Gene synthesis by circular assembly amplification

Transcription

1 Gene synthesis by circular assembly amplification Duhee Bang & George M Church Supplementary figures and text: Supplementary Figure 1. Dpo4 gene (1.05kb) construction by various methods. Supplementary Figure 2. Fraction of clones with incorrect full length Dpo4 sequence. Supplementary Figure 3. Circular assembly amplification efficiency for the construction of genes of various sizes. Supplementary Figure 4. Synthesis of a human minisatellite repeat sequence. Supplementary Figure 5. USER mediated-circular assembly amplification for the synthesis of tandem repeat Dpo4. Supplementary Table 1 Comparison of sequence errors generated by various methods for the synthesis of Dpo4. Supplementary Methods 1

2 Supplementary Figure 1. Dpo4 gene (1.05kb) construction by various methods. (a) Circular assembly amplification of 48 oligonucleotides. Use of insufficient exonuclease led to incomplete exonuclease degradation as shown in (a) lane 3 and lane 9 (compare to Figure 2a in the main text). (b) and (c) PCA reaction performed at two different annealing temperatures (65ºC for b and 70ºC for c); Lane 4 from both experiments was cloned.

3 Supplementary Figure 2. Fraction of clones with incorrect full length Dpo4 sequence made by various methods. Experiments are performed at annealing temperature (a) 65ºC and (b) 70ºC, respectively. Error bars denote a standard deviation (s.d.).

4 Supplementary Figure 3. Circular assembly amplification efficiency for the construction of genes of various sizes assessed by the synthesis of Pfu DNA polymerase gene fragments.

5 Supplementary Figure 4. Synthesis of a human minisatellite repeat sequence by circular assembly amplification. (a) Target DNA sequence (GenBank accession code: NT011515). (b) Lane 1-8; PCR products resulting from PCA reactions performed with different oligo concentrations. Lane 9 and 10; products resulting from PCR reactions on ligation mixtures. Lane 11 and 12; products resulting from PCR reactions on ligation mixtures treated with exonucleases. Lane 12 (red arrow) was used for further cloning and characterization of one clone shown in (c).

6 Supplementary Figure 5. USER mediated-circular assembly amplification for the synthesis of tandem repeat Dpo4. (a) Schematic representation of our strategy. (b) Verification of the order of tandem repeats of the Dpo4 genes using PCR amplification of the regions shown by crescent lines in the diagram below. Lanes on the gel are labeled according to the diagram below.

7 Supplementary Table 1 Comparison of sequence errors generated by various methods for the synthesis of Dpo4. (a) Number of errors per sequencing. A breakdown of the types of errors detected by sequencing is shown in parenthesis (deletion: insertion: transition: transversion). (b) Fraction of clones with incorrect full-length sequence.

8 Supplementary Methods. Experimental Procedure for Circular Assembly Amplification for the Construction of Dpo4 Target DNA sequence and oligonucleotide sequence design; DNA sequence (1052 base pairs) from Sulfolobus solfataricus P2 DNA polymerase IV was chosen as a target. Codon optimized 1056bp Dpo4 (Sulfolobus solfataricus P2 DNA polymerase IV) sequence was designed by using the Gene Design computer program < 24 plus strand oligonucleotides and 23 minus strand oligonucleotides were designed in such a way that, upon annealing, complementary oligos would overlap as a circular structure, leaving no gap to fill. In addition, one more guiding oligo (24th minus strand) was designed to join 5 and 3 ends of the Dpo4 sequence. The length of each oligo was approximately 40 to 50 base pairs, and the overlap of each oligo was designed to have a melting temperature of 60ºC by the nearestneighbor method. These oligonucleotides were synthesized by a commercial oligonucleotide synthesis company (Integrated DNA Technology). No purification step other than desalting was carried out. 5 phosphorylation of oligonucleotides; Each oligonucleotide was individually dissolved in water to 200 µm concentration. Equal volumes of the 24 plus strand oligo solutions were pooled together, and then the oligonucleotides are 5' phosphorylated by following procedures: 12 µl of the plus strand mixture was mixed with 120 µl of water, 12 µl of 10X T4 ligase buffer, and 6 µl of T4 polynucleotide kinase (10 U/µl, from New England Biolabs (NEB), Beverly, MA). The final concentration of each oligonucleotide was approximately 0.67 µm. This reaction mixture was incubated at 37 o C overnight, and stored at -20 o C. A pool of 23 minus strand oligos and 24 th minus strand oligo were 5' phosphorylated the same way. Annealing of 5' phosphorylated oligos, and circular ligation using thermostable ligase at 70ºC; The 5' phosphorylated oligos were annealed, and thermostable ligase was used to complete a circular double stranded-dna structure; 2.4 µl of Ampligase (100 units/µl, from Epicentre, Madison, WI) and 4.8 µl of 10X Ampligase buffer were mixed with 24 µl of a pool of 5'-phosphorylated 24 plus strand

9 oligos and 23 µl of a pool of 5 phosphorylated 23 minus strand oligos. This mixture was split to two batches, and 0.5 µl of a 5 phosphorylated 24 th minus strand oligo was added to the second batch. The concentration of each oligonucleotide in the ligation reaction was approximately 0.3 µm. The two batches of ligation mixture (with 47 oligonucleotides (as a control) & 48 oligonucleotides) were incubated at 95 o C for 3 minutes for melting, and were ramped to 70 o C at 0.1 o C/sec for annealing. The reaction mixture was incubated at 70 ºC for two hours for ligation, and was stored at 4 ºC. This thermo-reaction was carried out using a thermocycler. Exonuclease degradation of linear DNA; Exonucleases were used to degrade all but circularized DNA for the elimination of errors incurred during oligonucleotide synthesis. A typical exonuclease cocktail was prepared by mixing 36 µl of water, 5 µl of NEB Buffer 1, 6µl of exonuclease I (source from E. coli, 20 units/µl, NEB), 3µl of exonuclease III (100 units/µl, NEB), and 3µl of lambda exonuclease (100 units/µl, NEB). Then, typically 0.5µl aliquot from the circular assembly ligation reaction was mixed with 20µl of exonuclease cocktail, and incubated at 37ºC. Use of mismatch-cleaving endonuclease for the conversion of mismatch containing circular DNA to linear DNA; After four hours of exonuclease incubation, each reaction mixture was split to four batches (6 µl + 4 µl + 4 µl + 4 µl). The first batch (6µl) was incubated at 37ºC overnight without any treatment. The second batch (4 µl) was mixed with 0.5 µl of NEB buffer 4 and 1 µl of endonuclease V (100 units/µl, NEB). The third batch (4µl) was mixed with 1.5µl of aliquot from a cocktail made of 10µl of water +6 µl of NEB buffer µl of endonuclease V. The fourth batch was mixed with 1.5µl of aliquot of a cocktail made of 10µl of water +6µl of NEB buffer µl of endonuclease V. All these batches were incubated overnight at 37ºC. PCR amplification, Cloning & Sequencing; Outside primers were synthesized and purified using PAGE gel (Integrated DNA Technology). Using these primers, the double stranded DNA was amplified by PCR for cloning into a puc19 vector. A first primer (with an XbaI restriction site), a second primer (with a PstI restriction site), and iproof DNA polymerase (Bio-Rad, Hercules, CA) were used to amplify DNA sequence by PCR. PCR reaction was initiated by heating first at 95ºC for 3 min, followed by 38 cycles of the subsequent program: 95ºC for 30 s, 65ºC for 30 s, and 72ºC for 60 s. A final

10 extension at 72ºC was carried out for 10 min, and stored at 4ºC. Product band was excised, and extracted using QIAquick gel extraction column (Qiagen, Valencia, CA). The gel-purified Dpo4 gene products were cloned into puc19 vector (NEB), and transformed into T7 express competent E. coli cells (NEB). Individual colonies were picked and grown in Luria-Bertani broth containing carbenicillin antibiotics. Plasmids from grown colonies were purified, and sequenced using four different sequencing primers. Sequencing data was analyzed by using a DNA sequence analysis program, Lasergene (DNAstar, Madison, WI). Experimental Procedure for Polymerase Cycling Assembly (PCA); Each oligo was individually dissolved in water to 200 µm concentration. 24 plus strand oligos (oligo labeling from F1 to F24) and 23 minus strand oligos (oligo labeling from R1 to R23) were pooled together. 4 µl aliquot from the oligonucleotide pool was mixed with 32µl of water, and a series of six two-fold dilutions was carried out for the preparation of oligonucleotide pool concentrations ranging from 0.4µM to µM per each oligonucleotide. Aliquots of the dilution series were used for PCA reactions. Using PAGE gel purified outside primers, PCA was carried out. A first primer (with an XbaI restriction site), a second primer (with a PstI restriction site), and high-fidelity DNA polymerase (iproof DNA polymerase from BioRad, Hercules, CA) were used to amplify DNA sequence. PCA reaction was initiated by heating first at 95ºC for 3 min, followed by 35 cycles of the subsequent program: 95ºC for 30 s, 65ºC (or 70ºC) for 30 s, and 72ºC for 60 s. A final extension at 72ºC was carried out for 10 min, and stored at 4ºC. (see Supplementary Fig. 1b and c online for gel analysis of Dpo4 constructs from this PCA reaction). Product band was excised, and extracted using microcentrifuge column (Qiagen, Valencia, CA) for cloning into a puc19 vector. Transformation, sequencing, and sequence analysis were carried out as same procedures as Circular Assembly Amplification.

11 Experimental Procedures for Pfu DNA Polymerase Synthesis by USER-mediated Circular Assembly Amplification Protein sequence (775 codons) from Pfu DNA Polymerase (GenBank accession code for protein sequence: P61875) was chosen as a target. Codon optimized 2325bp sequence was designed by using the Gene Design computer program. The sequence was divided to three fragments (Pfu(1-811), Pfu( ), and Pfu( )). For each fragment, plus and minus strand oligonucleotides were designed in such a way that, upon annealing, complementary oligos would overlap as a circular structure, leaving no gap to fill. For the construction of each fragment, we largely followed a protocol for the synthesis of Dpo4 shown above except PCR amplification step that required primers containing deoxyuridines (three primer sets, see sequence information below). PfuTurboCx Hotstart DNA polymerase (Stratagene, CA) was used to amplify DNA sequence by PCR. 5% (volume) DMSO was added. PCR reaction was initiated by heating first at 95ºC for 3 min, followed by 28 cycles of the subsequent program: 95ºC for 30 s, 65ºC for 30 s, and 72ºC for 60s. A final extension at 72ºC was carried out for 10 min, and stored at 4ºC. Without a further purification step, 1 µl of aliquots from each PCR reaction was mixed with 1µl of USER TM (NEB), 1µl of 10X Thermopol buffer (NEB) and 9 µl of water. Each reaction mixture was incubated at 37 o C for 60 minutes. We mixed aliqout from the USER TM treated reactions (2 µl each from Pfu(1-811), Pfu( ), and Pfu( )), and added 0.5 µl of Ampligase and 1 µl of 10X Ampligase buffer, then incubated at 70 o C for two hours, and stored at 4 ºC. Exonuclease degradation of linear DNA was performed as described above. Using outside primers, we amplified full-length double stranded-dna. The PCR amplified DNA product was cloned into puc19 vector (NEB), and transformed into NEB 5-alpha Competent E. coli cells (NEB).

12 Experimental Procedures for the Synthesis of a Human Minisatellite Sequence by Circular Assembly Amplification; A human minisatellite region (GenBank accession code: NT011515) composed of tandem repeats of 45 bp with 97% homology was chosen as a target (see DNA sequence in Supplementary Fig. 4a). Plus strand oligonucleotides and minus strand oligonucleotides were designed, and 5 phosphorylated. Circular ligation reaction was performed as described above as an exception in the ligation temperature gradient to provide higher level of stringency for an annealing step; ligation mixtures were incubated at 95 o C for 3 minutes for melting, and were ramped to 80 o C at 0.1 o C/sec for annealing. The reaction mixture was incubated at 80 ºC for one hour, 75ºC for one hour, and 70ºC for one hour, and was stored at 4 ºC. Exonuclease degradation of linear DNA, PCR amplification, cloning and sequencing were performed as described above. Exceptions are; DNA amplification was performed by 22 PCR cycles; PCR amplicon was purified using Qiagen PCR purification column (Qiagen, Valencia, CA); transformation was performed using 5-alpha Competent E. coli cells (NEB).

13 Experimental Procedures for Tamdem Dpo4 Repeat Synthesis by USER-mediated Circular Assembly Amplification PCR amplification of Dpo4 and puc19 were performed using primers containing two deoxyu (four primer sets for Dpo4 amplification and one primer set for puc19 amplification, see sequence information below). PfuTurboCx Hotstart DNA polymerase (Stratagen, CA) was used to amplify DNA sequence by PCR. 5% (volume) DMSO was added. PCR reaction was initiated by heating first at 95ºC for 3 min, followed by cycles (30 for Dpo4, or 25 cycles for puc19) of the subsequent program: 95ºC for 30 s, 65ºC for 30 s, and 72ºC for 60s (for Dpo4), or 3min (for puc19). A final extension at 72ºC was carried out for 10 min, and stored at 4ºC. Without a further purification step, 1 µl of aliquots from each PCR reaction was mixed with 1µl of USER TM (NEB), 1µl of 10X Thermopol buffer (NEB) and 9 µl of water. Each reaction mixture was incubated at 37 o C for 60 minutes. Aliqouts from USER TM treated reaction (1µl each from four Dpo4 mixtures and 1µl from puc19 mixture) were mixed with 0.5 µl of Ampligase and 1 µl of 10X Ampligase buffer, 5 µl of water, and incubated at 70 o C for two hours for ligation. A resulting construct was transformed into NEB 5-alpha Competent E. coli cells (NEB). A colony was picked and characterized by using a set of PCR primers listed below.

14 List of target gene sequences and list of oligonucleotides Target Dpo4 sequence: ATGATCGTTCTGTTCGTTGACTTCGACTACTTCTACGCTCAGGTTGAAGAAGTTCTGAACCCGTCTCTGAAAGGTAAACCGGTTGTTGT TTGCGTTTTCTCTGGTCGTTTCGAAGACTCTGGTGCTGTTGCTACCGCTAACTACGAAGCTCGTAAATTCGGTGTTAAAGCTGGTATCC CGATCGTTGAAGCTAAAAAAATCCTGCCGAACGCTGTTTACCTGCCGATGCGTAAAGAAGTTTACCAGCAGGTTTCTTCTCGTATCATG AACCTGCTGCGTGAATACTCTGAAAAAATCGAAATCGCTTCTATCGACGAAGCTTACCTGGACATCTCTGACAAAGTTCGTGACTACCG TGAAGCTTACAACCTGGGTCTGGAAATCAAAAACAAAATCCTGGAAAAAGAAAAAATCACCGTTACCGTTGGTATCTCTAAAAACAAAG TTTTCGCTAAAATCGCTGCTGACATGGCTAAACCGAACGGTATCAAAGTTATCGACGACGAAGAAGTTAAACGTCTGATCCGTGAACTG GACATCGCTGACGTTCCGGGTATCGGTAACATCACCGCTGAAAAACTGAAAAAACTGGGTATCAACAAACTGGTTGACACCCTGTCTAT CGAATTCGACAAACTGAAAGGTATGATCGGTGAAGCTAAAGCTAAATACCTGATCTCTCTGGCTCGTGACGAATACAACGAACCGATCC GTACCCGTGTTCGTAAATCTATCGGTCGTATCGTTACCATGAAACGTAACTCTCGTAACCTGGAAGAAATCAAACCGTACCTGTTCCGT GCTATCGAAGAATCTTACTACAAACTGGACAAACGTATCCCGAAAGCTATCCACGTTGTTGCTGTTACCGAAGACCTGGACATCGTTTC TCGTGGTCGTACCTTCCCGCACGGTATCTCTAAAGAAACCGCTTACTCTGAATCTGTTAAACTGTTGCAAAAAATCCTGGAAGAAGACG AACGTAAAATCCGTCGTATCGGTGTTCGTTTCTCTAAATTCATCGAAGCTATCGGTCTGGACAAATTCTTCGACACC 24 plus strand oligonucleotides: F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 ATG ATC GTT CTG TTC GTT GAC TTC GAC TAC TTC TAC GCT CAG GTT GAA GAA GTT CTG AAC CCG TCT CTG AAA GGT AAA CCG GTT G TTG TTT GCG TTT TCT CTG GTC GTT TCG AAG ACT CTG GTG CT GTT GCT ACC GCT AAC TAC GAA GCT CGT AAA TTC GGT GTT AAA GC TGG TAT CCC GAT CGT TGA AGC TAA AAA AAT CCT GCC GAA CG CTG TTT ACC TGC CGA TGC GTA AAG AAG TTT ACC AGC AGG TTT CTT CTC GTA TCA TGA ACC TGC TGC GTG AAT ACT CTG AAA AAA TCG AAA TCG CTT CTA TCG ACG AAG CTT ACC TGG ACA TCT CTG ACA AAG TTC GTG ACT ACC GTG AAG CTT ACA ACC TGG GTC TGG AAA TC AAA AAC AAA ATC CTG GAA AAA GAA AAA ATC ACC GTT ACC GTT GGT ATC TC TAA AAA CAA AGT TTT CGC TAA AAT CGC TGC TGA CAT GGC TAA ACC G AAC GGT ATC AAA GTT ATC GAC GAC GAA GAA GTT AAA CGT CTG ATC C GTG AAC TGG ACA TCG CTG ACG TTC CGG GTA TCG GTA AC ATC ACC GCT GAA AAA CTG AAA AAA CTG GGT ATC AAC AAA CTG GTT GAC ACC CTG TCT ATC GAA TTC GAC AAA CTG AAA GGT ATG ATC GGT GAA GCT AAA GCT AAA TAC CTG ATC TCT CTG GCT CGT GAC GAA TAC AAC GAA CCG ATC CGT ACC CGT GTT CGT AAA TCT ATC GGT C GTA TCG TTA CCA TGA AAC GTA ACT CTC GTA ACC TGG AAG AAA TCA AAC CGT ACC TGT TCC GTG CTA TCG AAG AAT CTT ACT ACA AAC TGG ACA AA CGT ATC CCG AAA GCT ATC CAC GTT GTT GCT GTT ACC GAA GAC

15 F21 F22 F23 F24 CTG GAC ATC GTT TCT CGT GGT CGT ACC TTC CCG CAC GGT ATC TCT AAA GAA ACC GCT TAC TCT GAA TCT GTT AAA CTG TTG CAA AA AAT CCT GGA AGA AGA CGA ACG TAA AAT CCG TCG TAT CGG TGT TC GTT TCT CTA AAT TCA TCG AAG CTA TCG GTC TGG ACA AAT TCT TCG ACA CC 24 minus oligonucleotides: R24 (24th minus strand) is a guiding oligonucleotide designed to join 5 and 3 ends of the Dpo4 sequence. R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 ACG GGT TCA GAA CTT CTT CAA CCT GAG CGT AGA AGT AGT CGA GAC CAG AGA AAA CGC AAA CAA CAA CCG GTT TAC CTT TCA GAG TTC GTA GTT AGC GGT AGC AAC AGC ACC AGA GTC TTC GAA AC CTT CAA CGA TCG GGA TAC CAG CTT TAA CAC CGA ATT TAC GAG C GCA TCG GCA GGT AAA CAG CGT TCG GCA GGA TTT TTT TAG GCA GGT TCA TGA TAC GAG AAG AAA CCT GCT GGT AAA CTT CTT TAC GCT TCG TCG ATA GAA GCG ATT TCG ATT TTT TCA GAG TAT TCA CGC A GCT TCA CGG TAG TCA CGA ACT TTG TCA GAG ATG TCC AGG TAA GAT TTT TTC TTT TTC CAG GAT TTT GTT TTT GAT TTC CAG ACC CAG GTT GTA A GCG ATT TTA GCG AAA ACT TTG TTT TTA GAG ATA CCA ACG GTA ACG GT TCG TCG ATA ACT TTG ATA CCG TTC GGT TTA GCC ATG TCA GCA TCA GCG ATG TCC AGT TCA CGG ATC AGA CGT TTA ACT TCT TCG TTT TTC AGT TTT TCA GCG GTG ATG TTA CCG ATA CCC GGA ACG CGA ATT CGA TAG ACA GGG TGT CAA CCA GTT TGT TGA TAC CCA GT GAG ATC AGG TAT TTA GCT TTA GCT TCA CCG ATC ATA CCT TTC AGT TTG T GGT ACG GAT CGG TTC GTT GTA TTC GTC ACG AGC CAG A AGT TAC GTT TCA TGG TAA CGA TAC GAC CGA TAG ATT TAC GAA CAC G GAT AGC ACG GAA CAG GTA CGG TTT GAT TTC TTC CAG GTT ACG AG GTG GAT AGC TTT CGG GAT ACG TTT GTC CAG TTT GTA GTA AGA TTC TTC CAC GAG AAA CGA TGT CCA GGT CTT CGG TAA CAG CAA CAA C GTA AGC GGT TTC TTT AGA GAT ACC GTG CGG GAA GGT ACG AC TAC GTT CGT CTT CTT CCA GGA TTT TTT GCA ACA GTT TAA CAG ATT CAG A CGA TAG CTT CGA TGA ATT TAG AGA AAC GAA CAC CGA TAC GAC GGA TTT AGT CAA CGA ACA GAA CGA TCA TGG TGT CGA AGA ATT TGT CCA GAC PCR amplification Primers: Forward Primer (with XbaI site) GGTGGTTCTAGAATGATCGTTCTGTTCGTTGACTTCGACTACTTC Reverse Primer (with PstI site) GGTGGTCTGCAGTTAGGTGTCGAAGAATTTGTCCAGACCGATAGC Four Sequencing Primers:

16 S-Primer1 GCTGCAAGGCGATTAAGTTG S-Primer2 GGTATCTCTAAAAACAAAGTTTTCGC S-primer3 GTCAACCAGTTTGTTGATACCC S-primer4 CGGCTCGTATGTTGTGTGG Target Human Minisatellite Sequence (GenBank accession code: NT011515): CAGTTAGGGGTACACATCCTATCTGCAAATTCCTTTCCAATGGTTCAAGAAAGAATGG GCCTGGATCATGTGGGAAGACAGGTAATGAAGCCCTGAGGGTAGA GCCTGGATCATGTGGGAGGACAGGTCATGAAGCCCTGAGGGTAGA GCCTGGATCACGTGGGAGGACAGGTCATGAAGTCCTCAGGGTAGA GCCTGGATCATGTGGGAGGAAGGGTCATGAAGCCCTGAGGGTAGA GCCTGGATCACGTGGGAGGACAGGTCATGAAGCCC GGTAGGGTGCCACCCACTAAAGAATTCTAGTC (Italics sequence represents random sequence to add a reverse primer site for PCR amplification) 8 plus strand oligonucleotides for a human minisatellite sequence: F1 GGCAGTTAGGGGTACACATCCTATCTGCAAATTCCTTTCCAATGG F2 TTCAAGAAAGAATGGGCCTGGATCATGTGGGAAGACAGGTAATGA F3 AGCCCTGAGGGTAGAGCCTGGATCATGTGGGAGGACA F4 GGTCATGAAGCCCTGAGGGTAGAGCCTGGATCACGTG F5 GGAGGACAGGTCATGAAGTCCTCAGGGTAGAGCCTGGA F6 TCATGTGGGAGGAAGGGTCATGAAGCCCTGAGGGTAGA F7 GCCTGGATCACGTGGGAGGACAGGTCATGAAGCCC F8 GGTAGGGTGCCACCCACTAAAGAATTCTAGTCGAGAGCGC (Italics sequences were added to keep annealing temperature to be 60 degree C) 8 minus strand oligonucleotides for a human minisatellite sequence: R8 (8th minus strand) is a guiding oligonucleotide designed to join 5 and 3 ends of the tandem repeat sequence. R1 CAGGCCCATTCTTTCTTGAACCATTGGAAAGGAATTTGCAGATAG R2 GCTCTACCCTCAGGGCTTCATTACCTGTCTTCCCACATGATC R3 CCTCAGGGCTTCATGACCTGTCCTCCCACATGATCCAG R4 GACTTCATGACCTGTCCTCCCACGTGATCCAGGCTCTAC R5 ACCCTTCCTCCCACATGATCCAGGCTCTACCCTGAG R6 CCCACGTGATCCAGGCTCTACCCTCAGGGCTTCATG R7 GGGTGGCACCCTACCGGGCTTCATGACCTGTCCT R8 GATGTGTACCCCTAACTGCCGCGCTCTCGACTAGAATTCTTTAGT PCR amplification Primers for a human minisatellite sequence:

17 Forward Primer (with XbaI site) GGTGGTTCTAGACAGTTAGGGGTACACATCCTATC Reverse Primer (with PstI site) GGTGGTCTGCAGGACTAGAATTCTTTAGTGGGTGGC Tandem Dpo4 Repeat Synthesis Amplification primers (containing deoxyu, du) for tandem Dpo4 repeat synthesis Dpo4A-for AAGCAAAGCAdUCCTTGACCdUATGATCGTTCTGTTCGTTGACTTCGACTACTTC Dpo4A-rev AAATGGCAGAdUTCCGCACTdUGGTGTCGAAGAATTTGTCCAGACCGATAGC Dpo4B-for Dpo4B-rev AAGTGCGGAAdUCTGCCATTdUATGATCGTTCTGTTCGTTGACTTCGACTACTTC ACCCAGAGAAdUCGGTCCAGdUGGTGTCGAAGAATTTGTCCAGACCGATAGC Dpo4C-for Dpo4C-rev ACTGGACCGAdUTCTCTGGGdUATGATCGTTCTGTTCGTTGACTTCGACTACTTC ATTATTTCCAdUGCGCCGAGdUGGTGTCGAAGAATTTGTCCAGACCGATAGC Dpo4D-for Dpo4D-rev ACTCGGCGCAdUGGAAATAAdUATGATCGTTCTGTTCGTTGACTTCGACTACTTC ACTGTCGTCAdUGGAGCACAdUGGTGTCGAAGAATTTGTCCAGACCGATAGC puc19-for puc19-rev ATGTGCTCCAdUGACGACAGdUgcatgcaagcttggcgtaatc AGGTCAAGGAdUGCTTTGCTdUctgcaggtcgactctagagg Dpo4 tandem repeat connectivity verification primers: Amplicon 1 by puc19-seq-150f and puc19-seq-rev Amplicon 2 by puc19-seq-150f and Dpo4A-Seq-rev Amplicon 3 by Dpo4A-Seq-for and Dpo4A-Seq-rev Amplicon 4 by Dpo4B-Seq-for and Dpo4B-Seq-rev Amplicon 5 by Dpo4C-Seq-for and Dpo4C-Seq-rev Amplicon 6 by Dpo4D-Seq-for and Dpo4D-Seq-rev Amplicon 7 by puc19-seq-for and puc19-seq-rev Amplicon 8 by puc19-seq-for and puc19-seq-150r Dpo4A-Seq-for Dpo4A-Seq-rev Dpo4B-Seq-for Dpo4B-Seq-rev Dpo4C-Seq-for GGTGGTTCTAGAAAGCAAAGCATCCTTGACCT GGTGGTCTGCAGAAATGGCAGATTCCGCACTT GGTGGTTCTAGAAAGTGCGGAATCTGCCATTT GGTGGTCTGCAGACCCAGAGAATCGGTCCAGT GGTGGTTCTAGAACTGGACCGATTCTCTGGGT

18 Dpo4C-Seq-rev Dpo4D-Seq-for Dpo4D-Seq-rev puc19-seq-for puc19-seq-rev puc19-seq-150f puc19-seq-150r GGTGGTCTGCAGATTATTTCCATGCGCCGAGT GGTGGTTCTAGAACTCGGCGCATGGAAATAAT GGTGGTCTGCAGACTGTCGTCATGGAGCACAT GGTGGTTCTAGAATGTGCTCCATGACGACAGT GGTGGTCTGCAGAGGTCAAGGATGCTTTGCTT GGAGAAAATACCGCATCAGG GGCCGATTCATTAATGCAGC Target Pfu DNA polymerase sequence: ATGATCCTGGACGTTGACTACATCACCGAAGAAGGTAAACCGGTTATCCGTCTGTTCAAAAAAGAAAACGGTAAATTCAAAATCGAACA CGACCGTACCTTCCGTCCGTACATCTACGCTCTGCTGCGTGACGACTCTAAAATCGAAGAAGTTAAAAAAATCACCGGTGAACGTCACG GTAAAATCGTTCGTATCGTTGACGTTGAAAAAGTTGAAAAAAAATTCCTGGGTAAACCGATCACCGTTTGGAAACTGTACCTGGAACAC CCGCAGGACGTTCCGACCATCCGTGAAAAAGTTCGTGAACACCCGGCTGTTGTTGACATCTTCGAATACGACATCCCGTTCGCTAAACG TTACCTGATCGACAAAGGTCTGATCCCGATGGAAGGTGAAGAAGAACTGAAAATCCTGGCTTTCGACATCGAAACCCTGTACCACGAAG GTGAAGAATTCGGTAAAGGTCCGATCATCATGATCTCTTACGCTGACGAAAACGAAGCTAAAGTTATCACCTGGAAAAACATCGACCTG CCGTACGTTGAAGTTGTTTCTTCTGAACGTGAAATGATCAAACGTTTCCTGCGTATCATCCGTGAAAAAGACCCGGACATCATCGTTAC CTACAACGGTGACTCTTTCGACTTCCCGTACCTGGCTAAACGTGCTGAAAAACTGGGTATCAAACTGACCATCGGTCGTGACGGTTCTG AACCGAAAATGCAGCGTATCGGTGACATGACCGCTGTTGAAGTTAAAGGTCGTATCCACTTCGACCTGTACCACGTTATCACCCGTACC ATCAACCTGCCGACCTACACCCTGGAAGCTGTTTACGAAGCTATCTTCGGTAAACCGAAAGAAAAAGTTTACGCTGACGAAATCGCTAA AGCTTGGGAATCTGGTGAAAACCTGGAACGTGTTGCTAAATACTCTATGGAAGACGCTAAAGCTACCTACGAACTGGGTAAAGAATTCC TGCCGATGGAAATCCAGCTGTCTCGTCTGGTTGGTCAGCCGCTGTGGGACGTTTCTCGTTCTTCTACCGGTAACCTGGTTGAATGGTTC CTGCTGCGTAAAGCTTACGAACGTAACGAAGTTGCTCCGAACAAACCGTCTGAAGAAGAATACCAGCGTCGTCTGCGTGAATCTTACAC CGGTGGTTTCGTTAAAGAACCGGAAAAAGGTCTGTGGGAAAACATCGTTTACCTGGACTTCCGTGCTCTGTACCCGTCTATCATCATCA CCCACAACGTTTCTCCGGACACCCTGAACCTGGAAGGTTGCAAAAACTACGACATCGCTCCGCAGGTTGGTCACAAATTCTGCAAAGAC ATCCCGGGTTTCATCCCGTCTCTGCTGGGTCACCTGCTGGAAGAACGTCAGAAAATCAAAACCAAAATGAAAGAAACCCAGGACCCGAT CGAAAAAATCCTGCTGGACTACCGTCAGAAAGCTATCAAACTGCTGGCTAACTCTTTCTACGGTTACTACGGTTACGCTAAAGCTCGTT GGTACTGCAAAGAATGCGCTGAATCTGTTACCGCTTGGGGTCGTAAATACATCGAACTGGTTTGGAAAGAACTGGAAGAAAAATTCGGT TTCAAAGTTCTGTACATCGACACCGACGGTCTGTACGCTACCATCCCGGGTGGTGAATCTGAAGAAATCAAAAAAAAAGCTCTGGAATT CGTTAAATACATCAACTCTAAACTGCCGGGTCTGCTGGAACTGGAATACGAAGGTTTCTACAAACGTGGTTTCTTCGTTACCAAAAAAC GTTACGCTGTTATCGACGAAGAAGGTAAAGTTATCACCCGTGGTCTGGAAATCGTTCGTCGTGACTGGTCTGAAATCGCTAAAGAAACC CAGGCTCGTGTTCTGGAAACCATCCTGAAACACGGTGACGTTGAAGAAGCTGTTCGTATCGTTAAAGAAGTTATCCAGAAACTGGCTAA CTACGAAATCCCGCCGGAAAAACTGGCTATCTACGAACAGATCACCCGTCCGCTGCACGAATACAAAGCTATCGGTCCGCACGTTGCTG TTGCTAAAAAACTGGCTGCTAAAGGTGTTAAAATCAAACCGGGTATGGTTATCGGTTACATCGTTCTGCGTGGTGACGGTCCGATCTCT

19 AACCGTGCTATCCTGGCTGAAGAATACGACCCGAAAAAACACAAATACGACGCTGAATACTACATCGAAAACCAGGTTCTGCCGGCTGT TCTGCGTATCCTGGAAGGTTTCGGTTACCGTAAAGAAGACCTGCGTTACCAGAAAACCCGTCAGGTTGGTCTGACCTCTTGGCTGAACA TCAAAAAATCT 55 plus strand oligonucleotides: PfuF1 ATGATCCTGGACGTTGACTACATCACCGAAGAAGGTAAACCG PfuF2 GTTATCCGTCTGTTCAAAAAAGAAAACGGTAAATTCAAAATCGAACACGACC PfuF3 GTACCTTCCGTCCGTACATCTACGCTCTGCTGCGTG PfuF4 ACGACTCTAAAATCGAAGAAGTTAAAAAAATCACCGGTGAACGTCAC PfuF5 GGTAAAATCGTTCGTATCGTTGACGTTGAAAAAGTTGAAAAAAAATTCCTGGG PfuF6 TAAACCGATCACCGTTTGGAAACTGTACCTGGAACACCC PfuF7 GCAGGACGTTCCGACCATCCGTGAAAAAGTTCGTGAAC PfuF8 ACCCGGCTGTTGTTGACATCTTCGAATACGACATCCCG PfuF9 TTCGCTAAACGTTACCTGATCGACAAAGGTCTGATCCCGATGG PfuF10 AAGGTGAAGAAGAACTGAAAATCCTGGCTTTCGACATCGAAACCC PfuF11 TGTACCACGAAGGTGAAGAATTCGGTAAAGGTCCGATCATCATG PfuF12 ATCTCTTACGCTGACGAAAACGAAGCTAAAGTTATCACCTGGAAAAAC PfuF13 ATCGACCTGCCGTACGTTGAAGTTGTTTCTTCTGAACGTG PfuF14 AAATGATCAAACGTTTCCTGCGTATCATCCGTGAAAAAGACCCG PfuF15 GACATCATCGTTACCTACAACGGTGACTCTTTCGACTTCCCG PfuF16 TACCTGGCTAAACGTGCTGAAAAACTGGGTATCAAACTGACCA PfuF17 TCGGTCGTGACGGTTCTGAACCGAAAATGCAGCGTATC PfuF18 GGTGACATGACCGCTGTTGAAGTTAAAGGTCGTATCCACT PfuF19 TCGACCTGTACCACGTTATCACCCGTACCATCAACCTGC PfuF20 CGACCTACACCCTGGAAGCTGTTTACGAAGCTATCTTCGGT PfuF21 AAACCGAAAGAAAAAGTTTACGCTGACGAAATCGCTAAAGCTTGG PfuF22 GAATCTGGTGAAAACCTGGAACGTGTTGCTAAATACTCTATGGAAGAC PfuF23 GCTAAAGCTACCTACGAACTGGGTAAAGAATTCCTGCCGATGGA PfuF24 AATCCAGCTGTCTCGTCTGGTTGGTCAGCCGCTGTGG PfuF25 GACGTTTCTCGTTCTTCTACCGGTAACCTGGTTGAATGGTTCCT PfuF26 GCTGCGTAAAGCTTACGAACGTAACGAAGTTGCTCCGAAC PfuF27 AAACCGTCTGAAGAAGAATACCAGCGTCGTCTGCGTGA PfuF28 ATCTTACACCGGTGGTTTCGTTAAAGAACCGGAAAAAGGTCTG PfuF29 TGGGAAAACATCGTTTACCTGGACTTCCGTGCTCTGTACC PfuF30 CGTCTATCATCATCACCCACAACGTTTCTCCGGACACC PfuF31 CTGAACCTGGAAGGTTGCAAAAACTACGACATCGCTCCG PfuF32 CAGGTTGGTCACAAATTCTGCAAAGACATCCCGGGTTTCATC PfuF33 CCGTCTCTGCTGGGTCACCTGCTGGAAGAACGTCAG PfuF34 AAAATCAAAACCAAAATGAAAGAAACCCAGGACCCGATCGAAAAAATCC PfuF35 TGCTGGACTACCGTCAGAAAGCTATCAAACTGCTGGCTAA PfuF36 CTCTTTCTACGGTTACTACGGTTACGCTAAAGCTCGTTGGT

20 PfuF37 ACTGCAAAGAATGCGCTGAATCTGTTACCGCTTGGGGT PfuF38 CGTAAATACATCGAACTGGTTTGGAAAGAACTGGAAGAAAAATTCGGTT PfuF39 TCAAAGTTCTGTACATCGACACCGACGGTCTGTACGCTACC PfuF40 ATCCCGGGTGGTGAATCTGAAGAAATCAAAAAAAAAGCTCTGGAATTC PfuF41 GTTAAATACATCAACTCTAAACTGCCGGGTCTGCTGGAACTGGAATAC PfuF42 GAAGGTTTCTACAAACGTGGTTTCTTCGTTACCAAAAAACGTTACG PfuF43 CTGTTATCGACGAAGAAGGTAAAGTTATCACCCGTGGTCTGGA PfuF44 AATCGTTCGTCGTGACTGGTCTGAAATCGCTAAAGAAACCCA PfuF45 GGCTCGTGTTCTGGAAACCATCCTGAAACACGGTGACG PfuF46 TTGAAGAAGCTGTTCGTATCGTTAAAGAAGTTATCCAGAAACTGGCT PfuF47 AACTACGAAATCCCGCCGGAAAAACTGGCTATCTACGAACAG PfuF48 ATCACCCGTCCGCTGCACGAATACAAAGCTATCGGTCC PfuF49 GCACGTTGCTGTTGCTAAAAAACTGGCTGCTAAAGGTGT PfuF50 TAAAATCAAACCGGGTATGGTTATCGGTTACATCGTTCTGCGTGGT PfuF51 GACGGTCCGATCTCTAACCGTGCTATCCTGGCTGAAGAATACG PfuF52 ACCCGAAAAAACACAAATACGACGCTGAATACTACATCGAAAACCAG PfuF53 GTTCTGCCGGCTGTTCTGCGTATCCTGGAAGGTTTCG PfuF54 GTTACCGTAAAGAAGACCTGCGTTACCAGAAAACCCGTCAGG PfuF55 TTGGTCTGACCTCTTGGCTGAACATCAAAAAATCTTATTATTATAAGAAACGG (Italics sequences were added to keep annealing temperature to be 60 degree C) 55 minus oligonucleotides: PfuR1 GTTTTCTTTTTTGAACAGACGGATAACCGGTTTACCTTCTTCGGTGA PfuR2 GATGTACGGACGGAAGGTACGGTCGTGTTCGATTTTGAATTTACC PfuR3 TTTTTAACTTCTTCGATTTTAGAGTCGTCACGCAGCAGAGCGTA PfuR4 GTCAACGATACGAACGATTTTACCGTGACGTTCACCGGTGATT PfuR5 TCCAAACGGTGATCGGTTTACCCAGGAATTTTTTTTCAACTTTTTCAAC PfuR6 GGTCGGAACGTCCTGCGGGTGTTCCAGGTACAGTT PfuR7 GTCAACAACAGCCGGGTGTTCACGAACTTTTTCACGGAT PfuR8 CGATCAGGTAACGTTTAGCGAACGGGATGTCGTATTCGAAGAT PfuR9 AGGATTTTCAGTTCTTCTTCACCTTCCATCGGGATCAGACCTTTGT PfuR10 AATTCTTCACCTTCGTGGTACAGGGTTTCGATGTCGAAAGCC PfuR11 CGTTTTCGTCAGCGTAAGAGATCATGATGATCGGACCTTTACCG PfuR12 ACGTACGGCAGGTCGATGTTTTTCCAGGTGATAACTTTAGCTT PfuR13 CGCAGGAAACGTTTGATCATTTCACGTTCAGAAGAAACAACTTCA PfuR14 CCGTTGTAGGTAACGATGATGTCCGGGTCTTTTTCACGGATGATA PfuR15 CAGCACGTTTAGCCAGGTACGGGAAGTCGAAAGAGTCA PfuR16 AGAACCGTCACGACCGATGGTCAGTTTGATACCCAGTTTTT PfuR17 ACAGCGGTCATGTCACCGATACGCTGCATTTTCGGTTC PfuR18 TGATAACGTGGTACAGGTCGAAGTGGATACGACCTTTAACTTCA PfuR19 GCTTCCAGGGTGTAGGTCGGCAGGTTGATGGTACGGG PfuR20 AGCGTAAACTTTTTCTTTCGGTTTACCGAAGATAGCTTCGTAAACA

21 PfuR21 GTTCCAGGTTTTCACCAGATTCCCAAGCTTTAGCGATTTCGTC PfuR22 CCAGTTCGTAGGTAGCTTTAGCGTCTTCCATAGAGTATTTAGCAACAC PfuR23 CCAGACGAGACAGCTGGATTTCCATCGGCAGGAATTCTTTAC PfuR24 CGGTAGAAGAACGAGAAACGTCCCACAGCGGCTGACCAA PfuR25 GTTCGTAAGCTTTACGCAGCAGGAACCATTCAACCAGGTTAC PfuR26 TGGTATTCTTCTTCAGACGGTTTGTTCGGAGCAACTTCGTTAC PfuR27 CGAAACCACCGGTGTAAGATTCACGCAGACGACGC PfuR28 CCAGGTAAACGATGTTTTCCCACAGACCTTTTTCCGGTTCTTTAA PfuR29 TGTGGGTGATGATGATAGACGGGTACAGAGCACGGAAGT PfuR30 TGCAACCTTCCAGGTTCAGGGTGTCCGGAGAAACGT PfuR31 GCAGAATTTGTGACCAACCTGCGGAGCGATGTCGTAGTTTT PfuR32 TGACCCAGCAGAGACGGGATGAAACCCGGGATGTCTTT PfuR33 GGGTTTCTTTCATTTTGGTTTTGATTTTCTGACGTTCTTCCAGCAGG PfuR34 TCTGACGGTAGTCCAGCAGGATTTTTTCGATCGGGTCCT PfuR35 AACCGTAGTAACCGTAGAAAGAGTTAGCCAGCAGTTTGATAGCTT PfuR36 TCAGCGCATTCTTTGCAGTACCAACGAGCTTTAGCGT PfuR37 CCAAACCAGTTCGATGTATTTACGACCCCAAGCGGTAACAGAT PfuR38 GGTGTCGATGTACAGAACTTTGAAACCGAATTTTTCTTCCAGTTCTTT PfuR39 AGATTCACCACCCGGGATGGTAGCGTACAGACCGTC PfuR40 CGGCAGTTTAGAGTTGATGTATTTAACGAATTCCAGAGCTTTTTTTTTGATTTCTTC PfuR41 AAACCACGTTTGTAGAAACCTTCGTATTCCAGTTCCAGCAGACC PfuR42 ACTTTACCTTCTTCGTCGATAACAGCGTAACGTTTTTTGGTAACGAAG PfuR43 CCAGTCACGACGAACGATTTCCAGACCACGGGTGATA PfuR44 GGTTTCCAGAACACGAGCCTGGGTTTCTTTAGCGATTTCAGA PfuR45 ACGATACGAACAGCTTCTTCAACGTCACCGTGTTTCAGGAT PfuR46 CGGCGGGATTTCGTAGTTAGCCAGTTTCTGGATAACTTCTTTA PfuR47 GCAGCGGACGGGTGATCTGTTCGTAGATAGCCAGTTTTTC PfuR48 TTTAGCAACAGCAACGTGCGGACCGATAGCTTTGTATTCGT PfuR49 CGATAACCATACCCGGTTTGATTTTAACACCTTTAGCAGCCAGTTT PfuR50 CGGTTAGAGATCGGACCGTCACCACGCAGAACGATGTAAC PfuR51 GTCGTATTTGTGTTTTTTCGGGTCGTATTCTTCAGCCAGGATAGCA PfuR52 AGAACAGCCGGCAGAACCTGGTTTTCGATGTAGTATTCAGC PfuR53 CGCAGGTCTTCTTTACGGTAACCGAAACCTTCCAGGATACGC PfuR54 AGCCAAGAGGTCAGACCAACCTGACGGGTTTTCTGGTAA PfuR55 TGTAGTCAACGTCCAGGATCATCCGTTTCTTATAATAATAAGATTTTTTGATGTTC PfuR55 (55th minus strand) is a guiding oligonucleotide designed to join 5 and 3 ends of the Pfu DNA polymerase sequence. Guiding minus oligonucleotides for the circular ligations of different size Pfu DNA polymerase fragments:

22 GuidePfuSeqment(1-694) TGTAGTCAACGTCCAGGATCATTGGTCAGTTTGATACCCAGTTTTT (Complimentary to primers PfuF1 & PfuF16 for a ligation of 694bp circular DNA) GuidePfuSeqment(1-897) TGTAGTCAACGTCCAGGATCATCCAAGCTTTAGCGATTTCGTC (Complimentary to primers PfuF1 & PfuF21 for a ligation of 897bp circular DNA) GuidePfuSeqment(1-1148) TGTAGTCAACGTCCAGGATCATTCACGCAGACGACGC (Complimentary to primers PfuF1 & PfuF27 for a ligation of 1148bp circular DNA) GuidePfuSeqment(1-1386) TGTAGTCAACGTCCAGGATCATCTGACGTTCTTCCAGCAGG (Complimentary to primers PfuF1 & PfuF33 for a ligation of 1386bp circular DNA) GuidePfuSeqment(1-1603) TGTAGTCAACGTCCAGGATCATAACCGAATTTTTCTTCCAGTTCTTT (Complimentary to primers PfuF1 & PfuF38 for a ligation of 1603bp circular DNA) GuidePfuSeqment(1-1871) TGTAGTCAACGTCCAGGATCATTGGGTTTCTTTAGCGATTTCAGA (Complimentary to primers PfuF1 & PfuF44 for a ligation of 1871bp circular DNA) GuidePfuSeqment(1-2075) TGTAGTCAACGTCCAGGATCATACACCTTTAGCAGCCAGTTT (Complimentary to primers PfuF1 & PfuF49 for a ligation of 2075bp circular DNA) GuidePfuSeqment(1-2325) TGTAGTCAACGTCCAGGATCATCCGTTTCTTATAATAATAAGATTTTTTGATGTTC (Complimentary to primers PfuF1 & PfuF55 for a ligation of 2325bp circular DNA; GuidePfuSeqment(1-2325) is equal to oligo PfuR55) Pfu DNA polymerase by USER-mediated circular assembly amplification of three DNA fragements Guiding minus oligonucleotides for the circular ligations of Pfu DNA polymerase fragments: GuidePfuSeqment(1-811); TGTAGTCAACGTCCAGGATCATGCAGGTTGATGGTACGGG Complimentary to primers PfuF1 & PfuF19 for a ligation of circular DNA GuidePfuSeqment( ); GCTTCCAGGGTGTAGGTCGACCCCAAGCGGTAACAGAT Complimentary to primers PfuF20 & PfuF37 for a ligation of circular DNA GuidePfuSegment( );CCAAACCAGTTCGATGTATTTACGCCGTTTCTTATAATAATAAGATTTTTTGATGTTC Complimentary to primers PfuF38 & PfuF55 for a ligation of circular DNA Amplification primers (containing deoxyu, du) for PCR amplification of three segments Amplification of PfuSegment(1-811) PfuSegment(1-811)fdU ATTATTATAAGAAACGGAdUGATCCTGGACGdUTGACTACA PfuSegment(1-811)rdU AGGTCGGCAGGdUTGAdUGGTACGGG Amplification of PfuSeqment( ) PfuSeqment( )fdU ATCAACCdUGCCGACCdUACACCCTGGAAGC PfuSeqment( )rdU ATTdUACGACCCCAAGCGGdUAACAGAT

23 Amplification of PfuSegment( ) PfuSeqment( )fdU ACCGCTdUGGGGTCGTAAAdUACATCGAACTGGTTTGG PfuSeqment( )rdU ACGTCCAGGATCAdUCCGTTTCTTATAATAAdUAAGATTTTTTGATGTTC Pfu DNA polymerase PCR amplification Primers: PfuForward Primer (with XbaI site) GGTGGTTCTAGAATGATCCTGGACGTTGACTACATCAC PfuReverse Primer (with PstI site) GGTGGTCTGCAGAGATTTTTTGATGTTCAGCCAAGAGGTC Eight Sequencing Primers: Pfu-S-Primer1 Pfu-S-Primer2 Pfu-S-Primer3 Pfu-S-Primer4 Pfu-S-Primer5 Pfu-S-Primer6 Pfu-S-Primer7 Pfu-S-primer8 GCTGCAAGGCGATTAAGTTG ATCGACCTGCCGTACGT CAGCACGTTTAGCCAGGTA AAACCGTCTGAAGAAGAATACCA TGTGGGTGATGATGATAGACG GTTAAATACATCAACTCTAAACTGCCG CCAGTCACGACGAACGATT CGGCTCGTATGTTGTGTGG