-1 caggtcgactctagaggatcgcggccgcgaaaaaaagaac

40mer Seg 29-2 tattcctatatagccgaaaggttctttttttcgcggccgc

40mer Seg 29-3 ctttcggctatataggaatagtatgagcaataatgtctat

40mer Seg 29-4 caataaagcctagaaagccaatagacattattgctcatac

40mer Seg 29-5 tggctttctaggctttattgtatgagcccaccacatattc

40mer Seg 29-6 tctacatctaatcctactgtgaatatgtggtgggctcata

40mer Seg 29-7 acagtaggattagatgtagacacacgagcttactttacat

40mer Seg 29-8 tgcgataattatagtggctgatgtaaagtaagctcgtgtg

40mer Seg 29-9 cagccactataattatcgcaattcctaccggtgtcaaagt

40mer Seg 29-10 gggttgcaagtcagctaaatactttgacaccggtaggaat

40mer Seg 29-11 atttagctgacttgcaaccctacacggaggtaatattaaa

40mer Seg 29-12 catagtatagctggagatcatttaatattacctccgtgta

40mer Seg 29-13 tgatctccagctatactatgagccttaggctttattttct

40mer Seg 29-14 tagaccaccaactgtaaataagaaaataaagcctaaggct

40mer Seg 29-15 tatttacagttggtggtctaaccggaattgttgcggccgc

40mer Seg 29-16 gaattcgagctcggtacccggcggccgcaacaattccggt Supplementary Table 9. Oligos used to construct Mmus mtDNA Segment 29; Reverse complement of standard oligo design. Oligo name Oligo sequence

RC29-1 tattcctatatagccgaaaggttctttttttcgcggccgcgatcctctagagtcgacctg

RC29-2 ctttcggctatataggaatagtatgagcaataatgtctattggctttctaggctttattg

RC29-3 tctacatctaatcctactgtgaatatgtggtgggctcatacaataaagcctagaaagcca

RC29-4 acagtaggattagatgtagacacacgagcttactttacatcagccactataattatcgca

RC29-5 gggttgcaagtcagctaaatactttgacaccggtaggaattgcgataattatagtggctg

RC29-6 atttagctgacttgcaaccctacacggaggtaatattaaatgatctccagctatactatg

RC29-7 tagaccaccaactgtaaataagaaaataaagcctaaggctcatagtatagctggagatca

RC29-8 tatttacagttggtggtctaaccggaattgttgcggccgccgggtaccgagctcgaattc


MMusMito1-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 1 170 28 198 142 1-142MMusMito1-2 gtaactgccagggcggccacttgaggcgcgccctggatggaggcggataaagttgcaggMMusMito1-3 gtggccgccctggcagttacaactaggaacctctagcctgcaggtcctgcttatccacaaMMusMito1-4 CTACATTAACcacgtggtccacataaccgtgcgcaaaatgttgtggataagcaggacctMMusMito1-5 ggaccacgtgGTTAATGTAGCTTAATAACAAAGCAAAGCACTGAAAATGCTTAGATGGATMMusMito1-6 TAAGGCCAGGACCAAACCTTTGTGTTTATGGGATACAATTATCCATCTAAGCATTTTCAGMMusMito1-7 AAGGTTTGGTCCTGGCCTTATAATTAATTAGAGGTAAAATTACACATGCAAACCTCCATAMMusMito1-8 gaattcgagctcggtacccggcggccgcTTTACACCGGTCTATGGAGGTTTGCATGTGTAMMusMito2-1 caggtcgactctagaggatcgcggccgcGGTAAAATTACACATGCAAACCTCCATAGACC 2 28 28 56 284 103-386MMusMito2-2 CTCCTTAAATTTTAAGTAAATGTTTAAGGGATTTTACACCGGTCTATGGAGGTTTGCATGMMusMito2-3 TTTACTTAAAATTTAAGGAGAGGGTATCAAGCACATTAAAATAGCTTAAGACACCTTGCCMMusMito2-4 TTAATATTTATCACTGCTGAGTCCCGTGGGGGTGTGGCTAGGCAAGGTGTCTTAAGCTATMMusMito2-5 TCAGCAGTGATAAATATTAAGCAATAAACGAAAGTTTGACTAAGTTATACCTCTTAGGGTMMusMito2-6 GTTAATCGTATGACCGCGGTGGCTGGCACGAAATTTACCAACCCTAAGAGGTATAACTTAMMusMito2-7 ACCGCGGTCATACGATTAACCCAAACTAATTATCTTCGGCGTAAAACGTGTCAACTATAAMMusMito2-8 gaattcgagctcggtacccggcggccgcCTATTTATTTATTTATAGTTGACACGTTTTACMMusMito3-1 caggtcgactctagaggatcgcggccgcTCTTCGGCGTAAAACGTGTCAACTATAAATAA 3 28 28 56 284 347-630MMusMito3-2 ATGAATTTTCACATATAAGTTGGATTTTAATTCTATTTATTTATTTATAGTTGACACGTTMMusMito3-3 ACTTATATGTGAAAATTCATTGTTAGGACCTAAACTCAATAACGAAAGTAATTCTAGTCAMMusMito3-4 TCTAATCCCAGTTTGGGTCTTAGCTGTCGTGTATTATAAATGACTAGAATTACTTTCGTTMMusMito3-5 AGACCCAAACTGGGATTAGATACCCCACTATGCTTAGCCATAAACCTAAATAATTAAATTMMusMito3-6 TAAGCTATGGCTAGTAGTTCTCTGGCAAATAGTTTTGTTAAATTTAATTATTTAGGTTTAMMusMito3-7 GAACTACTAGCCATAGCTTAAAACTCAAAGGACTTGGCGGTACTTTATATCCATCTAGAGMMusMito3-8 gaattcgagctcggtacccggcggccgcTAGAACAGGCTCCTCTAGATGGATATAAAGTAMMusMito4-1 caggtcgactctagaggatcgcggccgcCTTGGCGGTACTTTATATCCATCTAGAGGAGC 4 28 28 56 284 591-874MMusMito4-2 AGATGGTGAGGTAGAGCGGGGTTTATCGATTATAGAACAGGCTCCTCTAGATGGATATAAMMusMito4-3 CCCGCTCTACCTCACCATCTCTTGCTAATTCAGCCTATATACCGCCATCTTCAGCAAACCMMusMito4-4 TTTATGTTTGATTCTTTTGCTTACTTTAATACCTTTTTAGGGTTTGCTGAAGATGGCGGTMMusMito4-5 GCAAAAGAATCAAACATAAAAACGTTAGGTCAAGGTGTAGCCAATGAAATGGGAAGAAATMMusMito4-6 AAAGGGTATAGTAATGTTCTTTTATAAGAAAATGTAGCCCATTTCTTCCCATTTCATTGGMMusMito4-7 AGAACATTACTATACCCTTTATGAAACTAAAGGACTAAGGAGGATTTAGTAGTAAATTAAMMusMito4-8 gaattcgagctcggtacccggcggccgcAGCTCTCTATTCTTAATTTACTACTAAATCCTMMusMito5-1 caggtcgactctagaggatcgcggccgcGACTAAGGAGGATTTAGTAGTAAATTAAGAAT 5 28 76 104 236 835-1070 ggcgcgccgcggtagtttatcacagttaaattgctaacgcagtcaMMusMito5-2 GTGTGTGCGTACTTCATTGCTCAATTCAATTAAGCTCTCTATTCTTAATTTACTACTAAAMMusMito5-3 GCAATGAAGTACGCACACACCGCCCGTCACCCTCCTCAAATTAAATTAAACTTAACATAAMMusMito5-4 ACGACTTATCTCCTCTCATAAACGGATGTCTAGAAATTAATTATGTTAAGTTTAATTTAAMMusMito5-5 TATGAGAGGAGATAAGTCGTAACAAGGTAAGCATACTGGAAAGTGTGCTTGGAATAATCAMMusMito5-6 TCTGGGTGTAGGCCAGATGCTTTAATATTAAGCTACACTATGATTATTCCAAGCACACTTMMusMito5-7 GCATCTGGCCTACACCCAGAAGATggcgcgccgcggtagtttatcacagttaaattgctaMMusMito5-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito6-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 6 76 28 104 236 1031-1266MMusMito6-2 CCAGATGCTTTAATATTAAGCTACggcgcgccctggatggaggcggataaagttgcaggMMusMito6-3 CTTAATATTAAAGCATCTGGCCTACACCCAGAAGATTTCATGACCAATGAACACTCTGAAMMusMito6-4 TAGTATAATTATATTTGTGTAGGGCTAGGGCTAGGATTAGTTCAGAGTGTTCATTGGTCAMMusMito6-5 ACACAAATATAATTATACTATTATATAAATCAAAACATTTATCCTACTAAAAGTATTGGAMMusMito6-6 GGTACTAGTTCTATAGCTCCTAGATGTACGAATTTCTTTCTCCAATACTTTTAGTAGGATMMusMito6-7 GGAGCTATAGAACTAGTACCGCAAGGGAAAGATGAAAGACTAATTAAAAGTAAGAACAAGMMusMito6-8 gaattcgagctcggtacccggcggccgcGTTTAATCTTTGCTTGTTCTTACTTTTAATTAMMusMito7-1 caggtcgactctagaggatcgcggccgcTGAAAGACTAATTAAAAGTAAGAACAAGCAAA 7 28 28 56 284 1227-1510MMusMito7-2 TTCTAGTTAGTTCATTATGCAAAAGGTACAAGGTTTAATCTTTGCTTGTTCTTACTTTTAMMusMito7-3 GCATAATGAACTAACTAGAAAACTTCTAACTAAAAGAATTACAGCTAGAAACCCCGAAACMMusMito7-4 ACGAGTTGATTCATAAAATTGTTTTTAGGTAGCTCGTTTGGTTTCGGGGTTTCTAGCTGTMMusMito7-5 AATTTTATGAATCAACTCGTCTATGTGGCAAAATAGTGAGAAGATTTTTAGGTAGAGGTGMMusMito7-6 TTTTTGGGTAACCAGCTATCACCAAGCTCGTTAGGCTTTTCACCTCTACCTAAAAATCTTMMusMito7-7 GATAGCTGGTTACCCAAAAAATGAATTTAAGTTCAATTTTAAACTTGCTAAAAAAACAACMMusMito7-8 gaattcgagctcggtacccggcggccgcCTTTTTGATTTTGTTGTTTTTTTAGCAAGTTTMMusMito8-1 caggtcgactctagaggatcgcggccgcTCAATTTTAAACTTGCTAAAAAAACAACAAAA 8 28 28 56 284 1471-1754MMusMito8-2 GCTGTCCCTCTTTTGGCTATAATCTAAACTTACTTTTTGATTTTGTTGTTTTTTTAGCAAMMusMito8-3 ATAGCCAAAAGAGGGACAGCTCTTCTGGAACGGAAAAAACCTTTAATAGTGAATAATTAAMMusMito8-4 GTGGCTGCTTTTAGGCCTACAATGGTTAAAAGCTGTTTTGTTAATTATTCACTATTAAAGMMusMito8-5 GTAGGCCTAAAAGCAGCCACCAATAAAGAAAGCGTTCAAGCTCAACATAAAATTTCAATTMMusMito8-6 CCAATTTTAAGTTTAGGAAGTTGGTGTAAATTATGGAATTAATTGAAATTTTATGTTGAGMMusMito8-7 CTTCCTAAACTTAAAATTGGGTTAATCTATAACTTTATAGATGCAACACTGTTAGTATGAMMusMito8-8 gaattcgagctcggtacccggcggccgcAATTCTTGTTACTCATACTAACAGTGTTGCATMMusMito9-1 caggtcgactctagaggatcgcggccgcCTTTATAGATGCAACACTGTTAGTATGAGTAA 9 28 28 56 284 1715-1998MMusMito9-2 GAGTTGTTATACGCGTATGCCTGGAGAATTGGAATTCTTGTTACTCATACTAACAGTGTTMMusMito9-3 GCATACGCGTATAACAACTCGGATAACCATTGTTAGTTAATCAGACTATAGGCAATAATCMMusMito9-4 TGGGTTAACAGAGAAGTTATAGGTGGATTATTTATAGTGTGATTATTGCCTATAGTCTGAMMusMito9-5 ATAACTTCTCTGTTAACCCAACACCGGAATGCCTAAAGGAAAGATCCAAAAAGATAAAAGMMusMito9-6 TGTTTTTGGTAAACAGGCGGGGTTCTTGTTTGCCGAGTTCCTTTTATCTTTTTGGATCTTMMusMito9-7 CCGCCTGTTTACCAAAAACATCACCTCTAGCATTACAAGTATTAGAGGCACTGCCTGCCCMMusMito9-8 gaattcgagctcggtacccggcggccgcACTTTAGTCACTGGGCAGGCAGTGCCTCTAATMMusMito10-1 caggtcgactctagaggatcgcggccgcTTACAAGTATTAGAGGCACTGCCTGCCCAGTG 10 28 76 104 236 1959-2194MMusMito10-2 CCTTTGCACGGTCAGGATACCGCGGCCGTTAAACTTTAGTCACTGGGCAGGCAGTGCCTCMMusMito10-3 GTATCCTGACCGTGCAAAGGTAGCATAATCACTTGTTCCTTAATTAGGGACTAGCATGAAMMusMito10-4 CTGATTAAAGATAAGAGACAGTTGGACCCTCGTTTAGCCGTTCATGCTAGTCCCTAATTAMMusMito10-5 TGTCTCTTATCTTTAATCAGTGAAATTGACCTTTCAGTGAAGAGGCTGAAATATAATAATMMusMito10-6 TAAGTTATATAATTTAAGCTCCATAGGGTCTTCTCGTCTTATTATTATATTTCAGCCTCTMMusMito10-7 AGCTTAAATTATATAACTTATCTAggcgcgccgcggtagtttatcacagttaaattgctaMMusMito10-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito11-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 11 76 28 104 236 2155-2390MMusMito11-2 TTTAAGCTCCATAGGGTCTTCTCGggcgcgccctggatggaggcggataaagttgcaggMMusMito11-3 AAGACCCTATGGAGCTTAAATTATATAACTTATCTATTTAATTTATTAAACCTAATGGCCMMusMito11-4 TCACCCCAACCGAAATTTCAAACTTATACTATAGTTTTTGGGCCATTAGGTTTAATAAATMMusMito11-5 TGAAATTTCGGTTGGGGTGACCTCGGAGAATAAAAAATCCTCCGAATGATTATAACCTAGMMusMito11-6 GGGTCAATAAGATATGTTGATTTTACTTTGACTTGTAAGTCTAGGTTATAATCATTCGGA

Supplementary Table 10. The design of the mouse mitochondrial genome at the nucleotide level.

caggtcgactctagaggatcgcggccgc ttacaagtattagaggcactgcctgcccagtgactaaagtttaacggccgcggtatcctgaccgtgcaaaggtagcataatcacttgttccttaattagggactagcatgaacggctaaacgagggtccaactgtctcttatctttaatcagtgaaattgacctttcagtgaagaggctgaaatataataataagacgagaagaccctatggagcttaaattatataacttatcta

ggcgcgccgcggtagtttatcacagttaaattgctaacgcagtcaggcgcggccgccgggtaccgagctcgaattc

caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcctccatccagggcgcgcc

cgagaagaccctatggagcttaaattatataacttatctatttaatttattaaacctaatggcccaaaaactatagtataagtttgaaatttcggttggggtgacctcggagaataaaaaatcctccgaatgattataacctagacttacaagtcaaagtaaaatcaacatatcttattgacccagatatattttgatcaacggaccaagttaccctagggataacagcgcaatcc

gcggccgccgggtaccgagctcgaattc

caggtcgactctagaggatcgcggccgc tcaattttaaacttgctaaaaaaacaacaaaatcaaaaagtaagtttagattatagccaaaagagggacagctcttctggaacggaaaaaacctttaatagtgaataattaacaaaacagcttttaaccattgtaggcctaaaagcagccaccaataaagaaagcgttcaagctcaacataaaatttcaattaattccataatttacaccaacttcctaaacttaaaattgggttaatctataactttatagatgcaacactgttagtatgagtaacaagaatt


caggtcgactctagaggatcgcggccgc ctttatagatgcaacactgttagtatgagtaacaagaattccaattctccaggcatacgcgtataacaactcggataaccattgttagttaatcagactataggcaataatcacactataaataatccacctataacttctctgttaacccaacaccggaatgcctaaaggaaagatccaaaaagataaaaggaactcggcaaacaagaaccccgcctgtttaccaaaaacatcacctctagcattacaagtattagaggcactgcctgcccagtgactaaagt



gtagcttaatattaaagcatctggcctacacccagaagatttcatgaccaatgaacactctgaactaatcctagccctagccctacacaaatataattatactattatataaatcaaaacatttatcctactaaaagtattggagaaagaaattcgtacatctaggagctatagaactagtaccgcaagggaaagatgaaagactaattaaaagtaagaacaagcaaagattaaac


caggtcgactctagaggatcgcggccgc tgaaagactaattaaaagtaagaacaagcaaagattaaaccttgtaccttttgcataatgaactaactagaaaacttctaactaaaagaattacagctagaaaccccgaaaccaaacgagctacctaaaaacaattttatgaatcaactcgtctatgtggcaaaatagtgagaagatttttaggtagaggtgaaaagcctaacgagcttggtgatagctggttacccaaaaaatgaatttaagttcaattttaaacttgctaaaaaaacaacaaaatcaaaaag


caggtcgactctagaggatcgcggccgc cttggcggtactttatatccatctagaggagcctgttctataatcgataaaccccgctctacctcaccatctcttgctaattcagcctatataccgccatcttcagcaaaccctaaaaaggtattaaagtaagcaaaagaatcaaacataaaaacgttaggtcaaggtgtagccaatgaaatgggaagaaatgggctacattttcttataaaagaacattactataccctttatgaaactaaaggactaaggaggatttagtagtaaattaagaatagagagct


caggtcgactctagaggatcgcggccgc gactaaggaggatttagtagtaaattaagaatagagagcttaattgaattgagcaatgaagtacgcacacaccgcccgtcaccctcctcaaattaaattaaacttaacataattaatttctagacatccgtttatgagaggagataagtcgtaacaaggtaagcatactggaaagtgtgcttggaataatcatagtgtagcttaatattaaagcatctggcctacacccagaagat

ggtaaaattacacatgcaaacctccatagaccggtgtaaaatcccttaaacatttacttaaaatttaaggagagggtatcaagcacattaaaatagcttaagacaccttgcctagccacacccccacgggactcagcagtgataaatattaagcaataaacgaaagtttgactaagttatacctcttagggttggtaaatttcgtgccagccaccgcggtcatacgattaacccaaactaattatcttcggcgtaaaacgtgtcaactataaataaataaatag


caggtcgactctagaggatcgcggccgc tcttcggcgtaaaacgtgtcaactataaataaataaatagaattaaaatccaacttatatgtgaaaattcattgttaggacctaaactcaataacgaaagtaattctagtcatttataatacacgacagctaagacccaaactgggattagataccccactatgcttagccataaacctaaataattaaatttaacaaaactatttgccagagaactactagccatagcttaaaactcaaaggacttggcggtactttatatccatctagaggagcctgttcta


# mtDNA bases synthesized

Total # extra bases

gttaatgtagcttaataacaaagcaaagcactgaaaatgcttagatggataattgtatcccataaacacaaaggtttggtcctggccttataattaattagaggtaaaattacacatgcaaacctccatagaccggtgtaaa

gcggccgccgggtaccgagctcgaattccaggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcctccatccagggcgcgcctcaagtggccgccctggcagttacaactaggaacctctagcctgcagg tcctgcttatccacaacattttgcgcacggttatgtggaccacgtg

caggtcgactctagaggatcgcggccgc

# extra bases on right

# extra bases on left

Segment #Oligonucleotide sequenceOligo name Vector overlaps and restriction sites on rightSynthesized mtDNA SequenceVector overlaps and restriction sites on leftSynthesized mtDNA

sequence coordinates


MMusMito11-7 TCAACATATCTTATTGACCCAGATATATTTTGATCAACGGACCAAGTTACCCTAGGGATAMMusMito11-8 gaattcgagctcggtacccggcggccgcGGATTGCGCTGTTATCCCTAGGGTAACTTGGTMMusMito12-1 caggtcgactctagaggatcgcggccgcATCAACGGACCAAGTTACCCTAGGGATAACAG 12 28 28 56 284 2351-2634MMusMito12-2 AAACCCTAATTGTCGATATGAACTCTTAAATAGGATTGCGCTGTTATCCCTAGGGTAACTMMusMito12-3 CATATCGACAATTAGGGTTTACGACCTCGATGTTGGATCAGGACATCCCAATGGTGTAGAMMusMito12-4 GTAGGACTTTAATCGTTGAACAAACGAACCATTAATAGCTTCTACACCATTGGGATGTCCMMusMito12-5 TTCAACGATTAAAGTCCTACGTGATCTGAGTTCAGACCGGAGCAATCCAGGTCGGTTTCTMMusMito12-6 TTCTCTTGTCCTTTCGTACTGGGAGAAATCGTAAATAGATAGAAACCGACCTGGATTGCTMMusMito12-7 AGTACGAAAGGACAAGAGAAATAGAGCCACCTTACAAATAAGCGCTCTCAACTTAATTTAMMusMito12-8 gaattcgagctcggtacccggcggccgcAGATTTTATTCATAAATTAAGTTGAGAGCGCTMMusMito13-1 caggtcgactctagaggatcgcggccgcTACAAATAAGCGCTCTCAACTTAATTTATGAA 13 28 28 56 284 2595-2878MMusMito13-2 TAGGTTAGAGGGTGTACGTATATATTTTATTTAGATTTTATTCATAAATTAAGTTGAGAGMMusMito13-3 TACGTACACCCTCTAACCTAGAGAAGGTTATTAGGGTGGCAGAGCCAGGAAATTGCGTAAMMusMito13-4 AGGGAGAGGATTTGAACCTCTGGGAACAAGGTTTTAAGTCTTACGCAATTTCCTGGCTCTMMusMito13-5 GAGGTTCAAATCCTCTCCCTAATAGTGTTCTTTATTAATATCCTAACACTCCTCGTCCCCMMusMito13-6 TGCGTTCTACTAATGTTAGGAAGGCTATGGCGATTAGAATGGGGACGAGGAGTGTTAGGAMMusMito13-7 CCTAACATTAGTAGAACGCAAAATCTTAGGGTACATACAACTACGAAAAGGCCCTAACATMMusMito13-8 gaattcgagctcggtacccggcggccgcTATGGACCAACAATGTTAGGGCCTTTTCGTAGMMusMito14-1 caggtcgactctagaggatcgcggccgcACATACAACTACGAAAAGGCCCTAACATTGTT 14 28 28 56 284 2839-3122MMusMito14-2 TTATGGCGTCTGCAAATGGTTGTAAAATGCCGTATGGACCAACAATGTTAGGGCCTTTTCMMusMito14-3 ACCATTTGCAGACGCCATAAAATTATTTATAAAAGAACCAATACGCCCTTTAACAACCTCMMusMito14-4 TGTGAGTGATAGGGTAGGTGCAATAATAAATAAGGATATAGAGGTTGTTAAAGGGCGTATMMusMito14-5 CACCTACCCTATCACTCACACTAGCATTAAGTCTATGAGTTCCCCTACCAATACCACACCMMusMito14-6 GCTAAAATAAATAAAATCCCTAGGTTTAAATTAATTAATGGGTGTGGTATTGGTAGGGGAMMusMito14-7 GGGATTTTATTTATTTTAGCAACATCTAGCCTATCAGTTTACTCCATTCTATGATCAGGAMMusMito14-8 gaattcgagctcggtacccggcggccgcGTTTGAGGCTCATCCTGATCATAGAATGGAGTMMusMito15-1 caggtcgactctagaggatcgcggccgcATCAGTTTACTCCATTCTATGATCAGGATGAG 15 28 76 104 236 3083-3318MMusMito15-2 GCTCGTAAAGCTCCGAATAGTGAGTATTTGGAGTTTGAGGCTCATCCTGATCATAGAATGMMusMito15-3 CTATTCGGAGCTTTACGAGCCGTAGCCCAAACAATTTCATATGAAGTAACCATAGCTATTMMusMito15-4 GAGAGTAGGATCCATTTATTAATAGAACTGATAAAAGGATAATAGCTATGGTTACTTCATMMusMito15-5 AATAAATGGATCCTACTCTCTACAAACACTTATTACAACCCAAGAACACATATGATTACTMMusMito15-6 TGAGATAAATCATATTATGGCTATGGGTCAGGCTGGCAGAAGTAATCATATGTGTTCTTGMMusMito15-7 CCATAATATGATTTATCTCAACCCggcgcgccgcggtagtttatcacagttaaattgctaMMusMito15-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito16-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 16 76 28 104 236 3279-3514MMusMito16-2 TATTATGGCTATGGGTCAGGCTGGggcgcgccctggatggaggcggataaagttgcaggMMusMito16-3 CCTGACCCATAGCCATAATATGATTTATCTCAACCCTAGCAGAAACAAACCGGGCCCCCTMMusMito16-4 AACCCTGATACTAATTCTGATTCTCCTTCTGTCAGGTCGAAGGGGGCCCGGTTTGTTTCTMMusMito16-5 TCAGAATTAGTATCAGGGTTTAACGTAGAATACGCAGCCGGCCCATTCGCGTTATTCTTTMMusMito16-6 TTAGGGCGTTTATTAGAATAATGTTAGTGTACTCTGCTATAAAGAATAACGCGAATGGGCMMusMito16-7 TATTCTAATAAACGCCCTAACAACTATTATCTTCCTAGGACCCCTATACTATATCAATTTMMusMito16-8 gaattcgagctcggtacccggcggccgcTAGAGTTCTGGTAAATTGATATAGTATAGGGGMMusMito17-1 caggtcgactctagaggatcgcggccgcTCCTAGGACCCCTATACTATATCAATTTACCA 17 28 28 56 284 3475-3758MMusMito17-2 GTAGTAGAGCTTCTATTATGAAGTTAGTTGAGTAGAGTTCTGGTAAATTGATATAGTATAMMusMito17-3 CATAATAGAAGCTCTACTACTATCATCAACATTCCTATGGATCCGAGCATCTTATCCACGMMusMito17-4 GTTTTTTCATAGAAGATGTATAAGTTGATCGTAACGGAAGCGTGGATAAGATGCTCGGATMMusMito17-5 TACATCTTCTATGAAAAAACTTTCTACCCCTAACACTAGCATTATGTATGTGACATATTTMMusMito17-6 TATATGTATGGTGGTACTCCCGCTGTAAAAATTGGTAAAGAAATATGTCACATACATAATMMusMito17-7 GGAGTACCACCATACATATAGAAATATGTCTGATAAAAGAATTACTTTGATAGAGTAAATMMusMito17-8 gaattcgagctcggtacccggcggccgcTGAACCTCTATAATTTACTCTATCAAAGTAATMMusMito18-1 caggtcgactctagaggatcgcggccgcATAAAAGAATTACTTTGATAGAGTAAATTATA 18 28 28 56 284 3719-4002MMusMito18-2 TCAATTCCTATTGTCCTAGAAATAAGAGGGCTTGAACCTCTATAATTTACTCTATCAAAGMMusMito18-3 TCTAGGACAATAGGAATTGAACCTACACTTAAGAATTCAAAATTCTCCGTGCTACCTAAAMMusMito18-4 CGATAGCTTAATTAGCTGACCTTACTATTAGGATAAGGTGTTTAGGTAGCACGGAGAATTMMusMito18-5 GTCAGCTAATTAAGCTATCGGGCCCATACCCCGAAAACGTTGGTTTAAATCCTTCCCGTAMMusMito18-6 TTGTGAAGTAGATGATGGCAAGGGTGATAGGATTTATTAGTACGGGAAGGATTTAAACCAMMusMito18-7 TGCCATCATCTACTTCACAATCTTCTTAGGTCCTGTAATCACAATATCCAGCACCAACCTMMusMito18-8 gaattcgagctcggtacccggcggccgcCATATTAGTATTAGGTTGGTGCTGGATATTGTMMusMito19-1 caggtcgactctagaggatcgcggccgcCTGTAATCACAATATCCAGCACCAACCTAATA 19 28 28 56 284 3963-4246MMusMito19-2 TAATTGCTAGTAGGCTGAATTCCAGGCCTACTCATATTAGTATTAGGTTGGTGCTGGATAMMusMito19-3 ATTCAGCCTACTAGCAATTATCCCCATACTAATCAACAAAAAAAACCCACGATCAACTGAMMusMito19-4 TGAGGCTGTTGCTTGTGTGACGAAGTATTTTGTTGCTGCTTCAGTTGATCGTGGGTTTTTMMusMito19-5 TCACACAAGCAACAGCCTCAATAATTATCCTCCTGGCCATCGTACTCAACTATAAACAACMMusMito19-6 ATAAGACCGTTTGTTTGTTGTTGAAATATTCATGTTCCTAGTTGTTTATAGTTGAGTACGMMusMito19-7 CAACAAACAAACGGTCTTATCCTTAACATAACATTAATAGCCCTATCCATAAAACTAGGCMMusMito19-8 gaattcgagctcggtacccggcggccgcGAATGGGGCGAGGCCTAGTTTTATGGATAGGGMMusMito20-1 caggtcgactctagaggatcgcggccgcATTAATAGCCCTATCCATAAAACTAGGCCTCG 20 28 76 104 236 4207-4442MMusMito20-2 ATCCCTTGAGTTACTTCTGGTAATCAGAAGTGGAATGGGGCGAGGCCTAGTTTTATGGATMMusMito20-3 CCAGAAGTAACTCAAGGGATCCCACTGCACATAGGACTTATTCTTCTTACATGACAAAAAMMusMito20-4 GTAGCGGGTAAATTTGAATTAAAATTGATAGGGGAGCAATTTTTTGTCATGTAAGAAGAAMMusMito20-5 AATTCAAATTTACCCGCTACTCAACTCTACTATCATTTTAATACTAGCAATTACTTCTATMMusMito20-6 TATTTGTGTTTGGTTAAGTCCTCCTCATGCCCCTATGAAAATAGAAGTAATTGCTAGTATMMusMito20-7 GACTTAACCAAACACAAATACGAAggcgcgccgcggtagtttatcacagttaaattgctaMMusMito20-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito21-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 21 76 28 104 236 4403-4638MMusMito21-2 GTTAAGTCCTCCTCATGCCCCTATggcgcgccctggatggaggcggataaagttgcaggMMusMito21-3 GGGCATGAGGAGGACTTAACCAAACACAAATACGAAAAATTATAGCCTATTCATCAATTGMMusMito21-4 GGGTTGTAAGGAAGAATTGCTAATATTCATCCTATGTGGGCAATTGATGAATAGGCTATAMMusMito21-5 GCAATTCTTCCTTACAACCCATCCCTCACTCTACTCAACCTCATAATCTATATTATTCTTMMusMito21-6 TAGAGTTATTTAGTATAAGTGCTATGAATATAGGGGCTGTAAGAATAATATAGATTATGAMMusMito21-7 ACTTATACTAAATAACTCTATAACCATCAACTCAATCTCACTTCTATGAAATAAAACTCCMMusMito21-8 gaattcgagctcggtacccggcggccgcGTTAGTATTGCTGGAGTTTTATTTCATAGAAGMMusMito22-1 caggtcgactctagaggatcgcggccgcCAATCTCACTTCTATGAAATAAAACTCCAGCA 22 28 28 56 284 4599-4882MMusMito22-2 CTCCTAGGGATAGTAATATCAGTGAGATTATAGTTAGTATTGCTGGAGTTTTATTTCATAMMusMito22-3 GATATTACTATCCCTAGGAGGCCTTCCACCACTAACAGGATTCTTACCAAAATGAATTATMMusMito22-4 TGCTATAATTAGACAGTTGTTTTTTATAAGTTCTGTGATGATAATTCATTTTGGTAAGAAMMusMito22-5 ACAACTGTCTAATTATAGCAACACTCATAGCAATAATAGCTCTACTAAACCTATTCTTTTMMusMito22-6 GGAAATATTGTTAGTGAAGTGGAATAAATTAGGCGAGTATAAAAGAATAGGTTTAGTAGAMMusMito22-7 ACTTCACTAACAATATTTCCAACCAACAATAACTCAAAAATAATAACTCACCAAACAAAAMMusMito22-8 gaattcgagctcggtacccggcggccgcGTTGGGTTTAGTTTTTGTTTGGTGAGTTATTAMMusMito23-1 caggtcgactctagaggatcgcggccgcCTCAAAAATAATAACTCACCAAACAAAAACTA 23 28 28 56 284 4843-5126MMusMito23-2 GTGCTTATGATAGCTAGGGTGGAAAATATTAGGTTGGGTTTAGTTTTTGTTTGGTGAGTT

caggtcgactctagaggatcgcggccgc caatctcacttctatgaaataaaactccagcaatactaactataatctcactgatattactatccctaggaggccttccaccactaacaggattcttaccaaaatgaattatcatcacagaacttataaaaaacaactgtctaattatagcaacactcatagcaataatagctctactaaacctattcttttatactcgcctaatttattccacttcactaacaatatttccaaccaacaataactcaaaaataataactcaccaaacaaaaactaaacccaac


caggtcgactctagaggatcgcggccgc ctcaaaaataataactcaccaaacaaaaactaaacccaacctaatattttccaccctagctatcataagcacaataacc


caggtcgactctagaggatcgcggccgc attaatagccctatccataaaactaggcctcgccccattccacttctgattaccagaagtaactcaagggatcccactgcacataggacttattcttcttacatgacaaaaaattgctcccctatcaattttaattcaaatttacccgctactcaactctactatcattttaatactagcaattacttctattttcataggggcatgaggaggacttaaccaaacacaaatacgaa



ataggggcatgaggaggacttaaccaaacacaaatacgaaaaattatagcctattcatcaattgcccacataggatgaatattagcaattcttccttacaacccatccctcactctactcaacctcataatctatattattcttacagcccctatattcatagcacttatactaaataactctataaccatcaactcaatctcacttctatgaaataaaactccagcaatactaac


caggtcgactctagaggatcgcggccgc ataaaagaattactttgatagagtaaattatagaggttcaagccctcttatttctaggacaataggaattgaacctacacttaagaattcaaaattctccgtgctacctaaacaccttatcctaatagtaaggtcagctaattaagctatcgggcccataccccgaaaacgttggtttaaatccttcccgtactaataaatcctatcacccttgccatcatctacttcacaatcttcttaggtcctgtaatcacaatatccagcaccaacctaatactaatatg


caggtcgactctagaggatcgcggccgc ctgtaatcacaatatccagcaccaacctaatactaatatgagtaggcctggaattcagcctactagcaattatccccatactaatcaacaaaaaaaacccacgatcaactgaagcagcaacaaaatacttcgtcacacaagcaacagcctcaataattatcctcctggccatcgtactcaactataaacaactaggaacatgaatatttcaacaacaaacaaacggtcttatccttaacataacattaatagccctatccataaaactaggcctcgccccattc



ccagcctgacccatagccataatatgatttatctcaaccctagcagaaacaaaccgggcccccttcgacctgacagaaggagaatcagaattagtatcagggtttaacgtagaatacgcagccggcccattcgcgttattctttatagcagagtacactaacattattctaataaacgccctaacaactattatcttcctaggacccctatactatatcaatttaccagaactcta


caggtcgactctagaggatcgcggccgc tcctaggacccctatactatatcaatttaccagaactctactcaactaacttcataatagaagctctactactatcatcaacattcctatggatccgagcatcttatccacgcttccgttacgatcaacttatacatcttctatgaaaaaactttctacccctaacactagcattatgtatgtgacatatttctttaccaatttttacagcgggagtaccaccatacatatagaaatatgtctgataaaagaattactttgatagagtaaattatagaggttca


caggtcgactctagaggatcgcggccgc acatacaactacgaaaaggccctaacattgttggtccatacggcattttacaaccatttgcagacgccataaaattatttataaaagaaccaatacgccctttaacaacctctatatccttatttattattgcacctaccctatcactcacactagcattaagtctatgagttcccctaccaataccacacccattaattaatttaaacctagggattttatttattttagcaacatctagcctatcagtttactccattctatgatcaggatgagcctcaaac


caggtcgactctagaggatcgcggccgc atcagtttactccattctatgatcaggatgagcctcaaactccaaatactcactattcggagctttacgagccgtagcccaaacaatttcatatgaagtaaccatagctattatccttttatcagttctattaataaatggatcctactctctacaaacacttattacaacccaagaacacatatgattacttctgccagcctgacccatagccataatatgatttatctcaaccc


caggtcgactctagaggatcgcggccgc atcaacggaccaagttaccctagggataacagcgcaatcctatttaagagttcatatcgacaattagggtttacgacctcgatgttggatcaggacatcccaatggtgtagaagctattaatggttcgtttgttcaacgattaaagtcctacgtgatctgagttcagaccggagcaatccaggtcggtttctatctatttacgatttctcccagtacgaaaggacaagagaaatagagccaccttacaaataagcgctctcaacttaatttatgaataaaatct


caggtcgactctagaggatcgcggccgc tacaaataagcgctctcaacttaatttatgaataaaatctaaataaaatatatacgtacaccctctaacctagagaaggttattagggtggcagagccaggaaattgcgtaagacttaaaaccttgttcccagaggttcaaatcctctccctaatagtgttctttattaatatcctaacactcctcgtccccattctaatcgccatagccttcctaacattagtagaacgcaaaatcttagggtacatacaactacgaaaaggccctaacattgttggtccata


aagttaccctagggataacagcgcaatcc


MMusMito23-3 ACCCTAGCTATCATAAGCACAATAACCCTACCCCTAGCCCCCCAACTAATTACCTAGAAGMMusMito23-4 TCTTAGGGCTTTGAAGGCTCGCGGACTAGTATATCCTAAACTTCTAGGTAATTAGTTGGGMMusMito23-5 GAGCCTTCAAAGCCCTAAGAAAACACACAAGTTTAACTTCTGATAAGGACTGTAAGACTTMMusMito23-6 TAATTAAAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCTTACAGTCCTTATCAMMusMito23-7 CAAATCAATTGCTTTAATTAAGCTAAGACCTCAACTAGATTGGCAGGAATTAAACCTACGMMusMito23-8 gaattcgagctcggtacccggcggccgcTAACTAAATTTTCGTAGGTTTAATTCCTGCCAMMusMito24-1 caggtcgactctagaggatcgcggccgcAACTAGATTGGCAGGAATTAAACCTACGAAAA 24 28 28 56 284 5087-5370MMusMito24-2 AGATTGAAGCCAGTAATAGGGTATTTAGCTGTTAACTAAATTTTCGTAGGTTTAATTCCTMMusMito24-3 CCTATTACTGGCTTCAATCTACTTCTACCGCCGAAAAAAAAAAATGGCGGTAGAAGTCTTMMusMito24-4 TGTCGAATTGCAAATTCGAAGGTGTAGAGAAATCTCTACTAAGACTTCTACCGCCATTTTMMusMito24-5 TTCGAATTTGCAATTCGACATGAATATCACCTTAAGACCTCTGGTAAAAAGAGGATTTAAMMusMito24-6 GGCTGAGTAAGCATTAGACTGTAAATCTAAACACAGAGGTTTAAATCCTCTTTTTACCAGMMusMito24-7 AGTCTAATGCTTACTCAGCCATTTTACCTATGTTCATTAATCGTTGATTATTCTCAACCAMMusMito24-8 gaattcgagctcggtacccggcggccgcTATCTTTGTGATTGGTTGAGAATAATCAACGAMMusMito25-1 caggtcgactctagaggatcgcggccgcTTCATTAATCGTTGATTATTCTCAACCAATCA 25 28 124 152 188 5331-5518MMusMito25-2 TCAGGCTCCGAATAGTAGATAGAGGGTTCCGATATCTTTGTGATTGGTTGAGAATAATCAMMusMito25-3 ATCTACTATTCGGAGCCTGAGCGGGAATAGTGGGTACTGCACTAAGTATTTTAATTCGAGMMusMito25-4 TGGTCATCTCCTAAAAGTGCACCTGGTTGACCTAATTCTGCTCGAATTAAAATACTTAGTMMusMito25-5 GCACTTTTAGGAGATGACCAAATTTACAATGTTATCGTAACTGCCCATGCTTTTGTcctgMMusMito25-6 aacttcgtatagcatacattatacgaagttatggaccctgcaggACAAAAGCATGGGCAGMMusMito25-7 aatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctMMusMito25-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito26-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 26 124 28 152 188 5479-5666MMusMito26-2 gtaactgccagggcggccacttgaggcgcgccctggatggaggcggataaagttgcaggMMusMito26-3 gtggccgccctggcagttacaactaggaacctctagcctgcaggACCAAATTTACAATGTMMusMito26-4 TATGAAGAAAATTATAACAAAAGCATGGGCAGTTACGATAACATTGTAAATTTGGTcctgMMusMito26-5 TTGTTATAATTTTCTTCATAGTAATACCAATAATAATTGGAGGCTTTGGAAACTGACTTGMMusMito26-6 CGTGGGAATGCTATATCTGGGGCTCCGATTATTAGTGGGACAAGTCAGTTTCCAAAGCCTMMusMito26-7 CCAGATATAGCATTCCCACGAATAAATAATATAAGTTTTTGACTCCTACCACCATCATTTMMusMito26-8 gaattcgagctcggtacccggcggccgcTAGGAGAAGGAGAAATGATGGTGGTAGGAGTCMMusMito27-1 caggtcgactctagaggatcgcggccgcAAGTTTTTGACTCCTACCACCATCATTTCTCC 27 28 28 56 284 5627-5910MMusMito27-2 GTTCCTGCTCCTGCTTCTACTATTGATGATGCTAGGAGAAGGAGAAATGATGGTGGTAGGMMusMito27-3 GTAGAAGCAGGAGCAGGAACAGGATGAACAGTCTACCCACCTCTAGCCGGAAATCTAGCMMusMito27-4 GAAGGGAGAAAATTGTTAGGTCTACTGATGCTCCTGCATGGGCTAGATTTCCGGCTAGAGMMusMito27-5 CCTAACAATTTTCTCCCTTCATTTAGCTGGAGTGTCATCTATTTTAGGTGCAATTAATTTMMusMito27-6 TGTTATGGCTGGGGGTTTCATGTTGATAATAGTGGTAATAAAATTAATTGCACCTAAAATMMusMito27-7 TGAAACCCCCAGCCATAACACAGTATCAAACTCCACTATTTGTCTGATCCGTACTTATTAMMusMito27-8 gaattcgagctcggtacccggcggccgcGCAGTACGGCTGTAATAAGTACGGATCAGACAMMusMito28-1 caggtcgactctagaggatcgcggccgcCCACTATTTGTCTGATCCGTACTTATTACAGC 28 28 28 56 284 5871-6154MMusMito28-2 GCCTGCGGCTAGCACTGGTAGTGATAATAGGAGCAGTACGGCTGTAATAAGTACGGATCAMMusMito28-3 TACCAGTGCTAGCCGCAGGCATTACTATACTACTAACAGACCGCAACCTAAACACAACTTMMusMito28-4 TGGTAGAGAATTGGGTCCCCTCCTCCAGCGGGATCAAAGAAAGTTGTGTTTAGGTTGCGGMMusMito28-5 GGGGACCCAATTCTCTACCAGCATCTGTTCTGATTCTTTGGGCACCCAGAAGTTTATATTMMusMito28-6 TAACTACATGTGAAATAATTCCAAATCCTGGGAGGATAAGAATATAAACTTCTGGGTGCCMMusMito28-7 AATTATTTCACATGTAGTTACTTACTACTCCGGAAAAAAAGAACCTTTCGGCTATATAGGMMusMito28-8 gaattcgagctcggtacccggcggccgcGCTCATACTATTCCTATATAGCCGAAAGGTTCMMusMito29-1 caggtcgactctagaggatcgcggccgcGAAAAAAAGAACCTTTCGGCTATATAGGAATA 29 28 28 56 284 6115-6398MMusMito29-2 CAATAAAGCCTAGAAAGCCAATAGACATTATTGCTCATACTATTCCTATATAGCCGAAAGMMusMito29-3 TGGCTTTCTAGGCTTTATTGTATGAGCCCACCACATATTCACAGTAGGATTAGATGTAGAMMusMito29-4 TGCGATAATTATAGTGGCTGATGTAAAGTAAGCTCGTGTGTCTACATCTAATCCTACTGTMMusMito29-5 CAGCCACTATAATTATCGCAATTCCTACCGGTGTCAAAGTATTTAGCTGACTTGCAACCCMMusMito29-6 CATAGTATAGCTGGAGATCATTTAATATTACCTCCGTGTAGGGTTGCAAGTCAGCTAAATMMusMito29-7 TGATCTCCAGCTATACTATGAGCCTTAGGCTTTATTTTCTTATTTACAGTTGGTGGTCTAMMusMito29-8 gaattcgagctcggtacccggcggccgcAACAATTCCGGTTAGACCACCAACTGTAAATAMMusMito30-1 caggtcgactctagaggatcgcggccgcTATTTTCTTATTTACAGTTGGTGGTCTAACCG 30 28 76 104 236 6359-6594MMusMito30-2 TGAAGCACGATGTCAAGGGATGAGTTGGATAAAACAATTCCGGTTAGACCACCAACTGTAMMusMito30-3 TCCCTTGACATCGTGCTTCACGATACATACTATGTAGTAGCCCATTTCCACTATGTTCTAMMusMito30-4 GAACAAATCCTGCTATGATAGCAAACACTGCTCCCATTGATAGAACATAGTGGAAATGGGMMusMito30-5 TATCATAGCAGGATTTGTTCACTGATTCCCATTATTTTCAGGCTTCACCCTAGATGACACMMusMito30-6 TACTCCTACGAATATGATGGCGAAGTGGGCTTTTGCTCATGTGTCATCTAGGGTGAAGCCMMusMito30-7 CCATCATATTCGTAGGAGTAAACAggcgcgccgcggtagtttatcacagttaaattgctaMMusMito30-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito31-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 31 76 28 104 236 6555-6790MMusMito31-2 TATGATGGCGAAGTGGGCTTTTGCggcgcgccctggatggaggcggataaagttgcaggMMusMito31-3 AAGCCCACTTCGCCATCATATTCGTAGGAGTAAACATAACATTCTTCCCTCAACATTTCCMMusMito31-4 GGGTAGTCTGAGTAGCGTCGTGGTATTCCTGAAAGGCCCAGGAAATGTTGAGGGAAGAATMMusMito31-5 CGACGCTACTCAGACTACCCAGATGCTTACACCACATGAAACACTGTCTCTTCTATAGGAMMusMito31-6 TAAAGATCATGATGAGAACAGCTGTTAGTGAAATAAATGATCCTATAGAAGAGACAGTGTMMusMito31-7 TGTTCTCATCATGATCTTTATAATTTGAGAGGCCTTTGCTTCAAAACGAGAAGTAATATCMMusMito31-8 gaattcgagctcggtacccggcggccgcGCATACGATACTGATATTACTTCTCGTTTTGAMMusMito32-1 caggtcgactctagaggatcgcggccgcCCTTTGCTTCAAAACGAGAAGTAATATCAGTA 32 28 28 56 284 6751-7034MMusMito32-2 GGCAGCCATGAAGTCATTCTAAATTTGTTGAAGCATACGATACTGATATTACTTCTCGTTMMusMito32-3 AGAATGACTTCATGGCTGCCCTCCACCATATCACACATTCGAGGAACCAACCTATGTAAAMMusMito32-4 CAATTTTAGGGGGTTCGATTCCTTCCTTTCTTATTTTACTTTTACATAGGTTGGTTCCTCMMusMito32-5 AATCGAACCCCCTAAAATTGGTTTCAAGCCAATCTCATATCCTATATGTCTTTCTCAATAMMusMito32-6 TAACTTTGACAAAGTTATGTAATTGATTTTACTAATATCTTATTGAGAAAGACATATAGGMMusMito32-7 ACATAACTTTGTCAAAGTTAAATTATAGATCAATAATCTATATATCTTATATGGCCTACCMMusMito32-8 gaattcgagctcggtacccggcggccgcCAAGTTGGAATGGGTAGGCCATATAAGATATAMMusMito33-1 caggtcgactctagaggatcgcggccgcATAATCTATATATCTTATATGGCCTACCCATT 33 28 28 56 284 6995-7278MMusMito33-2 TTCTATAATAGGGGATGTGGCGTCTTGTAGACCAAGTTGGAATGGGTAGGCCATATAAGAMMusMito33-3 CCACATCCCCTATTATAGAAGAGCTAATAAATTTCCATGATCACACACTAATAATTGTTTMMusMito33-4 ATTAGCGAGATGATATAGAGGACTAAGGAGCTAATTAGGAAAACAATTATTAGTGTGTGAMMusMito33-5 CTCTATATCATCTCGCTAATATTAACAACAAAACTAACACATACAAGCACAATAGATGCAMMusMito33-6 TTACAGCTGGTAGAATAGTTCAAATGGTTTCAACTTCTTGTGCATCTATTGTGCTTGTATMMusMito33-7 AACTATTCTACCAGCTGTAATCCTTATCATAATTGCTCTCCCCTCTCTACGCATTCTATAMMusMito33-8 gaattcgagctcggtacccggcggccgcTCGTCTATTATATATAGAATGCGTAGAGAGGGMMusMito34-1 caggtcgactctagaggatcgcggccgcTTGCTCTCCCCTCTCTACGCATTCTATATATA 34 28 28 56 284 7239-7522MMusMito34-2 TGGTTTTAACGGTTAATACGGGGTTGTTGATTTCGTCTATTATATATAGAATGCGTAGAGMMusMito34-3 CGTATTAACCGTTAAAACCATAGGGCACCAATGATACTGAAGCTACGAATATACTGACTAMMusMito34-4 GTTTGTTGGGATTATATATGAATCAAAGCATAGGTCTTCATAGTCAGTATATTCGTAGCTMMusMito34-5 CATATATAATCCCAACAAACGACCTAAAACCTGGTGAACTACGACTGCTAGAAGTTGATAMMusMito34-6 AATATACGGATTGGAAGTTCTATTGGCAGAACGACTCGGTTATCAACTTCTAGCAGTCGT

caggtcgactctagaggatcgcggccgc ttgctctcccctctctacgcattctatatataatagacgaaatcaacaaccccgtattaaccgttaaaaccatagggcaccaatgatactgaagctacgaatatactgactatgaagacctatgctttgattcatatataatcccaacaaacgacctaaaacctggtgaactacgactgctagaagttgataaccgagtcgttctgccaatagaacttccaatccgtatattaatttcatctgaaga


caggtcgactctagaggatcgcggccgc cctttgcttcaaaacgagaagtaatatcagtatcgtatgcttcaacaaatttagaatgacttcatggctgccctccaccatatcacacattcgaggaaccaacctatgtaaaagtaaaataagaaaggaaggaatcgaaccccctaaaattggtttcaagccaatctcatatcctatatgtctttctcaataagatattagtaaaatcaattacataactttgtcaaagttaaattatagatcaataatctatatatcttatatggcctacccattccaacttg


caggtcgactctagaggatcgcggccgc ataatctatatatcttatatggcctacccattccaacttggtctacaagacgccacatcccctattatagaagagctaataaatttccatgatcacacactaataattgttttcctaattagctccttagtcctctatatcatctcgctaatattaacaacaaaactaacacatacaagcacaatagatgcacaagaagttgaaaccatttgaactattctaccagctgtaatccttatcataattgctctcccctctctacgcattctatatataatagacga


caggtcgactctagaggatcgcggccgc tattttcttatttacagttggtggtctaaccggaattgttttatccaactcatcccttgacatcgtgcttcacgatacatactatgtagtagcccatttccactatgttctatcaatgggagcagtgtttgctatcatagcaggatttgttcactgattcccattattttcaggcttcaccctagatgacacatgagcaaaagcccacttcgccatcatattcgtaggagtaaaca



gcaaaagcccacttcgccatcatattcgtaggagtaaacataacattcttccctcaacatttcctgggcctttcaggaataccacgacgctactcagactacccagatgcttacaccacatgaaacactgtctcttctataggatcatttatttcactaacagctgttctcatcatgatctttataatttgagaggcctttgcttcaaaacgagaagtaatatcagtatcgtatgc


caggtcgactctagaggatcgcggccgc ccactatttgtctgatccgtacttattacagccgtactgctcctattatcactaccagtgctagccgcaggcattactatactactaacagaccgcaacctaaacacaactttctttgatcccgctggaggaggggacccaattctctaccagcatctgttctgattctttgggcacccagaagtttatattcttatcctcccaggatttggaattatttcacatgtagttacttactactccggaaaaaaagaacctttcggctatataggaatagtatgagc


caggtcgactctagaggatcgcggccgc gaaaaaaagaacctttcggctatataggaatagtatgagcaataatgtctattggctttctaggctttattgtatgagcccaccacatattcacagtaggattagatgtagacacacgagcttactttacatcagccactataattatcgcaattcctaccggtgtcaaagtatttagctgacttgcaaccctacacggaggtaatattaaatgatctccagctatactatgagccttaggctttattttcttatttacagttggtggtctaaccggaattgtt


caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcctccatccagggcgcgcctcaagtggccgccctggcagttacaactaggaacctctagcctgcagg

accaaatttacaatgttatcgtaactgcccatgcttttgttataattttcttcatagtaataccaataataattggaggctttggaaactgacttgtcccactaataatcggagccccagatatagcattcccacgaataaataatataagtttttgactcctaccaccatcatttctccttctccta


caggtcgactctagaggatcgcggccgc aagtttttgactcctaccaccatcatttctccttctcctagcatcatcaatagtagaagcaggagcaggaacaggatgaacagtctacccacctctagccggaaatctagcccatgcaggagcatcagtagacctaacaattttctcccttcatttagctggagtgtcatctattttaggtgcaattaattttattaccactattatcaacatgaaacccccagccataacacagtatcaaactccactatttgtctgatccgtacttattacagccgtactgc


caggtcgactctagaggatcgcggccgc aactagattggcaggaattaaacctacgaaaatttagttaacagctaaataccctattactggcttcaatctacttctaccgccgaaaaaaaaaaatggcggtagaagtcttagtagagatttctctacaccttcgaatttgcaattcgacatgaatatcaccttaagacctctggtaaaaagaggatttaaacctctgtgtttagatttacagtctaatgcttactcagccattttacctatgttcattaatcgttgattattctcaaccaatcacaaagata


caggtcgactctagaggatcgcggccgc ttcattaatcgttgattattctcaaccaatcacaaagatatcggaaccctctatctactattcggagcctgagcgggaatagtgggtactgcactaagtattttaattcgagcagaattaggtcaaccaggtgcacttttaggagatgaccaaatttacaatgttatcgtaactgcccatgcttttgt

cctgcagggtccataacttcgtataatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctaacgcagtcaggcgcggccgccgggtaccgagctcgaattc

g gctacccctagccccccaactaattacctagaagtttaggatatactagtccgcgagccttcaaagccctaagaaaacacacaagtttaacttctgataaggactgtaagacttcatcctacatctattgaatgcaaatcaattgctttaattaagctaagacctcaactagattggcaggaattaaacctacgaaaatttagtta


MMusMito34-7 GAACTTCCAATCCGTATATTAATTTCATCTGAAGACGTCCTCCACTCATGAGCAGTCCCCMMusMito34-8 gaattcgagctcggtacccggcggccgcAAGTCCTAGGGAGGGGACTGCTCATGAGTGGAMMusMito35-1 caggtcgactctagaggatcgcggccgcAGACGTCCTCCACTCATGAGCAGTCCCCTCCC 35 28 76 104 236 7483-7718MMusMito35-2 TGATTTAGTCGGCCTGGGATGGCATCAGTTTTAAGTCCTAGGGAGGGGACTGCTCATGAGMMusMito35-3 ATCCCAGGCCGACTAAATCAAGCAACAGTAACATCAAACCGACCAGGGTTATTCTATGGCMMusMito35-4 GCATAAAGCTATGGTTAGATCCACAAATTTCAGAGCATTGGCCATAGAATAACCCTGGTCMMusMito35-5 ATCTAACCATAGCTTTATGCCCATTGTCCTAGAAATGGTTCCACTAAAATATTTCGAAAAMMusMito35-6 CTTAGCTTCATAGTGAAATTAAATTATTGAAGCAGATCAGTTTTCGAAATATTTTAGTGGMMusMito35-7 AATTTCACTATGAAGCTAAGAGCGggcgcgccgcggtagtttatcacagttaaattgctaMMusMito35-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito36-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 36 76 28 104 236 7679-7914MMusMito36-2 GTGAAATTAAATTATTGAAGCAGAggcgcgccctggatggaggcggataaagttgcaggMMusMito36-3 CTTCAATAATTTAATTTCACTATGAAGCTAAGAGCGTTAACCTTTTAAGTTAAAGTTAGAMMusMito36-4 GTATCTAGTTGTGGCATATCACTATGGAGATTTTAAGGTCTCTAACTTTAACTTAAAAGGMMusMito36-5 GATATGCCACAACTAGATACATCAACATGATTTATCACAATTATCTCATCAATAATTACCMMusMito36-6 ATGTTTGTGATGAGACTTTTAGTTGAAATAAGATAAATAGGGTAATTATTGATGAGATAAMMusMito36-7 AAAAGTCTCATCACAAACATTCCCACTGGCACCTTCACCAAAATCACTAACAACCATAAAMMusMito36-8 gaattcgagctcggtacccggcggccgcGGGGTTTTTACTTTTATGGTTGTTAGTGATTTMMusMito37-1 caggtcgactctagaggatcgcggccgcCTTCACCAAAATCACTAACAACCATAAAAGTA 37 28 28 56 284 7875-8158MMusMito37-2 GCAAATAGATTTTCGTTCATTTTAATTCTCAAGGGGTTTTTACTTTTATGGTTGTTAGTGMMusMito37-3 ATGAACGAAAATCTATTTGCCTCATTCATTACCCCAACAATAATAGGATTCCCAATCGTTMMusMito37-4 AGGATGGGAATAGGATTGAAGGAAATATAATGATGGCTACAACGATTGGGAATCCTATTAMMusMito37-5 TTCAATCCTATTCCCATCCTCAAAACGCCTAATCAACAACCGTCTCCATTCTTTCCAACAMMusMito37-6 GATTAGCATTATTTGTTTGATAATAAGTTTAACTAGTCAGTGTTGGAAAGAATGGAGACGMMusMito37-7 TCAAACAAATAATGCTAATCCACACACCAAAAGGACGAACATGAACCCTAATAATTGTTTMMusMito37-8 gaattcgagctcggtacccggcggccgcATATGATTAGGGAAACAATTATTAGGGTTCATMMusMito38-1 caggtcgactctagaggatcgcggccgcGGACGAACATGAACCCTAATAATTGTTTCCCT 38 28 28 56 284 8119-8402MMusMito38-2 TAAAAGGCCTAGGAGATTTGTTGATCCAATAAATATGATTAGGGAAACAATTATTAGGGTMMusMito38-3 CAAATCTCCTAGGCCTTTTACCACATACATTTACACCTACTACCCAACTATCCATAAATCMMusMito38-4 GTAATTACGGCTCCAGCTCATAGTGGAATGGCTATACTTAGATTTATGGATAGTTGGGTAMMusMito38-5 TGAGCTGGAGCCGTAATTACAGGCTTCCGACACAAACTAAAAAGCTCACTTGCCCACTTCMMusMito38-6 TAAGTATTGGAATTAGTGAAATTGGAGTTCCTTGTGGAAGGAAGTGGGCAAGTGAGCTTTMMusMito38-7 TTCACTAATTCCAATACTTATTATTATTGAAACAATTAGCCTATTTATTCAACCAATGGCMMusMito38-8 gaattcgagctcggtacccggcggccgcCGGACTGCTAATGCCATTGGTTGAATAAATAGMMusMito39-1 caggtcgactctagaggatcgcggccgcCAATTAGCCTATTTATTCAACCAATGGCATTA 39 28 28 56 284 8363-8646MMusMito39-2 ATAAGTGTCCTGCAGTAATGTTAGCTGTAAGCCGGACTGCTAATGCCATTGGTTGAATAAMMusMito39-3 CATTACTGCAGGACACTTATTAATACACCTAATCGGAGGAGCTACTCTAGTATTAATAAAMMusMito39-4 TAAAATAATAAATGTAATGGTAGCTGTTGGTGGGCTAATATTTATTAATACTAGAGTAGCMMusMito39-5 CCATTACATTTATTATTTTACTTCTACTCACAATTCTAGAATTTGCAGTAGCATTAATTCMMusMito39-6 TGTAGATATAGGCTTACTAGGAGGGTGAATACGTAGGCTTGAATTAATGCTACTGCAAATMMusMito39-7 CTAGTAAGCCTATATCTACATGATAATACATAATGACCCACCAAACTCATGCATATCACAMMusMito39-8 gaattcgagctcggtacccggcggccgcTTGGATTAACTATGTGATATGCATGAGTTTGGMMusMito40-1 caggtcgactctagaggatcgcggccgcATGACCCACCAAACTCATGCATATCACATAGT 40 28 76 104 236 8607-8842MMusMito40-2 GGCTGAAAAGGCTCCAGTTAATGGTCATGGACTTGGATTAACTATGTGATATGCATGAGTMMusMito40-3 TAACTGGAGCCTTTTCAGCCCTCCTTCTAACATCAGGTCTAGTAATATGATTTCACTATAMMusMito40-4 ATATTGGTGAGTAGGCCAAGGGTTAATAGTGTAATTGAATTATAGTGAAATCATATTACTMMusMito40-5 CTTGGCCTACTCACCAATATCCTCACAATATATCAATGATGACGAGACGTAATTCGTGAAMMusMito40-6 CTTTTTGTACAATAGGAGTGTGGTGGCCTTGGTAGGTTCCTTCACGAATTACGTCTCGTCMMusMito40-7 CACTCCTATTGTACAAAAAGGACTggcgcgccgcggtagtttatcacagttaaattgctaMMusMito40-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito41-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 41 76 28 104 236 8803-9038MMusMito41-2 TAGGAGTGTGGTGGCCTTGGTAGGggcgcgccctggatggaggcggataaagttgcaggMMusMito41-3 CCAAGGCCACCACACTCCTATTGTACAAAAAGGACTACGATATGGTATAATTCTATTCATMMusMito41-4 CGCTCAGAAGAATCCTGCAAAGAAAAATACTTCCGAGACGATGAATAGAATTATACCATAMMusMito41-5 TTGCAGGATTCTTCTGAGCGTTCTATCATTCTAGCCTCGTACCAACACATGATCTAGGAGMMusMito41-6 AGAGGGTTAAGTGGTGAAATTCCTGTTGGAGGTCAGCAGCCTCCTAGATCATGTGTTGGTMMusMito41-7 ATTTCACCACTTAACCCTCTAGAAGTCCCACTACTTAATACTTCAGTACTTCTAGCATCAMMusMito41-8 gaattcgagctcggtacccggcggccgcAATTGAAACACCTGATGCTAGAAGTACTGAAGMMusMito42-1 caggtcgactctagaggatcgcggccgcACTTAATACTTCAGTACTTCTAGCATCAGGTG 42 28 28 56 284 8999-9282MMusMito42-2 TTACCTTCTATAAGGCTATGATGAGCTCATGTAATTGAAACACCTGATGCTAGAAGTACTMMusMito42-3 CATAGCCTTATAGAAGGTAAACGAAACCACATAAATCAAGCCCTACTAATTACCATTATAMMusMito42-4 AGTATTCTGAAGCTTGGAGGATGGTGAAGTAAAGTCCTAGTATAATGGTAATTAGTAGGGMMusMito42-5 CCTCCAAGCTTCAGAATACTTTGAAACATCATTCTCCATTTCAGATGGTATCTATGGTTCMMusMito42-6 TACATGGAGTCCATGGAATCCAGTAGCCATGAAGAATGTAGAACCATAGATACCATCTGAMMusMito42-7 GATTCCATGGACTCCATGTAATTATTGGATCAACATTCCTTATTGTTTGCCTACTACGACMMusMito42-8 gaattcgagctcggtacccggcggccgcGAAATTTTAGTTGTCGTAGTAGGCAAACAATAMMusMito43-1 caggtcgactctagaggatcgcggccgcACATTCCTTATTGTTTGCCTACTACGACAACT 43 28 28 56 284 9243-9526MMusMito43-2 TTCAAATCCGAAGTGATGTTTTGATGTGAAGTGAAATTTTAGTTGTCGTAGTAGGCAAACMMusMito43-3 AACATCACTTCGGATTTGAAGCCGCAGCATGATACTGACATTTTGTAGACGTAGTCTGACMMusMito43-4 GAGTAAGATCCTCATCAATAAATGGAGACGTATAGGAAAAGTCAGACTACGTCTACAAAAMMusMito43-5 TATTGATGAGGATCTTACTCCCTTAGTATAATTAATATAACTGACTTCCAATTAGTAGATMMusMito43-6 CAGTGTACAGGTTAATTACTCTCTTCTGGGTTTATTCAGAATCTACTAATTGGAAGTCAGMMusMito43-7 AGTAATTAACCTGTACACTGTTATCTTCATTAATATTTTATTATCCCTAACGCTAATTCTMMusMito43-8 gaattcgagctcggtacccggcggccgcCAGAATGCAACTAGAATTAGCGTTAGGGATAAMMusMito44-1 caggtcgactctagaggatcgcggccgcATATTTTATTATCCCTAACGCTAATTCTAGTT 44 28 28 56 284 9487-9770MMusMito44-2 CTTTTTCTGAGTACAGATTTATTTGGGGGAGTCAGAATGCAACTAGAATTAGCGTTAGGGMMusMito44-3 AAATCTGTACTCAGAAAAAGCAAATCCATATGAATGCGGATTCGACCCTACAAGCTCTGCMMusMito44-4 AATTGCTACCAAGAAAAATTTTATTGAGAATGGTAGACGTGCAGAGCTTGTAGGGTCGAAMMusMito44-5 AATTTTTCTTGGTAGCAATTACATTTCTATTATTTGACCTAGAAATTGCTCTTCTACTTCMMusMito44-6 ATAGTAGAGGTTTTAATTGTTTGAATTGCTCATGGTAGTGGAAGTAGAAGAGCAATTTCTMMusMito44-7 ACAATTAAAACCTCTACTATAATAATTATAGCCTTTATTCTAGTCACAATTCTATCTCTAMMusMito44-8 gaattcgagctcggtacccggcggccgcATATGCTAGGCCTAGAGATAGAATTGTGACTAMMusMito45-1 caggtcgactctagaggatcgcggccgcCTTTATTCTAGTCACAATTCTATCTCTAGGCC 45 28 76 104 236 9731-9966MMusMito45-2 TCTGTTCATTCTAATCCTTTTTGTGTTCATTCATATGCTAGGCCTAGAGATAGAATTGTGMMusMito45-3 AAAGGATTAGAATGAACAGAGTAAATGGTAATTAGTTTAAAAAAAATTAATGATTTCGACMMusMito45-4 TGGCATATTGGTAATTATGAACATCATCATAATCTAATGAGTCGAAATCATTAATTTTTTMMusMito45-5 TCATAATTACCAATATGCCATCTACCTTCTTCAACCTCACCATAGCCTTCTCACTATCACMMusMito45-6 GTGGATATTAGGTGAGAGCGAAATATAAGTGTCCCTAGAAGTGATAGTGAGAAGGCTATGMMusMito45-7 CGCTCTCACCTAATATCCACATTAggcgcgccgcggtagtttatcacagttaaattgctaMMusMito45-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito46-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 46 76 28 104 236 9927-10162MMusMito46-2 TGAGAGCGAAATATAAGTGTCCCTggcgcgccctggatggaggcggataaagttgcagg


agggacacttatatttcgctctcacctaatatccacattactatgcctggaaggcatagtattatccttatttattataacttca


caggtcgactctagaggatcgcggccgc atattttattatccctaacgctaattctagttgcattctgactcccccaaataaatctgtactcagaaaaagcaaatccatatgaatgcggattcgaccctacaagctctgcacgtctaccattctcaataaaatttttcttggtagcaattacatttctattatttgacctagaaattgctcttctacttccactaccatgagcaattcaaacaattaaaacctctactataataattatagcctttattctagtcacaattctatctctaggcctagcatat


caggtcgactctagaggatcgcggccgc ctttattctagtcacaattctatctctaggcctagcatatgaatgaacacaaaaaggattagaatgaacagagtaaatggtaattagtttaaaaaaaattaatgatttcgactcattagattatgatgatgttcataattaccaatatgccatctaccttcttcaacctcaccatagccttctcactatcacttctagggacacttatatttcgctctcacctaatatccacatta


caggtcgactctagaggatcgcggccgc acttaatacttcagtacttctagcatcaggtgtttcaattacatgagctcatcatagccttatagaaggtaaacgaaaccacataaatcaagccctactaattaccattatactaggactttacttcaccatcctccaagcttcagaatactttgaaacatcattctccatttcagatggtatctatggttctacattcttcatggctactggattccatggactccatgtaattattggatcaacattccttattgtttgcctactacgacaactaaaatttc


caggtcgactctagaggatcgcggccgc acattccttattgtttgcctactacgacaactaaaatttcacttcacatcaaaacatcacttcggatttgaagccgcagcatgatactgacattttgtagacgtagtctgacttttcctatacgtctccatttattgatgaggatcttactcccttagtataattaatataactgacttccaattagtagattctgaataaacccagaagagagtaattaacctgtacactgttatcttcattaatattttattatccctaacgctaattctagttgcattctg


caggtcgactctagaggatcgcggccgc atgacccaccaaactcatgcatatcacatagttaatccaagtccatgaccattaactggagccttttcagccctccttctaacatcaggtctagtaatatgatttcactataattcaattacactattaacccttggcctactcaccaatatcctcacaatatatcaatgatgacgagacgtaattcgtgaaggaacctaccaaggccaccacactcctattgtacaaaaaggact



cctaccaaggccaccacactcctattgtacaaaaaggactacgatatggtataattctattcatcgtctcggaagtatttttctttgcaggattcttctgagcgttctatcattctagcctcgtaccaacacatgatctaggaggctgctgacctccaacaggaatttcaccacttaaccctctagaagtcccactacttaatacttcagtacttctagcatcaggtgtttcaatt


caggtcgactctagaggatcgcggccgc ggacgaacatgaaccctaataattgtttccctaatcatatttattggatcaacaaatctcctaggccttttaccacatacatttacacctactacccaactatccataaatctaagtatagccattccactatgagctggagccgtaattacaggcttccgacacaaactaaaaagctcacttgcccacttccttccacaaggaactccaatttcactaattccaatacttattattattgaaacaattagcctatttattcaaccaatggcattagcagtccg


caggtcgactctagaggatcgcggccgc caattagcctatttattcaaccaatggcattagcagtccggcttacagctaacattactgcaggacacttattaatacacctaatcggaggagctactctagtattaataaatattagcccaccaacagctaccattacatttattattttacttctactcacaattctagaatttgcagtagcattaattcaagcctacgtattcaccctcctagtaagcctatatctacatgataatacataatgacccaccaaactcatgcatatcacatagttaatccaa



tctgcttcaataatttaatttcactatgaagctaagagcgttaaccttttaagttaaagttagagaccttaaaatctccatagtgatatgccacaactagatacatcaacatgatttatcacaattatctcatcaataattaccctatttatcttatttcaactaaaagtctcatcacaaacattcccactggcaccttcaccaaaatcactaacaaccataaaagtaaaaacccc


caggtcgactctagaggatcgcggccgc cttcaccaaaatcactaacaaccataaaagtaaaaaccccttgagaattaaaatgaacgaaaatctatttgcctcattcattaccccaacaataataggattcccaatcgttgtagccatcattatatttccttcaatcctattcccatcctcaaaacgcctaatcaacaaccgtctccattctttccaacactgactagttaaacttattatcaaacaaataatgctaatccacacaccaaaaggacgaacatgaaccctaataattgtttccctaatcatat


gccaatagaacttccaatccgtatattaatttcatctgaagacgtcctccactcatgagcagtcccctccctaggactt

caggtcgactctagaggatcgcggccgc agacgtcctccactcatgagcagtcccctccctaggacttaaaactgatgccatcccaggccgactaaatcaagcaacagtaacatcaaaccgaccagggttattctatggccaatgctctgaaatttgtggatctaaccatagctttatgcccattgtcctagaaatggttccactaaaatatttcgaaaactgatctgcttcaataatttaatttcactatgaagctaagagcg



MMusMito46-3 ACACTTATATTTCGCTCTCACCTAATATCCACATTACTATGCCTGGAAGGCATAGTATTAMMusMito46-4 TGGAGTTTAGGGAAGTTACTGAAGTTATAATAAATAAGGATAATACTATGCCTTCCAGGCMMusMito46-5 AGTAACTTCCCTAAACTCCAACTCCATAAGCTCCATACCAATCCCCATCACCATCTTAGTMMusMito46-6 TAGTAGGGCTAGTCCTACAGCTGCTTCGCAGGCTGCGAAAACTAAGATGGTGATGGGGATMMusMito46-7 CTGTAGGACTAGCCCTACTAGTAAAAGTTTCAAACACGTACGGAACAGATTACGTCCAAAMMusMito46-8 gaattcgagctcggtacccggcggccgcGTAGGTTGAGATTTTGGACGTAATCTGTTCCGMMusMito47-1 caggtcgactctagaggatcgcggccgcAACACGTACGGAACAGATTACGTCCAAAATCT 47 28 28 56 284 10123-10406MMusMito47-2 TTAGTGAGGGAAGAATAATTTTTAGCATTGTAGTAGGTTGAGATTTTGGACGTAATCTGTMMusMito47-3 AATTATTCTTCCCTCACTAATGCTACTACCACTAACCTGACTATCAAGCCCTAAAAAAACMMusMito47-4 TAAACTAATTAGAAAACTATATGAGGTTACGTTTGTTCAGGTTTTTTTAGGGCTTGATAGMMusMito47-5 ATAGTTTTCTAATTAGTTTAACCAGCCTAACACTTCTATGACAAACCGACGAAAATTATAMMusMito47-6 GTGGATAGGGGGTCTGAGGAGAATATATTTGAAAAGTTTTTATAATTTTCGTCGGTTTGTMMusMito47-7 TCCTCAGACCCCCTATCCACACCATTAATTATTTTAACAGCCTGATTACTGCCACTAATAMMusMito47-8 gaattcgagctcggtacccggcggccgcGCTAGCTATTAATATTAGTGGCAGTAATCAGGMMusMito48-1 caggtcgactctagaggatcgcggccgcTTTAACAGCCTGATTACTGCCACTAATATTAA 48 28 28 56 284 10367-10650MMusMito48-2 AGTACGTTATTATCTTTTTTTAGGTGGTTTTGGCTAGCTATTAATATTAGTGGCAGTAATMMusMito48-3 AAAAAAGATAATAACGTACTACAAAAACTCTACATCTCAATACTAATCAGCTTACAAATTMMusMito48-4 ATATAATTAGTTCAGTTGCTGAAAAGGTTATGATTAGGAGAATTTGTAAGCTGATTAGTAMMusMito48-5 AGCAACTGAACTAATTATATTTTATATTTTATTTGAAGCAACCTTAATCCCAACACTTATMMusMito48-6 GTTTAGGCGTTCAGTTTGGTTCCCTCATCGGGTAATAATAATAAGTGTTGGGATTAAGGTMMusMito48-7 ACCAAACTGAACGCCTAAACGCAGGGATTTATTTCCTATTTTATACCCTAATCGGTTCTAMMusMito48-8 gaattcgagctcggtacccggcggccgcTTAGCAGTGGAATAGAACCGATTAGGGTATAAMMusMito49-1 caggtcgactctagaggatcgcggccgcTTCCTATTTTATACCCTAATCGGTTCTATTCC 49 28 28 56 284 10611-10894MMusMito49-2 TCCTACATGGTTTTGGATTAAGATGAGGGCAATTAGCAGTGGAATAGAACCGATTAGGGTMMusMito49-3 TAATCCAAAACCATGTAGGAACCCTAAACCTCATAATTTTATCATTCACAACACACACCTMMusMito49-4 CATGCCAACCATAGTAAGTTGTTAGATCATGAAGCGTCTAAGGTGTGTGTTGTGAATGATMMusMito49-5 AACTTACTATGGTTGGCATGCATAATAGCATTTCTTATTAAAATACCATTATATGGAGTTMMusMito49-6 CAATTGGAGCTTCAACATGGGCTTTTGGTAGTCATAGGTGAACTCCATATAATGGTATTTMMusMito49-7 CCATGTTGAAGCTCCAATTGCTGGGTCAATAATTCTAGCAGCTATTCTTCTAAAATTAGGMMusMito49-8 gaattcgagctcggtacccggcggccgcATTCCGTAACTACCTAATTTTAGAAGAATAGCMMusMito50-1 caggtcgactctagaggatcgcggccgcTTCTAGCAGCTATTCTTCTAAAATTAGGTAGT 50 28 124 152 188 10855-11042MMusMito50-2 TTAGTGGGTCTAGAATAATGGAGATGCGAATTATTCCGTAACTACCTAATTTTAGAAGAAMMusMito50-3 CATTATTCTAGACCCACTAACAAAATATATAGCATACCCCTTCATCCTTCTCTCCCTATGMMusMito50-4 TGTTTGGCGTAAGCAGATTGAGCTAGTTATAATTATTCCTCATAGGGAGAGAAGGATGAAMMusMito50-5 CAATCTGCTTACGCCAAACAGATTTAAAATCACTAATCGCCTACTCCTCAGTTAGCcctgMMusMito50-6 aacttcgtatagcatacattatacgaagttatggaccctgcaggGCTAACTGAGGAGTAGMMusMito50-7 aatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctMMusMito50-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito51-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 51 124 28 152 188 11003-11190MMusMito51-2 gtaactgccagggcggccacttgaggcgcgccctggatggaggcggataaagttgcaggMMusMito51-3 gtggccgccctggcagttacaactaggaacctctagcctgcaggAACAGATTTAAAATCAMMusMito51-4 TAACAAGTGCTATGTGGCTAACTGAGGAGTAGGCGATTAGTGATTTTAAATCTGTTcctgMMusMito51-5 TAGCCACATAGCACTTGTTATTGCATCAATCATAATCCAAACTCCATGAAGCTTCATAGGMMusMito51-6 TGATGATGTGAGGCCATGTGCGATTATTAGTATTGTTGCTCCTATGAAGCTTCATGGAGTMMusMito51-7 CACATGGCCTCACATCATCACTCCTATTCTGCCTAGCAAACTCCAACTACGAACGGATCCMMusMito51-8 gaattcgagctcggtacccggcggccgcTAGTACGGCTGTGGATCCGTTCGTAGTTGGAGMMusMito52-1 caggtcgactctagaggatcgcggccgcCTAGCAAACTCCAACTACGAACGGATCCACAG 52 28 28 56 284 11151-11434MMusMito52-2 GAAGACCATTTGAAGTCCTCGGGCCATGATTATAGTACGGCTGTGGATCCGTTCGTAGTTMMusMito52-3 GAGGACTTCAAATGGTCTTCCCACTTATAGCCACATGATGACTGATAGCAAGTCTAGCTAMMusMito52-4 AATTCTCCTATTAGATTGATTGAAGGGGGTAGAGCTAGATTAGCTAGACTTGCTATCAGTMMusMito52-5 ATCAATCTAATAGGAGAATTATTCATTACCATATCATTATTTTCTTGATCAAACTTTACCMMusMito52-6 ATATACCTGTAATAATAATGTTAATTCCTATAAGAATAATGGTAAAGTTTGATCAAGAAAMMusMito52-7 CATTATTATTACAGGTATATACTCAATATACATAATTATTACCACCCAACGCGGCAAACTMMusMito52-8 gaattcgagctcggtacccggcggccgcATATGGTTGGTTAGTTTGCCGCGTTGGGTGGTMMusMito53-1 caggtcgactctagaggatcgcggccgcTAATTATTACCACCCAACGCGGCAAACTAACC 53 28 28 56 284 11395-11678MMusMito53-2 GTTCTCGTGTGTGTGAGGGTTGGAGGTTAATTATATGGTTGGTTAGTTTGCCGCGTTGGGMMusMito53-3 ACCCTCACACACACGAGAACTAACACTAATAGCCCTTCACATAATTCCACTTATTCTTCTMMusMito53-4 ACATATTGTCAGGCCTGTAATTAGTTTTGGACTGGTAGTTAGAAGAATAAGTGGAATTATMMusMito53-5 TTACAGGCCTGACAATATGTGAATATAGTTTACAAAAAACATTAGACTGTGAATCTGACAMMusMito53-6 TTGCAATCTTTCTTGGTGAATAAGGAGGTTTATTTCCTGTTGTCAGATTCACAGTCTAATMMusMito53-7 TTCACCAAGAAAGATTGCAAGAACTGCTAATTCATGCTTCCATGTTTAAAAACATGGCTTMMusMito53-8 gaattcgagctcggtacccggcggccgcATAAAAGTAAGAAAGCCATGTTTTTAAACATGMMusMito54-1 caggtcgactctagaggatcgcggccgcCATGCTTCCATGTTTAAAAACATGGCTTTCTT 54 28 28 56 284 11639-11922MMusMito54-2 GGTTCCTAAGACCAATGGATTACTATTATCCTATAAAAGTAAGAAAGCCATGTTTTTAAAMMusMito54-3 ATCCATTGGTCTTAGGAACCAAAAACCTTGGTGCAAATCCAAATAAAAGTAATCAATATTMMusMito54-4 ATAGTAGAAGAATGAAGATTAATAAGATTGAGGTTGTGAAAATATTGATTACTTTTATTTMMusMito54-5 AATCTTCATTCTTCTACTATCCCCAATCCTAATTTCAATATCAAACCTAATTAAACACATMMusMito54-6 GGAGAATTTGATTGATGTGGTGGTGTACAGTGGGAAGTTGATGTGTTTAATTAGGTTTGAMMusMito54-7 CCACATCAATCAAATTCTCCTTCATTATTAGCCTCTTACCCCTATTAATATTTTTCCACAMMusMito54-8 gaattcgagctcggtacccggcggccgcATTCTATATTATTGTGGAAAAATATTAATAGGMMusMito55-1 caggtcgactctagaggatcgcggccgcCTCTTACCCCTATTAATATTTTTCCACAATAA 55 28 76 104 236 11883-12118MMusMito55-2 TATGGTGACTCAGTGCCAGGTTGTAATTATATATTCTATATTATTGTGGAAAAATATTAAMMusMito55-3 CCTGGCACTGAGTCACCATAAATTCAATAGAACTTAAAATAAGCTTCAAAACTGACTTTTMMusMito55-4 CATGTGACAAAAAGGGCTACAGATGTAAACAGGATAGAGAAAAAGTCAGTTTTGAAGCTTMMusMito55-5 GTAGCCCTTTTTGTCACATGATCAATTATACAATTCTCTTCATGATATATACACTCAGACMMusMito55-6 GGAATAGTGTAAGATATTTAATGAATCGATTGATGTTTGGGTCTGAGTGTATATATCATGMMusMito55-7 TAAATATCTTACACTATTCCTGATggcgcgccgcggtagtttatcacagttaaattgctaMMusMito55-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito56-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 56 76 28 104 236 12079-12314MMusMito56-2 GATATTTAATGAATCGATTGATGTggcgcgccctggatggaggcggataaagttgcaggMMusMito56-3 CAATCGATTCATTAAATATCTTACACTATTCCTGATTACCATGCTTATCCTCACCTCAGCMMusMito56-4 CACCCCTTCTCAGCCAATGAAAAGTTGAAATATGTTGTTGGCTGAGGTGAGGATAAGCATMMusMito56-5 TCATTGGCTGAGAAGGGGTGGGAATTATATCTTTCCTACTAATTGGATGATGGTACGGACMMusMito56-6 TAGAGGATTGCTTGTAGGGCTGCAGTATTTGCGTCTGTTCGTCCGTACCATCATCCAATTMMusMito56-7 GCCCTACAAGCAATCCTCTATAACCGCATCGGAGACATCGGATTCATTTTAGCTATAGTTMMusMito56-8 gaattcgagctcggtacccggcggccgcTAGGGAAAATCAAACTATAGCTAAAATGAATCMMusMito57-1 caggtcgactctagaggatcgcggccgcAGACATCGGATTCATTTTAGCTATAGTTTGAT 57 28 28 56 284 12275-12558MMusMito57-2 ATAATCTGTTGAAGTTCTCATGAGTTTATGTTTAGGGAAAATCAAACTATAGCTAAAATGMMusMito57-3 TGAGAACTTCAACAGATTATATTCTCCAACAACAACGACAATCTAATTCCACTTATAGGCMMusMito57-4 GGCCAAATTGTGCTGATTTTCCTGTAGCTGCGATTAATAGGCCTATAAGTGGAATTAGATMMusMito57-5 AAAATCAGCACAATTTGGCCTCCACCCATGACTACCATCAGCAATAGAAGGCCCTACACCMMusMito57-6 TGCAACTACTATTGTACTTGAGTGTAGTAGTGCTGAAACTGGTGTAGGGCCTTCTATTGC


acatcaatcgattcattaaatatcttacactattcctgattaccatgcttatcctcacctcagccaacaacatatttcaacttttcattggctgagaaggggtgggaattatatctttcctactaattggatgatggtacggacgaacagacgcaaatactgcagccctacaagcaatcctctataaccgcatcggagacatcggattcattttagctatagtttgattttcccta


caggtcgactctagaggatcgcggccgc agacatcggattcattttagctatagtttgattttccctaaacataaactcatgagaacttcaacagattatattctccaacaacaacgacaatctaattccacttataggcctattaatcgcagctacaggaaaatcagcacaatttggcctccacccatgactaccatcagcaatagaaggccctacaccagtttcagcactactacactcaagtacaatagtagttgcaggaattttcctac


caggtcgactctagaggatcgcggccgc catgcttccatgtttaaaaacatggctttcttacttttataggataatagtaatccattggtcttaggaaccaaaaaccttggtgcaaatccaaataaaagtaatcaatattttcacaacctcaatcttattaatcttcattcttctactatccccaatcctaatttcaatatcaaacctaattaaacacatcaacttcccactgtacaccaccacatcaatcaaattctccttcattattagcctcttacccctattaatatttttccacaataatatagaat


caggtcgactctagaggatcgcggccgc ctcttacccctattaatatttttccacaataatatagaatatataattacaacctggcactgagtcaccataaattcaatagaacttaaaataagcttcaaaactgactttttctctatcctgtttacatctgtagccctttttgtcacatgatcaattatacaattctcttcatgatatatacactcagacccaaacatcaatcgattcattaaatatcttacactattcctgat


caggtcgactctagaggatcgcggccgc ctagcaaactccaactacgaacggatccacagccgtactataatcatggcccgaggacttcaaatggtcttcccacttatagccacatgatgactgatagcaagtctagctaatctagctctacccccttcaatcaatctaataggagaattattcattaccatatcattattttcttgatcaaactttaccattattcttataggaattaacattattattacaggtatatactcaatatacataattattaccacccaacgcggcaaactaaccaaccatat


caggtcgactctagaggatcgcggccgc taattattaccacccaacgcggcaaactaaccaaccatataattaacctccaaccctcacacacacgagaactaacactaatagcccttcacataattccacttattcttctaactaccagtccaaaactaattacaggcctgacaatatgtgaatatagtttacaaaaaacattagactgtgaatctgacaacaggaaataaacctccttattcaccaagaaagattgcaagaactgctaattcatgcttccatgtttaaaaacatggctttcttacttttat


caggtcgactctagaggatcgcggccgc ttctagcagctattcttctaaaattaggtagttacggaataattcgcatctccattattctagacccactaacaaaatatatagcataccccttcatccttctctccctatgaggaataattataactagctcaatctgcttacgccaaacagatttaaaatcactaatcgcctactcctcagttagc

cctgcagggtccataacttcgtataatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctaacgcagtcaggcgcggccgccgggtaccgagctcgaattc

caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcctccatccagggcgcgcctcaagtggccgccctggcagttacaactaggaacctctagcctgcagg

aacagatttaaaatcactaatcgcctactcctcagttagccacatagcacttgttattgcatcaatcataatccaaactccatgaagcttcataggagcaacaatactaataatcgcacatggcctcacatcatcactcctattctgcctagcaaactccaactacgaacggatccacagccgtacta


caggtcgactctagaggatcgcggccgc tttaacagcctgattactgccactaatattaatagctagccaaaaccacctaaaaaaagataataacgtactacaaaaactctacatctcaatactaatcagcttacaaattctcctaatcataaccttttcagcaactgaactaattatattttatattttatttgaagcaaccttaatcccaacacttattattattacccgatgagggaaccaaactgaacgcctaaacgcagggatttatttcctattttataccctaatcggttctattccactgctaa


caggtcgactctagaggatcgcggccgc ttcctattttataccctaatcggttctattccactgctaattgccctcatcttaatccaaaaccatgtaggaaccctaaacctcataattttatcattcacaacacacaccttagacgcttcatgatctaacaacttactatggttggcatgcataatagcatttcttattaaaataccattatatggagttcacctatgactaccaaaagcccatgttgaagctccaattgctgggtcaataattctagcagctattcttctaaaattaggtagttacggaat


g g ggg g g g gg gg ggtaacttccctaaactccaactccataagctccataccaatccccatcaccatcttagttttcgcagcctgcgaagcagctgtaggactagccctactagtaaaagtttcaaacacgtacggaacagattacgtccaaaatctcaacctac

caggtcgactctagaggatcgcggccgc aacacgtacggaacagattacgtccaaaatctcaacctactacaatgctaaaaattattcttccctcactaatgctactaccactaacctgactatcaagccctaaaaaaacctgaacaaacgtaacctcatatagttttctaattagtttaaccagcctaacacttctatgacaaaccgacgaaaattataaaaacttttcaaatatattctcctcagaccccctatccacaccattaattattttaacagcctgattactgccactaatattaatagctagc



MMusMito57-7 CAAGTACAATAGTAGTTGCAGGAATTTTCCTACTGGTCCGATTCCACCCCCTCACGACTAMMusMito57-8 gaattcgagctcggtacccggcggccgcTAAAGTTATTATTAGTCGTGAGGGGGTGGAATMMusMito58-1 caggtcgactctagaggatcgcggccgcCTGGTCCGATTCCACCCCCTCACGACTAATAA 58 28 28 56 284 12519-12802MMusMito58-2 TAGGGCTCCGAGGCAAAGTATAGTTGTTAAAATAAAGTTATTATTAGTCGTGAGGGGGTGMMusMito58-3 TACTTTGCCTCGGAGCCCTAACCACATTATTTACAGCTATTTGTGCTCTCACCCAAAACGMMusMito58-4 AGTTGGCTTGATGTAGAGAAGGCAATGATTTTTTTGATGTCGTTTTGGGTGAGAGCACAAMMusMito58-5 TTCTCTACATCAAGCCAACTAGGCCTGATAATAGTGACGCTAGGAATAAACCAACCACACMMusMito58-6 CTTTGAAGAATGCGTGGGTACAGATGTGTAGGAATGCTAGGTGTGGTTGGTTTATTCCTAMMusMito58-7 TACCCACGCATTCTTCAAAGCTATACTCTTTATATGCTCTGGCTCAATCATTCATAGCCTMMusMito58-8 gaattcgagctcggtacccggcggccgcTGTTCGTCTGCCAGGCTATGAATGATTGAGCCMMusMito59-1 caggtcgactctagaggatcgcggccgcTATGCTCTGGCTCAATCATTCATAGCCTGGCA 59 28 28 56 284 12763-13046MMusMito59-2 TTTTTGTGATGTTTCCTATTTTTCGGATGTCTTGTTCGTCTGCCAGGCTATGAATGATTGMMusMito59-3 AATAGGAAACATCACAAAAATCATACCATTCACATCATCATGCCTAGTAATCGGAAGCCTMMusMito59-4 TGAGTAGAACCCTGTTAGGAATGGTATTCCTGTGAGGGCGAGGCTTCCGATTACTAGGCAMMusMito59-5 TCCTAACAGGGTTCTACTCAAAAGACCTAATTATTGAAGCAATTAATACCTGCAACACCAMMusMito59-6 ATAGAAGTGGCGATTAGTGTAATTAGTAGGGCTCAGGCGTTGGTGTTGCAGGTATTAATTMMusMito59-7 ACACTAATCGCCACTTCTATAACAGCTATGTACAGCATACGAATCATTTACTTCGTAACAMMusMito59-8 gaattcgagctcggtacccggcggccgcCGGTTTTGTTATTGTTACGAAGTAAATGATTCMMusMito60-1 caggtcgactctagaggatcgcggccgcCAGCATACGAATCATTTACTTCGTAACAATAA 60 28 76 104 236 13007-13242MMusMito60-2 TTTTCGTTAATGGAGATTAGGGGGGGAAAACGCGGTTTTGTTATTGTTACGAAGTAAATGMMusMito60-3 CTAATCTCCATTAACGAAAATGACCCAGACCTCATAAACCCAATCAAACGCCTAGCATTCMMusMito60-4 GAATATTATATGAGATGACAAATCCTGCAAAGATGCTTCCGAATGCTAGGCGTTTGATTGMMusMito60-5 TGTCATCTCATATAATATTCCACCAACCAGCATTCCAGTCCTCACAATACCATGATTTTTMMusMito60-6 TAGGAATCCTAATACTGAAATAATTAGGGCTGTGGTTTTTAAAAATCATGGTATTGTGAGMMusMito60-7 TTTCAGTATTAGGATTCCTAATCGggcgcgccgcggtagtttatcacagttaaattgctaMMusMito60-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito61-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 61 76 28 104 236 13203-13438MMusMito61-2 TACTGAAATAATTAGGGCTGTGGTggcgcgccctggatggaggcggataaagttgcaggMMusMito61-3 CAGCCCTAATTATTTCAGTATTAGGATTCCTAATCGCACTAGAACTAAACAACCTAACCAMMusMito61-4 AAGGATGAATATGGATTTGCTTTATTTATTGATAGTTTTATGGTTAGGTTGTTTAGTTCTMMusMito61-5 GCAAATCCATATTCATCCTTCTCAACTTTACTGGGGTTTTTCCCATCTATTATTCACCGCMMusMito61-6 ATGTTTTTAGGCTTAGGTTGAGAGATTTTATGGGTGTAATGCGGTGAATAATAGATGGGAMMusMito61-7 CAACCTAAGCCTAAAAACATCCCTAACTCTCCTAGACTTGATCTGGTTAGAAAAAACCATMMusMito61-8 gaattcgagctcggtacccggcggccgcGTGGATTTTGGGATGGTTTTTTCTAACCAGATMMusMito62-1 caggtcgactctagaggatcgcggccgcTAGACTTGATCTGGTTAGAAAAAACCATCCCA 62 28 28 56 284 13399-13682MMusMito62-2 TTAAAGTGGTTATGTTTGTGTGAAGAGTTGAGGTGGATTTTGGGATGGTTTTTTCTAACCMMusMito62-3 CACAAACATAACCACTTTAACAACCAACCAAAAAGGCTTAATTAAATTGTACTTTATATCMMusMito62-4 TGAGTATAAGATAATAATTAAGATGATGTTAATTAGGAATGATATAAAGTACAATTTAATMMusMito62-5 TAATTATTATCTTATACTCAATTAATCTCGAGTAATCTCGATAATAATAAAAATACCCGCMMusMito62-6 TGCTACTTGAATGATGGTAGTAGCTGGGTGATCTTTGTTTGCGGGTATTTTTATTATTATMMusMito62-7 CTACCATCATTCAAGTAGCACAACTATATATTGCCGCTACCCCAATCCCTCCTTCCAACAMMusMito62-8 gaattcgagctcggtacccggcggccgcATGTTGGAGTTATGTTGGAAGGAGGGATTGGGMMusMito63-1 caggtcgactctagaggatcgcggccgcGCCGCTACCCCAATCCCTCCTTCCAACATAAC 63 28 28 56 284 13643-13926MMusMito63-2 TTTGGGAGATTGGTTGATGTATGAGGTTGATGATGTTGGAGTTATGTTGGAAGGAGGGATMMusMito63-3 ACATCAACCAATCTCCCAAACCATCAAGATTAATTACTCCAACTTCATCATAATAATTAAMMusMito63-4 GTATTGGGGGTGATTATAGAGGTTTTTTTAATTTGTGTGCTTAATTATTATGATGAAGTTMMusMito63-5 TCTATAATCACCCCCAATACTAAAAAACCCAAAATTAATCAGTTAGATCCCCAAGTCTCTMMusMito63-6 TGTTTGGATATACGACTGCTATAGCTACTGAGGAATATCCAGAGACTTGGGGATCTAACTMMusMito63-7 AGCAGTCGTATATCCAAACACAACCAACATCCCCCCTAAATAAATTAAAAAAACTATTAAMMusMito63-8 gaattcgagctcggtacccggcggccgcTCGTTTTTAGGTTTAATAGTTTTTTTAATTTAMMusMito64-1 caggtcgactctagaggatcgcggccgcCCCCTAAATAAATTAAAAAAACTATTAAACCT 64 28 28 56 284 13887-14170MMusMito64-2 TTGGTTGTTTAATGGTTTTAGGGTTTGGTGGATCGTTTTTAGGTTTAATAGTTTTTTTAAMMusMito64-3 TAAAACCATTAAACAACCAACAAACCCACTAACAATTAAACCTAAACCTCCATAAATAGGMMusMito64-4 ATTTTTGGTTGGTTGTCTTGGGTTAGCATTAAAGCCTTCACCTATTTATGGAGGTTTAGGMMusMito64-5 CAAGACAACCAACCAAAAATAATGAACTTAAAACAAAAATATAATTATTCATTATTTCTAMMusMito64-6 CGATGATTTTTCATGTCATTGGTCGCAGTTGAATGCTGTGTAGAAATAATGAATAATTATMMusMito64-7 AATGACATGAAAAATCATCGTTGTAATTCAACTACAGAAACACCTAATGACAAACATACGMMusMito64-8 gaattcgagctcggtacccggcggccgcGGGTGTGTTTTTCGTATGTTTGTCATTAGGTGMMusMito65-1 caggtcgactctagaggatcgcggccgcTACAGAAACACCTAATGACAAACATACGAAAA 65 28 76 104 236 14131-14366MMusMito65-2 CAATGAATGAGTGGTTAATAATTTTAAATAATGGGTGTGTTTTTCGTATGTTTGTCATTAMMusMito65-3 TATTAACCACTCATTCATTGACCTACCTGCCCCATCCAACATTTCATCATGATGAAACTTMMusMito65-4 AATGATTTGGACTATTAGGCAGACTCCTAGAAGGGACCCAAAGTTTCATCATGATGAAATMMusMito65-5 GCCTAATAGTCCAAATCATTACAGGTCTTTTCTTAGCCATACACTACACATCAGATACAAMMusMito65-6 ACGTCTCGACAAATGTGTGTTACTGATGAAAAGGCTGTTATTGTATCTGATGTGTAGTGTMMusMito65-7 ACACACATTTGTCGAGACGTAAATggcgcgccgcggtagtttatcacagttaaattgctaMMusMito65-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito66-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 66 76 28 104 236 14327-14562MMusMito66-2 ATGTGTGTTACTGATGAAAAGGCTggcgcgccctggatggaggcggataaagttgcaggMMusMito66-3 TTTTCATCAGTAACACACATTTGTCGAGACGTAAATTACGGGTGACTAATCCGATATATAMMusMito66-4 GGAATAAGCAAATAAAAAATATTGAGGCTCCGTTTGCGTGTATATATCGGATTAGTCACCMMusMito66-5 ATTTTTTATTTGCTTATTCCTTCATGTCGGACGAGGCTTATATTATGGATCATATACATTMMusMito66-6 TGCGAACAGTAGAAGTACTCCAATGTTTCAGGTTTCTATAAATGTATATGATCCATAATAMMusMito66-7 GAGTACTTCTACTGTTCGCAGTCATAGCCACAGCATTTATAGGCTACGTCCTTCCATGAGMMusMito66-8 gaattcgagctcggtacccggcggccgcATGATATTTGTCCTCATGGAAGGACGTAGCCTMMusMito67-1 caggtcgactctagaggatcgcggccgcGCATTTATAGGCTACGTCCTTCCATGAGGACA 67 28 28 56 284 14523-14806MMusMito67-2 TAGGAGGTTTGTAATAACTGTGGCACCTCAGAATGATATTTGTCCTCATGGAAGGACGTAMMusMito67-3 CAGTTATTACAAACCTCCTATCAGCCATCCCATATATTGGAACAACCCTAGTCGAATGAAMMusMito67-4 CGGGTCAAGGTGGCTTTGTCTACTGAGAAGCCCCCTCAAATTCATTCGACTAGGGTTGTTMMusMito67-5 GACAAAGCCACCTTGACCCGATTCTTCGCTTTCCACTTCATCTTACCATTTATTATCGCGMMusMito67-6 CTGTTTCGTGGAGGAAGAGGAGGTGAACGATTGCTAGGGCCGCGATAATAAATGGTAAGAMMusMito67-7 CCTCTTCCTCCACGAAACAGGATCAAACAACCCAACAGGATTAAACTCAGATGCAGATAAMMusMito67-8 gaattcgagctcggtacccggcggccgcTGAAATGGAATTTTATCTGCATCTGAGTTTAAMMusMito68-1 caggtcgactctagaggatcgcggccgcCAACAGGATTAAACTCAGATGCAGATAAAATT 68 28 28 56 284 14767-15050MMusMito68-2 TACCTAGGATATCTTTGATTGTATAGTAGGGGTGAAATGGAATTTTATCTGCATCTGAGTMMusMito68-3 AATCAAAGATATCCTAGGTATCCTAATCATATTCTTAATTCTCATAACCCTAGTATTATTMMusMito68-4 TGGTATGTAGTTGTCTGGGTCTCCTAGTATGTCTGGGAAAAATAATACTAGGGTTATGAGMMusMito68-5 ACCCAGACAACTACATACCAGCTAATCCACTAAACACCCCACCCCATATTAAACCCGAATMMusMito68-6 GGGATTGAGCGTAGAATGGCGTATGCAAATAGGAAATATCATTCGGGTTTAATATGGGGTMMusMito68-7 GCCATTCTACGCTCAATCCCCAATAAACTAGGAGGTGTCCTAGCCTTAATCTTATCTATCMMusMito68-8 gaattcgagctcggtacccggcggccgcGGCTAAAATTAGGATAGATAAGATTAAGGCTAMMusMito69-1 caggtcgactctagaggatcgcggccgcAGGTGTCCTAGCCTTAATCTTATCTATCCTAA 69 28 28 56 284 15011-15294MMusMito69-2 CGTTGCTTTGAGGTATGAAGGAAAGGTATTAGGGCTAAAATTAGGATAGATAAGATTAAG

caggtcgactctagaggatcgcggccgc caacaggattaaactcagatgcagataaaattccatttcacccctactatacaatcaaagatatcctaggtatcctaatcatattcttaattctcataaccctagtattatttttcccagacatactaggagacccagacaactacataccagctaatccactaaacaccccaccccatattaaacccgaatgatatttcctatttgcatacgccattctacgctcaatccccaataaactaggaggtgtcctagccttaatcttatctatcctaattttagcc


caggtcgactctagaggatcgcggccgc aggtgtcctagccttaatcttatctatcctaattttagccctaatacctttccttcatacctcaaagcaacgaagcctaatattc



agccttttcatcagtaacacacatttgtcgagacgtaaattacgggtgactaatccgatatatacacgcaaacggagcctcaatattttttatttgcttattccttcatgtcggacgaggcttatattatggatcatatacatttatagaaacctgaaacattggagtacttctactgttcgcagtcatagccacagcatttataggctacgtccttccatgaggacaaatatcat


caggtcgactctagaggatcgcggccgc gcatttataggctacgtccttccatgaggacaaatatcattctgaggtgccacagttattacaaacctcctatcagccatcccatatattggaacaaccctagtcgaatgaatttgagggggcttctcagtagacaaagccaccttgacccgattcttcgctttccacttcatcttaccatttattatcgcggccctagcaatcgttcacctcctcttcctccacgaaacaggatcaaacaacccaacaggattaaactcagatgcagataaaattccatttca


caggtcgactctagaggatcgcggccgc cccctaaataaattaaaaaaactattaaacctaaaaacgatccaccaaaccctaaaaccattaaacaaccaacaaacccactaacaattaaacctaaacctccataaataggtgaaggctttaatgctaacccaagacaaccaaccaaaaataatgaacttaaaacaaaaatataattattcattatttctacacagcattcaactgcgaccaatgacatgaaaaatcatcgttgtaattcaactacagaaacacctaatgacaaacatacgaaaaacacaccc


caggtcgactctagaggatcgcggccgc tacagaaacacctaatgacaaacatacgaaaaacacacccattatttaaaattattaaccactcattcattgacctacctgccccatccaacatttcatcatgatgaaactttgggtcccttctaggagtctgcctaatagtccaaatcattacaggtcttttcttagccatacactacacatcagatacaataacagccttttcatcagtaacacacatttgtcgagacgtaaat


caggtcgactctagaggatcgcggccgc tagacttgatctggttagaaaaaaccatcccaaaatccacctcaactcttcacacaaacataaccactttaacaaccaaccaaaaaggcttaattaaattgtactttatatcattcctaattaacatcatcttaattattatcttatactcaattaatctcgagtaatctcgataataataaaaatacccgcaaacaaagatcacccagctactaccatcattcaagtagcacaactatatattgccgctaccccaatccctccttccaacataactccaacat


caggtcgactctagaggatcgcggccgc gccgctaccccaatccctccttccaacataactccaacatcatcaacctcatacatcaaccaatctcccaaaccatcaagattaattactccaacttcatcataataattaagcacacaaattaaaaaaacctctataatcacccccaatactaaaaaacccaaaattaatcagttagatccccaagtctctggatattcctcagtagctatagcagtcgtatatccaaacacaaccaacatcccccctaaataaattaaaaaaactattaaacctaaaaacga


caggtcgactctagaggatcgcggccgc cagcatacgaatcatttacttcgtaacaataacaaaaccgcgttttccccccctaatctccattaacgaaaatgacccagacctcataaacccaatcaaacgcctagcattcggaagcatctttgcaggatttgtcatctcatataatattccaccaaccagcattccagtcctcacaataccatgatttttaaaaaccacagccctaattatttcagtattaggattcctaatcg



accacagccctaattatttcagtattaggattcctaatcgcactagaactaaacaacctaaccataaaactatcaataaataaagcaaatccatattcatccttctcaactttactggggtttttcccatctattattcaccgcattacacccataaaatctctcaacctaagcctaaaaacatccctaactctcctagacttgatctggttagaaaaaaccatcccaaaatccac


caggtcgactctagaggatcgcggccgc ctggtccgattccaccccctcacgactaataataactttattttaacaactatactttgcctcggagccctaaccacattatttacagctatttgtgctctcacccaaaacgacatcaaaaaaatcattgccttctctacatcaagccaactaggcctgataatagtgacgctaggaataaaccaaccacacctagcattcctacacatctgtacccacgcattcttcaaagctatactctttatatgctctggctcaatcattcatagcctggcagacgaaca


caggtcgactctagaggatcgcggccgc tatgctctggctcaatcattcatagcctggcagacgaacaagacatccgaaaaataggaaacatcacaaaaatcataccattcacatcatcatgcctagtaatcggaagcctcgccctcacaggaataccattcctaacagggttctactcaaaagacctaattattgaagcaattaatacctgcaacaccaacgcctgagccctactaattacactaatcgccacttctataacagctatgtacagcatacgaatcatttacttcgtaacaataacaaaaccg


tactacactcaagtacaatagtagttgcaggaattttcctactggtccgattccaccccctcacgactaataataacttta


MMusMito69-3 CTTCATACCTCAAAGCAACGAAGCCTAATATTCCGCCCAATCACACAAATTTTGTACTGAMMusMito69-4 CCCCAATTCAGGTTAAGATAAGTAGGTTGGCTACTAGGATTCAGTACAAAATTTGTGTGAMMusMito69-5 TATCTTAACCTGAATTGGGGGCCAACCAGTAGAACACCCATTTATTATCATTGGCCAACTMMusMito69-6 TATAAGAATTAAGATGATTGAGAAGTATGAGATGGAGGCTAGTTGGCCAATGATAATAAAMMusMito69-7 CAATCATCTTAATTCTTATACCAATCTCAGGAATTATCGAAGACAAAATACTAAAATTATMMusMito69-8 gaattcgagctcggtacccggcggccgcCAAGACATGGATATAATTTTAGTATTTTGTCTMMusMito70-1 caggtcgactctagaggatcgcggccgcATTATCGAAGACAAAATACTAAAATTATATCC 70 28 76 104 236 15255-15490MMusMito70-2 GGTTTACAAGACCAGAGTAATGTTTATACTATCAAGACATGGATATAATTTTAGTATTTTMMusMito70-3 TTACTCTGGTCTTGTAAACCTGAAATGAAGATCTTCTCTTCTCAAGACATCAAGAAGAAGMMusMito70-4 TAGAATACCAGCTTTGGGTGCTGGTGGTGGGGAGTAGCTCCTTCTTCTTGATGTCTTGAGMMusMito70-5 CACCCAAAGCTGGTATTCTAATTAAACTACTTCTTGAGTACATAAATTTACATAGTACAAMMusMito70-6 GGGGAAAATAGTTTAATGTACGATATACATAAATGTACTGTTGTACTATGTAAATTTATGMMusMito70-7 TACATTAAACTATTTTCCCCAAGCggcgcgccgcggtagtttatcacagttaaattgctaMMusMito70-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataaMMusMito71-1 caggtcgactctagaggatcgcggccgcgcgcagaagtggtcctgcaactttatccgcc 71 76 28 104 236 15451-15686MMusMito71-2 TTAATGTACGATATACATAAATGTggcgcgccctggatggaggcggataaagttgcaggMMusMito71-3 TTATGTATATCGTACATTAAACTATTTTCCCCAAGCATATAAGCTAGTACATTAAATCAAMMusMito71-4 ATATTGATTTATGTTGATGATTATTTTATGACCTGAACCATTGATTTAATGTACTAGCTTMMusMito71-5 TCATCAACATAAATCAATATATATACCATGAATATTATCTTAAACACATTAAACTAATGTMMusMito71-6 CTGTATGGTGTATGTCAGATAACACAGATATGTCCTTATAACATTAGTTTAATGTGTTTAMMusMito71-7 ATCTGACATACACCATACAGTCATAAACTCTTCTCTTCCATATGACTATCCCCTTCCCCAMMusMito71-8 gaattcgagctcggtacccggcggccgcTAATAGACCAAATGGGGAAGGGGATAGTCATAMMusMito72-1 caggtcgactctagaggatcgcggccgcCTCTTCCATATGACTATCCCCTTCCCCATTTG 72 28 28 56 284 15647-15930MMusMito72-2 GCGGGTTGTTGGTTTCACGGAGGATGGTAGATTAATAGACCAAATGGGGAAGGGGATAGTMMusMito72-3 CCGTGAAACCAACAACCCGCCCACCAATGCCCCTCTTCTCGCTCCGGGCCCATTAAACTTMMusMito72-4 AGAACCAGATGTCTGATAAAGTTTCAGTTTAGCTACCCCCAAGTTTAATGGGCCCGGAGCMMusMito72-5 TTTATCAGACATCTGGTTCTTACTTCAGGGCCATCAAATGCGTTATCGCCCATACGTTCCMMusMito72-6 GCTGATTAGACCCGATACCATCGAGATGTCTTATTTAAGGGGAACGTATGGGCGATAACGMMusMito72-7 TGGTATCGGGTCTAATCAGCCCATGACCAACATAACTGTGGTGTCATGCATTTGGTATCTMMusMito72-8 gaattcgagctcggtacccggcggccgcGCCAAAATAAAAAGATACCAAATGCATGACACMMusMito73-1 caggtcgactctagaggatcgcggccgcTAACTGTGGTGTCATGCATTTGGTATCTTTTT 73 28 28 56 284 15891-16174MMusMito73-2 TCATGCCTTGACGGCTATGTTGATGAAAGTAGGCCAAAATAAAAAGATACCAAATGCATGMMusMito73-3 ACATAGCCGTCAAGGCATGAAAGGACAGCACACAGTCTAGACGCACCTACGGTGAAGAATMMusMito73-4 GAATAATTAGCCTTAGGTGATTGGGTTTTGCGGACTAATGATTCTTCACCGTAGGTGCGTMMusMito73-5 TCACCTAAGGCTAATTATTCATGCTTGTTAGACATAAATGCTACTCAATACCAAATTTTAMMusMito73-6 GGTTTGGCATTAAGAGGAGGGGGTGGGGGGTTTGGAGAGTTAAAATTTGGTATTGAGTAGMMusMito73-7 CCTCCTCTTAATGCCAAACCCCAAAAACACTAAGAACTTGAAAGACATATAATATTAACTMMusMito73-8 gaattcgagctcggtacccggcggccgcATAGGGTTTGATAGTTAATATTATATGTCTTTMMusMito74-1 caggtcgactctagaggatcgcggccgcAGAACTTGAAAGACATATAATATTAACTATCA 74 28 28 56 284 16135-16418MMusMito74-2 ATATTTTGGGAACTACTAGAATTGATCAGGACATAGGGTTTGATAGTTAATATTATATGTMMusMito74-3 TCTAGTAGTTCCCAAAATATGACTTATATTTTAGTACTTGTAAAAATTTTACAAAATCATMMusMito74-4 TTGCGTAATAGAGTATGATTAGAGTTTTGGTTCACGGAACATGATTTTGTAAAATTTTTAMMusMito74-5 AATCATACTCTATTACGCAATAAACATTAACAAGTTAATGTAGCTTAATAACAAAGCAAAMMusMito74-6 GTTTATGGGATACAATTATCCATCTAAGCATTTTCAGTGCTTTGCTTTGTTATTAAGCTAMMusMito74-7 GATAATTGTATCCCATAAACACAAAGGTTTGGTCCTGGCCTTATAATTAATTAGAGGTAAMMusMito74-8 gaattcgagctcggtacccggcggccgcGCATGTGTAATTTTACCTCTAATTAATTATAAMMusMito75-1 caggtcgactctagaggatcgcggccgcTCCTGGCCTTATAATTAATTAGAGGTAAAATT 75 28 170 198 142 16379-16520MMusMito75-2 TTAAGGGATTTTACACCGGTCTATGGAGGTTTGCATGTGTAATTTTACCTCTAATTAATTMMusMito75-3 ACCGGTGTAAAATCCCTTAAACATTTACTTAAAATTTAAGGAGAGGGTATCAAGCACATTMMusMito75-4 gccccacgtgGTGGCTAGGCAAGGTGTCTTAAGCTATTTTAATGTGCTTGATACCCTCTCMMusMito75-5 GCCTAGCCACcacgtggggccgtcgaccaattctcatgtttgacagcttatcatcgcctgMMusMito75-6 aacttcgtatagcatacattatacgaagttatggaccctgcaggcgatgataagctgtcMMusMito75-7 aatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctMMusMito75-8 gaattcgagctcggtacccggcggccgcgcctgactgcgttagcaatttaactgtgataa

caggtcgactctagaggatcgcggccgc agaacttgaaagacatataatattaactatcaaaccctatgtcctgatcaattctagtagttcccaaaatatgacttatattttagtacttgtaaaaattttacaaaatcatgttccgtgaaccaaaactctaatcatactctattacgcaataaacattaacaagttaatgtagcttaataacaaagcaaagcactgaaaatgcttagatggataattgtatcccataaacacaaaggtttggtcctggccttataattaattagaggtaaaattacacatgc


caggtcgactctagaggatcgcggccgc tcctggccttataattaattagaggtaaaattacacatgcaaacctccatagaccggtgtaaaatcccttaaacatttacttaaaatttaaggagagggtatcaagcacattaaaatagcttaagacaccttgcctagccac

cacgtggggccgtcgaccaattctcatgtttgacagcttatcatcgcctgcagggtccataacttcgtataatgtatgctatacgaagttatttggcgcgccgcggtagtttatcacagttaaattgctaacgcagtcaggcgcggccgccgggtaccgagctcgaattc

caggtcgactctagaggatcgcggccgc ctcttccatatgactatccccttccccatttggtctattaatctaccatcctccgtgaaaccaacaacccgcccaccaatgcccctcttctcgctccgggcccattaaacttgggggtagctaaactgaaactttatcagacatctggttcttacttcagggccatcaaatgcgttatcgcccatacgttccccttaaataagacatctcgatggtatcgggtctaatcagcccatgaccaacataactgtggtgtcatgcatttggtatctttttattttggc


caggtcgactctagaggatcgcggccgc taactgtggtgtcatgcatttggtatctttttattttggcctactttcatcaacatagccgtcaaggcatgaaaggacagcacacagtctagacgcacctacggtgaagaatcattagtccgcaaaacccaatcacctaaggctaattattcatgcttgttagacataaatgctactcaataccaaattttaactctccaaaccccccaccccctcctcttaatgccaaaccccaaaaacactaagaacttgaaagacatataatattaactatcaaaccctat


caggtcgactctagaggatcgcggccgc attatcgaagacaaaatactaaaattatatccatgtcttgatagtataaacattactctggtcttgtaaacctgaaatgaagatcttctcttctcaagacatcaagaagaaggagctactccccaccaccagcacccaaagctggtattctaattaaactacttcttgagtacataaatttacatagtacaacagtacatttatgtatatcgtacattaaactattttccccaagc



acatttatgtatatcgtacattaaactattttccccaagcatataagctagtacattaaatcaatggttcaggtcataaaataatcatcaacataaatcaatatatataccatgaatattatcttaaacacattaaactaatgttataaggacatatctgtgttatctgacatacaccatacagtcataaactcttctcttccatatgactatccccttccccatttggtctatta


g g gcgcccaatcacacaaattttgtactgaatcctagtagccaacctacttatcttaacctgaattgggggccaaccagtagaacacccatttattatcattggccaactagcctccatctcatacttctcaatcatcttaattcttataccaatctcaggaattatcgaagacaaaatactaaaattatatccatgtcttg

The 600 oligonucleotide sequences used to construct the mouse mitochondrial genome are shown. Mouse mitochondrial genome sequence is shown in uppercase. Vector sequences and restriction sites are shown in lowercase. Also included are the sequences of each of the 75 first stage assemblies. The synthesized mouse mitochondrial genome sequence (orange) is flanked by extra bases on the left and right. These bases include vector overlaps and restriction sites (NotI, AscI, SbfI, and PmlI). The pUC19 sequences (red), pBR322 sequences (purple), pSMART sequences (blue), and pCC1BAC sequences (green) are shown. The synthesized genome sequence coordinates are given and are relative to the 16,520-bp sequence (i.e. contains the 221-bp repeat).


Supplementary Table 11. The number of colonies (CFU) produced following individual transformation of each of the 75 first-stage assemblies. Segment CFUs Segment CFUs Segment CFUs

1 933 26 491 51 5352 401 27 716 52 2963 691 28 917 53 5734 1259 29 1645 54 8155 1542 30 666 55 8126 500 31 1390 56 4397 1194 32 442 57 5228 896 33 1173 58 4559 649 34 539 59 794

10 642 35 622 60 95711 425 36 968 61 59312 977 37 1624 62 52513 1231 38 457 63 60914 1361 39 1004 64 40415 654 40 809 65 69516 649 41 706 66 41417 769 42 572 67 46218 1182 43 340 68 157119 1343 44 119 69 12720 702 45 819 70 37021 1122 46 52 71 43022 1219 47 656 72 97723 1137 48 807 73 33724 51 49 785 74 37225 365 50 306 75 357

No DNA 57


Supplementary Table 12. Cloning efficiencies and synthesis error rates for each of the 75 mouse mitochondrial genome segments.

1 6 2 33.3 9 0.00528 0-0.014082 5 0 0.0 10 0.00704 0.00352-0.014493 6 3 50.0 2 0.00117 0-0.003534 6 1 16.7 15 0.00880 0-0.017605 6 4 66.7 2 0.00117 0-0.007046 8 1 12.5 2 0.00088 0-0.003527 5 2 40.0 4 0.00282 0-0.007048 5 2 40.0 3 0.00211 0-0.007069 8 2 25.0 17 0.00748 0-0.0281610 3 2 66.7 1 0.00117 0-0.0035211 7 2 28.6 14 0.00704 0-0.0316912 7 3 42.9 6 0.00302 0-0.0140813 8 2 25.0 14 0.00616 0-0.0246414 7 3 42.9 10 0.00503 0-0.0252715 8 4 50.0 12 0.00528 0-0.0275816 7 2 28.6 7 0.00352 0-0.0176017 8 4 50.0 4 0.00176 0-0.0035218 3 1 33.3 3 0.00352 0-0.0070119 6 4 66.7 6 0.00352 0-0.0140820 8 2 25.0 6 0.00264 0-0.0070421 6 3 50.0 3 0.00176 0-0.0070422 5 4 80.0 1 0.00070 0-0.0035223 8 5 62.5 4 0.00176 0-0.0105624 3 0 0.0 5 0.00587 0.00352-0.0105625 8 4 50.0 10 0.00440 0-0.0140826 7 4 57.1 0 0.00000 027 8 4 50.0 4 0.00176 0-0.0070428 7 3 42.9 1 0.00050 0-0.0035229 7 2 28.6 4 0.00201 0-0.0070430 2 1 50.0 4 0.00704 0-0.0142831 8 3 37.5 5 0.00220 0-0.0105632 6 2 33.3 4 0.00235 0-0.0105633 8 5 62.5 8 0.00352 0-0.0175434 5 3 60.0 2 0.00141 0-0.0035235 7 1 14.3 12 0.00604 0-0.0173036 6 2 33.3 11 0.00646 0-0.0246437 7 6 85.7 1 0.00050 0-0.0035238 8 4 50.0 3 0.00132 0-0.0035239 7 3 42.9 4 0.00201 0-0.0070440 6 3 50.0 3 0.00176 0-0.0035241 5 0 0.0 12 0.00845 0.00352-0.0176042 4 3 75.0 7 0.00616 0-0.0246443 6 1 16.7 6 0.00352 0-0.0070444 5 3 60.0 2 0.00141 0-0.0035245 8 5 62.5 3 0.00132 0-0.0070446 5 1 20.0 9 0.00634 0-0.0211247 8 3 37.5 6 0.00264 0-0.0070448 7 5 71.4 4 0.00201 0-0.0070449 8 4 50.0 7 0.00308 0-0.0105650 7 4 57.1 5 0.00252 0-0.0070451 8 4 50.0 4 0.00176 0-0.0035252 6 2 33.3 5 0.00293 0-0.0105653 7 1 14.3 8 0.00402 0-0.0105654 4 2 50.0 4 0.00352 0-0.0070455 5 4 80.0 5 0.00352 0-0.0176056 8 3 37.5 14 0.00616 0-0.0364957 7 4 57.1 7 0.00352 0-0.0105658 6 4 66.7 3 0.00176 0-0.0070459 8 5 62.5 4 0.00176 0-0.0070460 6 3 50.0 4 0.00235 0-0.0070461 8 4 50.0 6 0.00264 0-0.0070462 7 4 57.1 5 0.00252 0-0.0105663 4 2 50.0 3 0.00264 0-0.0070464 6 2 33.3 4 0.00235 0-0.0035265 6 5 83.3 1 0.00059 0-0.0035266 5 2 40.0 3 0.00211 0-0.0035267 6 3 50.0 11 0.00646 0-0.0281668 7 4 57.1 3 0.00151 0-0.0035269 4 3 75.0 0 0.00000 070 7 4 57.1 5 0.00252 0-0.0105671 7 5 71.4 1 0.00050 0-0.0035272 7 6 85.7 1 0.00050 0-0.0035273 5 4 80.0 2 0.00141 0-0.0070474 7 3 42.9 12 0.00604 0-0.0316975 8 3 37.5 6 0.00264 0-0.00709

Range (errors/bp)Assembly #Number of clones

analyzedNumber of

correct clones% correct clones

Number of errors

Error rate (errors/ bp)


Supplementary Table 13. DNA sequence characterization of four E. coli clones containing the complete mitochondrial genome following four rounds of assembly and 45 cycles of PCR amplification.

Clone #

31 1470T-C 1775G-A 3758A 5764C-T 8843A-G 9039A-G 13795T-A 14807C-G 16125A 16378G-C

35 2838T-C 3082T-C 3758A 9283A-G 10408A 11435A-G

40 4598T-C 7036T-C 8843A-G 9283A-G 9527A-G 10481C-A 13927T-G 15450T-C 16176T-C

43

Errors

Perfect Match Deletions () and substitutions (-) are noted. DNA sequences that exactly matched the designed genome sequence are listed as “Perfect Match”. Supplementary Table 14. Additional oligos used in this study. Oligo name Oligo sequence

Not1 + 8 For agaggatcgcggccgc

Not1 + 8 Rev ggtacccggcggccgc

pSMART-VNTI-CPCR-For ccgccctggcagttacaact

pSMART-VNTI-CPCR-Rev acgaagttatggaccctgca

pBR322 CPCR 20bp-Clone FOR gcgcagaagtggtcctgcaa

pBR322 CPCR 20bp-Clone REV gcctgactgcgttagcaatt

pUC19 CPCR FOR caggtcgactctagaggatc

pUC19 CPCR REV gaattcgagctcggtacccg

pCC1BAC Assembly For gtccacataaccgtgcgcaaaatgttgtgg

pCC1BAC Assembly Rev gggccgtcgaccaattctcatgtttgacag

M13 Universal For tgtaaaacgacggccagt

M13 Universal Rev caggaaacagctatgacc

puc19Univ-synthesisNONot1 For gatcctctagagtcgacctg

puc19Univ-synthesisNONot1 Rev cgggtaccgagctcgaattc

Seq pCC1BAC Ass Vector FOR caagggcaagtattgacatgtcgt

Seq pCC1BAC Ass Vector REV ggcagttattggtgcccttaaacg


Supplementary Table 15. DNA sequence characterization of four E. coli clones containing the complete mitochondrial genome following four rounds of assembly and 80 cycles of PCR amplification.

Clone #

16 1470T-C 1755C-G 3758A 5172A 10505A 13027 14807C-G

20 1755C-G 7036T-C 6750G-C 9283A-G 9527A-G 11679A-G

33 1755C-G 5169C-T 9283A-G 9527A-G 11679A-G 13927T-G

59 1755C-G 2838T-C 3758A-G 4598T-C 5371T-G 15358A-G

Errors

Deletions () and substitutions (-) are noted. Position 1755 appears to be a mutation “hot spot”, as each of the four clones has a C-G change.

SUPPLEMENTARY NOTE

We assembled and cloned into E. coli two different 460-bp dsDNA fragments from 12 overlapping 60-mer oligonucleotides. Sequencing of more than 70 clones of each assembly revealed that 55% and 59% of the clones contained the intended sequence (Supplementary Table 1 online). Average error rates for these two assemblies were 0.00111 errors/bp (1 error per 901 bp, range from 0.0 to 0.00217 errors/bp) and 0.00128 errors/bp (1 error per 781 bp, range from 0.0 to 0.00434 errors/bp), respectively. Typically, a large number of clones result from this method. Most incorrect assemblies are due to point mutations or other issues with the oligos themselves and misassembly events are infrequent. Limitations and potential improvements Different assemblies have different error rates (see above, Results, and Supplementary Table 12). This may be caused by a combination of events including sequence-dependent chemical synthesis errors (e.g. double G coupling), polymerization errors (e.g. homopolymer runs), and differences in the capacity for E. coli to fix mistakes. Colony counts from the individually transformed mitochondrial genome sub-assemblies closely reflected the ease with which the correct sequences were obtained from the pooled assemblies (Compare Supplementary Table 11 to Fig. 2b). If such information could be gleaned without the associated labor, it could prove useful for appropriately skewing pooled populations before automated sequencing. This way, over-sequencing of some constructs and under-sequencing of others could be avoided. Although a complete set of clones was not immediately identified (Fig. 2), missing sub-assemblies were quickly recovered. In the first case, a new library was formed from the original 11 sub-assembly reactions not recovered during the first round of sequencing from the pooled approach (Fig. 2a). The reduced pool was transformed and submitted for automated colony picking, plasmid DNA preparation, and 16 x over-sequencing. In the second case, additional colonies from the 3 missing assemblies from the individual reaction approach (Fig. 2b) were screened by colony PCR for full-length assemblies, and then sequenced. Both approaches provided a useful means for recovering the missing sub-assemblies. Although the


complete set of sequence-verified sub-assemblies was obtained without difficulty, further process integration and automation could eliminate the need for more than one sequencing cycle. We repeated the genome construction process from the 75 sequence-verified assemblies but used 80 cycles of PCR-amplification (Supplementary Fig. 5 online). Surprisingly, the error-rate was identical (Supplementary Table 15 online). Thus, one does not necessarily have to carefully limit the number of PCR cycles if this mode of amplification is taken. This way, fragments can be amplified to saturation to produce equal concentrations of the DNA to be assembled. Prior to assembling DNA molecules, the PCR products are cleaved with restriction enzymes to remove the non-complementary sequence and expose the overlapping regions. We found that this restriction digestion step is unnecessary so long as the non-complementary region is kept to a minimum. This can be accomplished by using short 16-mer primers for amplification (Supplementary Fig. 6 online). However, best results will be achieved when a restriction-digestion step is included. Previously we have always amplified the vector constituent of an assembly reaction with primers that contain 5' extensions homologous to the termini of the corresponding assembly and with restriction sites to release the inserts1, 2. Here we chose to include these sequences within the initial oligos themselves (Supplementary Table 10 online). This meant that only three primer sets, and not 93, were required to amplify all of the assembly intermediates. This simplified the process and made it more amenable to automation. However, a limitation of this approach is that restriction sites cannot be recycled. Therefore, as the DNA fragments get larger, additional different restriction sites will be required and they may become unavailable. However, because our method can resolve long non-complementary sequences (Supplementary Fig. 6 online) homing endonucleases restriction sites, which are longer and extremely rare, can also be used. Our previously described approach of producing unique PCR-amplified vectors for each assembly should still be considered. It provides more flexibility since assembly schemes can be changed during the building process and restriction sites can be reused. The assembly system is performed at 50 °C, which is not likely to be the optimal annealing temperature for all of the oligos being assembled in a particular set. This could reduce the efficiency of obtaining a particular clone if no assembly or mis-assembly occurs with a particular oligo. One solution is to anneal the oligos under more stringent conditions prior to assembly at 50 °C (see Online Methods). Alternatively, software could be used to determine melting temperatures at the overlaps and they could be adjusted accordingly. The assembly system relies on cloning in E. coli to filter out the oligonucleotide errors. E. coli growth can be time consuming and occasionally DNA molecules will not propagate or get mutated in E. coli. It should be possible to isolate single assembled molecules and amplify them in vitro by, for example, emulsion PCR. We have assembled the entire mouse mitochondrial genome to demonstrate the generality of our one-step assembly reaction. Although more oligonucleotides can be assembled at once with our yeast-based system3, the simplicity and rapidity of the one-step method normally offsets this difference. Moreover, due to the error rate of chemical synthesis, unwanted mistakes occur


frequently as oligo assemblies become larger and larger4. Length limitations also arise because Sanger sequencing reads are restricted to about 750 nucleotides. While reading from either end of an assembly would cover products approaching 1.5 kb, primer walking or library construction would introduce unnecessary complications and expense beyond this size. The method described here does not require any error-correction procedures. Ultimately, small sub-assemblies are easily joined into giant constructs in a few assembly cycles1. Thus, there is little practical difference when beginning with short or long oligonucleotide assemblies. Indeed, once all 284-bp sub-assemblies were at hand, it took one person about 48 h to complete the synthesis of the mouse mitochondrial genome (Supplementary Fig. 1 online). Many laboratories, including ours, have outsourced the synthesis of DNA fragments1, 5. This is mainly because less work is involved—DNA sequences are simply uploaded to a manufacturer. However, with the simple method described here, DNA fragments can be quickly made and at a reasonable cost. We estimate that 70% of our synthesis expenditures were for the oligonucleotides. A ten-fold reduction in the price of oligos would obviously provide significant and immediate savings. Such advancement does not seem unrealistic as less than 0.01% of each purchased oligo was needed to form the 75 sub-assemblies. Microfluidics research could address this problem6. Another important consideration is oligonucleotide quality. The chemical synthesis of oligonucleotides remains an imperfect process. Thus, time-consuming and costly DNA sequencing quality-control steps are required during the DNA synthesis process. Perfect oligonucleotide sequence would dramatically accelerate progress in synthetic biology. Using synthetic genomics to better understand mitochondrial disease. We synthesized an organellar genome for the first time. A synthetic genomics approach could be used to restore respiratory competence to mammalian cells with mitochondrial deficiencies. Mutations in mitochondrial DNA have been associated with the pathogenesis of neurodegenerative disease, blindness, deafness, various cancers, type II diabetes, and the aging process7. Here we offer powerful new tools for the design and synthesis of complete mitochondrial chromosomes, which may then be expressed once installed in mitochondria. Reiterative synthesis and transplantation of many different versions of mitochondrial genomes should help accelerate progress in determining the genetic defects associated with mitochondrial diseases. And, when a combinatorial approach to synthesizing mitochondrial genomes is used, hundreds or thousands of different synthetic mitochondria can be produced at once. The structure and function of the resulting synthetic mitochondria can then be studied. This would allow for gene therapy strategies and therapeutic drugs to be more logically engineered. One route for testing the synthetic mouse mitochondrial genome for function could be to install it into mouse cells with mitochondria devoid of DNA (rho0) and then rescue the deficiencies (conversion to rho+). Rho0 mammalian cells do not undergo aerobic respiration so have uridine and pyruvate auxotrophies8, 9. This would allow for selection of mitochondria that have taken up a genome. Alternatively, these cells could be selected by resistance to chloramphenicol9, 10.

1. Gibson, D.G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215-1220 (2008).

2. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345 (2009).


3. Gibson, D.G. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37, 6984-6990 (2009).

4. Tian, J.D. et al. Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432, 1050-1054 (2004).

5. Gibson, D.G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52-56 (2010).

6. Melin, J. & Quake, S.R. Microfluidic large-scale integration: the evolution of design rules for biological automation. Annu Rev Biophys Biomol Struct 36, 213-231 (2007).

7. Taylor, R.W. & Turnbull, D.M. Mitochondrial DNA mutations in human disease. Nat Rev Genet 6, 389-402 (2005).

8. Kukat, A. et al. Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses. Nucleic Acids Res 36, e44 (2008).

9. King, M.P. & Attardi, G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246, 500-503 (1989).

10. King, M.P. & Attardi, G. Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA. Cell 52, 811-819 (1988).


chemical synthesis of the mouse mitochondrial genome · mitochondrial genome sequence as it exists...

Documents