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EcoFlex – User GuideTo provide the user with details for customizable modification of plasmids, we provide details of cloning procedures used.ORFsORFs can be amplified by PCR from plasmid or genomic DNA, or synthesised to remove rare codons and excluded restriction sites (NdeI, BamHI, BsaI, BsmBI). Alternatively, genes with internal NdeI and BamHI sites can be PCR amplified with primers containing flanking BsaI sites or sub-cloned using an optional NcoI (CCATGG) site located in pBP-ORF, which also provides a start codon. Additionally, if BamHI is present within a gene, the 3’ primer can incorporate a BglII site, which forms cohesive overhangs with BamHI.BiopartsUsers can customize the library with alternative DNA parts (promoters, RBS, tags etc) using the pBP-lacZ plasmid. Whilst we provide N-terminal His6 and Strep(II) tags; further fusion tags such as C-terminal extensions and fluorescence or probe tags can be custom built.Plasmid purification and sequencingSingle colonies were grown overnight with antibiotic in 3-5 mL LB in a 50 mL Falcon. All plasmids were purified with a QIAprep Spin Miniprep kit and eluted with 50-100 μL of sterile (pre-heated 65°C for plasmids larger than 10 kb) distilled water to obtain ~60-200 ng/ul dsDNA. The yield varies depending on insert size, whilst decreased yields can be obtained with the promoter parts (SJM901 and SJM911) that exhibit toxicity from the GFP reporter.Oligo annealingAnnealing primers were prepared at 20 M in 25 ? of 1 × T4 DNA ligase buffer (10 × NEB), heated to 90°C for 1 min, before cooling on ice. Annealed primers were phosphorylated with T4 Polynucleotide kinase for 1 hour at 37°C before dilution to 200 nM and ligation into the backbone plasmid. Standard ligations were prepared in 2 × Rapid Ligation buffer (Promega) and T4 DNA ligase (Promega) at room temperature for 30 min. Ligation mixtures were transformed into DH10, JM109 or KRX chemically competent cells. For routine Level 1 transformations, DH10 competent cells were prepared with the calcium chloride method. For Level 2 transformations, commercial competent cells were used (competency 108 CFU/g).Golden Gate Reaction – Level 120 units of BsaI-HF (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and cycled 15 times with 5 min at 37 °C and 10 min 16 °C. This was followed by 5 min incubations at 50°C and 80°C.Golden Gate Reaction – Level 210 units of BsmBI (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and incubated at 37°C for 16 hours. This was followed by a 5 min incubation at 80°C. Alternatively a 30 cycle protocol equivalent to Level 1 assembly can be used. This increases the total number of colonies obtained, but the percentage of correct clones decreases when using GFP as a positive marker for a 5 TU assembly.Golden Gate Reaction – Level 320 units of BsaI-HF (NEB), 1-3 units of T4 DNA ligase (Promega) was combined with 10X ligase buffer (Promega) and 1 mg mL-1 BSA. 50 ng of “destination vector” and 100 ng of “entry vectors” were combined in a one-pot reaction and cycled 30 times with 5 min at 37°C and 10 min 16°C. This was followed by 5 min incubations at 50°C and 80°C.Secondary module cloningA Level 2 unit containing 2, 3, 4 or 5 TUs can be sub-cloned with BsaI into two cohesive BpiI sites located in pTUS-A, -a and –b. The assembled plasmid is then ready for standard Level 2 assembly to accommodate up to 10 TUs.EcoFlex kit plasmids – Assembly plasmids and “Destination Vectors”Level 0pBP-lacZ (#72948)Promoters, RBS, Tags and terminators ligated between NdeI and SphIATCTAGAGACGCATATG-lacZ-GCATGCCGTCTCATTAGpBP-ORF (#72949)Open reading frames cloned between NdeI and BamHIGGTCTCACATATGGGTTCCATGGGGCACGGATCCTCGAAGAGACCLevel 1 -[NM]- Negative marker rfp or lacZ fragmentpTU1-A (#72935 and #72939)CGTCTCAATCTCTATAGAGACC-[NM]-GGTCTCATGTTTGCCAGAGACGpTU1-B (#72936 and #72940)CGTCTCATGCCCTATAGAGACC-[NM]-GGTCTCATGTTCCGGAGAGACGpTU1-C (#72937 and #72941)CGTCTCACCGGCTATAGAGACC-[NM]-GGTCTCATGTTGAAGAGAGACGpTU1-D (#72938 and #72942)CGTCTCAGAAGCTATAGAGACC-[NM]-GGTCTCATGTTTTAGAGAGACGpTU1-D1 (#72943)CGTCTCAGAAGCTATAGAGACC-[NM]-GGTCTCATGTTCTTCAGAGACG pTU1-E (#72944)CGTCTCACTTCCTATAGAGACC-[NM]-GGTCTCATGTTTTAGAGAGACGLevel 2pTU2-A (#72950 and #72954)GGTCTCACTATATCTAGAGACG-[NM]-CGTCTCATTAGGTACAGAGACCpTU2-B (#72951 and #72955)GGTCTCAGTACATCTAGAGACG-[NM]-CGTCTCATTAGGGACAGAGACCpTU2-C (#72952 and #72956)GGTCTCAGGACATCTAGAGACG-[NM]-CGTCTCATTAGTCGAAGAGACCpTU2-D (#72953 and #72957)GGTCTCATCGAATCTAGAGACG-[NM]-CGTCTCATTAGTGTTAGAGACCpTU2-a (#72958) GGTCTCACTATATCTAGAGACG-[RFP]-CGTCTCACCGGGTACAGAGACCpTU2-b (#72959)GGTCTCACTATATCTAGAGACG-[RFP]-CGTCTCAGAAGGTACAGAGACCLevel 3pTU3-A – 2 fragments from Level 2 (#72945)CGTCTCAATCTCTATAGAGACC-[RFP]-GGTCTCAGGACTTAGAGAGACGpTU3-B – 4 fragments from Level 4 (#72946)CGTCTCAATCTCTATAGAGACC-[RFP]-GGTCTCATGTTTTAGAGAGACGSecondary module (Level 2) site – (pTU2S-a, -b and –A)#73010 – pTU2S-a (pMB1 origin)Accepts 2 TUs#73011 – pTU2S-b (pMB1 origin)Accepts 3 TUsPlaced between AatI and EcoRI sites in pTU2–A, pTU2-a and pTU2-b. Modules from pTU2-A are sub-cloned (by gel extraction) as a BsaI fragment into the compatible BpiI sites. NdeI and SphI are placed between the BpiI sites for further modification and spacing between the restriction sites.CTATAAGTCTTCCATATGCACGCATGCGAAGACAAGTACBiological PartsPromotersJ23100 (#72963)CTATTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGTACJ23108 (#72964)CTATCTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTACJ23114 (#72965)CTATTTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCGTACSJM901 (#72966)CTATTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTACSJM902 (#72967)CTATTTTACAGCTAGCTCAGTCCTAGGGATTATGCTAGCGTACSJM903 (#72968)CTATCTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGCGTACSJM905 (#72969)CTATTTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGCGTACSJM906 (#72970)CTATTTGATGGCTAGCTCAGTCCTAGGGATTGTGCTAGCGTACSJM908 (#72971)CTATTTTATAGCTAGCTCAGCCCTTGGTATTATGCTAGCGTACSJM910 (#72972)CTATTTGATGGCTAGCTCAGTCCTTGGTATTATGCTAGCGTACSJM911 (#72973)CTATTTGACAGCTAGCTCAGTCCTTGGTACTGTGCTAGCGTAC SJM912 (#72974)CTATTTGATAGCTAGCTCAGTCCTAGGTACTATGCTAGCGTACSJM915 (#72976)CTATTTTATGGCTAGCTCAGTCCTTGGTATTATGCTAGCGTACT7 consensus (#72977)CTATTAATACGACTCACTATAGGGAGAGTACT7 promoter and PET RBS combinationsT7 promoter, lac operator and PET RBS (#72978)CTATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATAT7 promoter, lac operator, PET RBS, His6-tag and thrombin site (#72989)CTATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATARBS, linkers and tagsBBa_B0034 (#72980)GTACAAAGAGGAGAAACATAPET RBS (#72981)GTACTTTAACTTTAAGAAGGAGATATACATAN-terminus tag-linker (#72982)GTACTTTAACTTTAAGAAGGAGATATATAAAHexahistidine (#72983)TAAATGCACCATCACCATCACCATAStrep(II) (#72984)TAAATGTGGAGCCACCCGCAGTTCGAAAAACCGCATATL1 (#72985)GTACAGATCTAATAATTTTGTTTAACTTTGGGGGGATACATATL2 (#72986)GTACAGATCTAATAATTTTGTTTAACTTTGGGAGGATACATATL3 (#72987)GTACAGATCTAATAATTTTGTTTAACTTTGGGGGAATACATATL4 (#72988)GTACAGATCTAATAATTTTGTTTAACTTTAAGGAGATACATATL5 (#72989)GTACAGATCTAATAATTTTGTTTAACTTTGGAGAAATACATATL6 (#72990)GTACAGATCTAATAATTTTGTTTAACTTTGGGGAAATACATATL7 (#72991)GTACAGATCTAATAATTTTGTTTAACTTTAGGGGAATACATATL8 (#72992)GTACAGATCTAATAATTTTGTTTAACTTTAAAGAGATACATATL9 (#72993)GTACAGATCTAATAATTTTGTTTAACTTTAAAAAGATACATATL10 (#72994)GTACAGATCTAATAATTTTGTTTAACTTTGAAAAAATACATATL11 (#72995)GTACAGATCTAATAATTTTGTTTAACTTTAAAGGGATACATATL12 (#72996)GTACAGATCTAATAATTTTGTTTAACTTTGGGAAGATACATAOpen reading framesGFP (#72960)CATATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGAGCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATCGACFP (#72961)CATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCTAAGGATCCTCGAmCherry (#72962)CATATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGGATCCTCGAiGEM terminatorsBBa_B0012 (#72997)TCGATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATGTTBBa_B0015 (#72998)TCGACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATGTTSynthetic terminatorsL3S2P21 (#72999)TCGACTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCTGTTL3S1P51 (#73000)TCGAAAAAAAAAAAAAGGCCTCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAATGTTL3S1P32 (#73001) – BsmBI site underlinedTCGAGACGAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTATTTTTCAACAAAATGTTL3S1P11 (#73002) – BsmBI site underlinedTCGAGACGAACAATAAGGCCTCCCTTCGGGGGGGCCTTTTTTATTGATAACAAAATGTTL2U8H11 (#73003)TCGATAGCGTGCTAACCACGCACGCTATTGTTGTATTGTTL2U5H11 (#73004)TCGATAGCGTGCGAACAGCACGCTATTGTTGTATTGTTL2U5H08 (#73005)TCGATGCCGGGAGAACATCCCGGCATTGTTGTATTGTTL2U3H03 (#73006)TCGATAGCGTGACCGGCGCATCGGTCACGCTATTTGTTGAGTGTTL2U2H09 (#73007)TCGAACGGCCCTCGCAAGGGCCGTTTTTTTGTATGTTL1U1H09 (#73008)TCGACGACGATGTTCGCATCGTCGTTTTTTTTTTGTTL1U1H08 (#73009)TCGACCCGCATGTTCGCATGCGGGTTTTTTTTTTGTTIndividual plasmidsSecondary module (Level 2) site – (pTU2S-a, -b and –A)#74088 – pTU2S-a (p15A origin)Accepts 2 TUs#74089 – pTU2S-b (p15A origin)Accepts 3 TUs#74090 – pTU2S-A (p15A origin)Accepts 4-5 TUs#74091 – pTU2S-a (colE1 origin)Accepts 2 TUs#74092 – pTU2S-b (colE1 origin)Accepts 3 TUs#74093 – pTU2S-A (colE1 origin)Accepts 4-5 TUsLactate promoter - Bba_K822000 (#74094)CTATCACATTCCTATAGGCCGAGTAAGGTGTTCACGCCGCATCCGGCAAGATAAGGCGCTCTGGATCAACAACCTAAGGGCAATTCTCTGATGAGGATTGCCCTTTTCTTTACCAGACATCTCCCCCCACAAGAATTGGCCCTACCAATTCTTCGCTTATCTGACCTCTGGTTCACAATTTCCCAATTAAAACTCACATCAATGTTGCCAATACATAACATTTAGTTAACCATTCATTGTCATTATCCCTACACAACACAATTGGCAGTGCCACTTTTACACAACGTGTGACAAGGAGATGAGCAACAGACTCATTACACGATGTGCGTGGACTCCGTAClldR – E. coli MG1655 (#74095)CATATGATTGTTTTACCCAGACGCCTGTCAGACGAGGTTGCCGATCGTGTGCGGGCGCTGATTGATGAAAAAAACCTGGAAGCGGGCATGAAGTTGCCCGCTGAGCGCCAACTGGCGATGCAACTCGGCGTATCACGTAATTCACTGCGCGAGGCGCTGGCAAAACTGGTGAGTGAAGGCGTGCTGCTCAGTCGACGCGGCGGCGGGACGTTTATTCGCTGGCGTCATGACACATGGTCGGAGCAAAACATCGTCCAGCCGCTAAAAACACTGATGGCCGATGATCCGGATTACAGTTTCGATATTCTGGAAGCCCGCTACGCCATTGAAGCCAGCACCGCATGGCATGCGGCAATGCGCGCCACACCTGGCGACAAAGAAAAGATTCAGCTTTGCTTTGAAGCAACGCTAAGTGAAGACCCGGATATCGCCTCACAAGCGGACGTTCGTTTTCATCTGGCGATTGCCGAAGCCTCACATAACATCGTGCTGCTGCAAACCATGCGCGGTTTCTTCGATGTCCTGCAATCCTCAGTGAAGCATAGCCGTCAGCGGATGTATCTGGTGCCACCGGTTTTTTCACAACTGACCGAACAACATCAGGCTGTCATTGACGCCATTTTTGCCGGTGATGCTGACGGGGCGCGTAAAGCAATGATGGCGCACCTTAGTTTTGTTCACACCACCATGAAACGATTCGATGAAGATCAGGCTCGCCACGCACGGATTACCCGCCTGCCCGGTGAGCATAATGAGCATTCGAGGGAGAAAAACGCATAAGGATCCTCGAT7 RNA polymerase (#74096)CATATGaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaTCGA 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