Nupack code
This code was entered into Nupack Design mode. Feel free to copy this code if you want to check or expand upon our designs. It should work via copy and pasting but errors can occur if going via word processing programs.
Tact is the structure of our switch in the translationally active state.
Toff is the structure in the translationally inactive state.
Apt1 is the structure of our trigger sequence.
A . represents a single unpaired nucleotide.
( and ) represent two nucleotides in a base pair (can be separated by other paired or unpair nculeotides.
+ is used to separate strands
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10x Buffer recipe
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50mM NaCl: 29.22g, 58.44g/mol
10mM MgCl2: 20.33g, 203.3g/mol
10mM Tris: 12.114g, 121.14g/mol
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Made up to 1L with MilliQ water then adjusted to pH8 using HCl and NaOH.
All of our data can be found with this link: https://drive.google.com/open?id=0B6B6-qDOpcS_TjZOUlpJc2VKcVk
This includes all of our Nupack code, diagram components, cropped and contrasted gels and plate reader data.
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5’ FAM fluorophore-labelled oligo:
/56-FAM/CCTAAATTGTTATCCGCTCA
3’ BHQ Quencher-labelled oligo:
CTCACTCACCATATCGTATC/3BHQ_1/
5’ FAM fluorophore labelled “Mimic” oligo:
/56-FAM/TATCTCCTCT
Homo Sym 8:
TGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTACAAGAATAGGAGTAGAGGAGATACGATATGGTGAGTGAG
NC homo:
TCCTCTACTCCTATTCTTGTACTCCTATTCTTGTAGAGGA
1*:
ACTCCTATTCTTGT
1*1*:
ACTCCTATTCTTGTACTCCTATTCTTGT
Hetero sym 8:
TGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTGAGGGTAAGACAGTAGAGGAGATACGATATGGTGAGTGAG
2*:
ACTGTCTTACCCTC
NC hetero:
TCCTCTACTGTCTTACCCTCACTCCTATTCTTGTAGAGGA
2*1*:
ACTGTCTTACCCTCACTCCTATTCTTGT
Single binding site:
TGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTGTAGAGGAGATACGATATGGTGAGTGAG
NC single binding site:
TCCTCTACACTCCTATTCTTGTAGAGGA
aabb20:
TGAGCGGATAACAATTTAGGCAAGACAAAGACAAGACAAAGACGAAACGAGAACAAGACAACGGAGACATAGAGGAGACATAGAGATACGATATGGTGAGTGAG
acdb20:
TGAGCGGATAACAATTTAGGCAAGACAAAGACGAAACATAGACGAAACGAGAACAAGACAACCACAGACCAGAGGAGACATAGAGATACGATATGGTGAGTGAG
a*b*:
TCTTTGTCTTGTCTATGTCTCC
c*d*:
TCTATGTTTCGTCTGGTCTGTG
a*a*b*b*:
TCTTTGTCTTGTCTTTGTCTTGTCTATGTCTCCTCTATGTCTCC
c*a*b*d*:
TCTATGTTTCGTCTTTGTCTTGTCTATGTCTCCTCTGGTCTGTG
Vrigg:
TCTACTCCTATTCTTGTAGA
Mimic switch:
TCCTCTACAAGAATAGGAGTACAAGAATAGGAGTAGAGGAGATAGATACGATATGGTGAGTGAG
Supplementary Information
Plasmid sequences for in vivo testing
Homo Sym 8:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGACAAGAATAGGAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTACAAGAATAGGAGTAGAGGAGATACGATATGGTGAGTGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Homo Sym 12:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGACAAGAATAGGAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTGTGCACAAGAATAGGAGTACAAGAATAGGAGTGCACAGAGGAGATGGAATATGGTGAGTGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Homo Sym 18:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGACAAGAATAGGAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTATCTTCGTGCACAAGAATAGGAGTACAAGAATAGGAGTGCACGAAGATAGAGGAGATGGAGTATGGTGAGTGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Homo Sym Long:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTGTGACACCTTTATCACTGATATCGATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGGTAAATGGAGATAGGGTTACATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGCATCGTTTATCTCCTCTGAAAATGGAGATAGGGTTACGTAAATGGAGATAGGGTTACAGAGGAGATAAACGATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Asym:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTGATCGCAGAATGGTATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGCTAGGACAATAACTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTAGGACAATAACTCTAGGACAATAACTAGAGGAGACGAACGATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Single Binding Site:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGACAAGAATAGGAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTGTAGAGGAGATACGATATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
aabb50:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTGTGACACCTTAATCACTGATACTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAAGCGGAGACATAGACAAGACAAAGAATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGCAAGACAAAGACAAGACAAAGAAACAATCATAAACATAACACTAACATCACTACAACTATCAATAACTAACAGGAGACATAGAGGAGACATAGAATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
aabb20:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTGTGACACCTTAATCACTGATACTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAAGCGGAGACATAGACAAGACAAAGAATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGCAAGACAAAGACAAGACAAAGACGAAACGAGAACAAGACAACGGAGACATAGAGGAGACATAGAATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
aabb6:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTGTGACACCTTAATCACTGATACTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAAGCGGAGACATAGACAAGACAAAGAATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGCAAGACAAAGACAAGACAAAGATACAACGGAGACATAGAGGAGACATAGAATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
AB:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACAGGTTAATGAGTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGCTCGGGATGGGAAGACAAAGACATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAGAAACGAAGACAAAGACACAGAGGAGACTCGGGATGGGGGTGAGCGAGCTAATCAAAGAAAATATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
AABB:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACAGGTTAATGAGTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGCTCGGGATGGGAAGACAAAGACATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAGAAACGAAGACAAAGACAAGACAAAGACACAGAGGAGACTCGGGATGGGCTCGGGATGGGGAGTGAGCGAACTAATCAAAGAAAATATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
AAABBB:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACAGGTTAATGAGTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGCTCGGGATGGGAAGACAAAGACATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAGAAACGAAGACAAAGACAAGACAAAGACAAGACAAAGACACAGAGGAGACTCGGGATGGGCTCGGGATGGGCTCGGGATGGGGTGAGCGAACTAATCAAAGAAAATATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Hetero 1-2 Ribozyme:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAATCACTGATAGTGTGCTCTGTCTTACCCTCGGTATATCTCCTTCGTGGAATCCAGGATCCATCCCTACGCTGGCATTATCCAGATCAGGTGATACGGGTATTTCTCAGCCTTCACGCAGAAGGGCACCCCGAGGATTTCGTCCTATTTGGGACTCATCAGCTGGATGTACCGAAGGAGAAATGCCTGGTTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAAATATACAAGAATAGGAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTCCTCTACAAGAATAGGAGTGAGGGTAAGACAGTAGAGGAGATACGATATGGTGAGTGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
OffTrig:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGTCTGTGTGAGAGGTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGATAGAGGAGACAAATGATGGATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATTGAGCTCAAGCTTAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGACGAAGACAAAGACAAAGATACAAAGACAAAGATAGAGGAGACAAATGATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
TripTrig:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCAGTGATACTAATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGTATTAACACGAATAATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGTGTCCTATTAACACGAATACTTTTATTAACACGAATATATTAACACGAATAAAAGAGGAGAAATAGGACATGGTGTCCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
ToeTrig:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCAACTATCTTATTCACTTATCAACATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAATCGACGCGTCAGGACTTATATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATGAGCTCAAGCTTAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAATGGGACGCGTCAGGACTTATGCGAAACAGAGGAGAATAAGTATGGACGCGTCAGGACTTATTGTGAGCGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
Positive Control:
GAATTCGCGGCCGCTTCTAGAGGCGAAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGACTGTCTGACACCTTAATCACTGATAGTCATTGCCATGAATGATCCCGAAGGATCATCAGAGTATGTGGGAGCCCACACTCTACTCGACAGATACGAATATCTGGACCCGACCGTCTCCCACATACACATGGCAACGACGAGAATAGTAGTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTGCGAAGCGGCATGCATAAGCTTGGCTCACCTTTAGGTGAGCCTTTCTGCGAATGTGCCTGTCAAATTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTAGGACGAAGACAAAGACAAAGATACAAAGACAAAGATAGAGGAGACAAATGATGGTGAGTGAGTTGATTAAAGAAAACATGCACATGAAGCTGTACATGGAAGGCACCGTCAATAACCATCACTTTAAGTGTACATCCGAGGGAGAGGGCAAGCCTTACGAGGGTACGCAGACTATGCGTATCAAAGCTGTTGAAGGTGGGCCTCTTCCATTCGCATTCGATATTTTGGCAACTTCTTTCATGTACGGATCAAAGACATTCATCAATCACACTCAAGGTATTCCTGACTTTTTTAAGCAGAGCTTTCCAGAGGGTTTTACGTGGGAGCGTGTCACAACATACGAAGACGGTGGAGTCTTAACCGCGACTCAGGATACATCGCTTCAAGATGGTTGCCTTATCTACAATGTTAAGATCCGTGGTGTGAACTTCCCGTCTAACGGACCCGTCATGCAAAAGAAAACACTGGGTTGGGAGGCAAGCACTGAAACGTTGTATCCAGCAGATGGTGGTTTGGAGGGTCGTGCTGACATGGCTTTAAAGTTGGTTGGTGGTGGACATCTTATCTGTAACCTTAAAACGACGTACCGTTCTAAGAAGCCCGCTAAGAATCTGAAGATGCCTGGCGTATATTACGTCGATCGTCGCTTAGAGCGCATCAAGGAGGCAGATAAAGAGACTTATGTAGAACAGCATGAAGTGGCAGTGGCTCGCTATTGCGATTTACCCTCTAAGTTAGGTCACCGTTGATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTATAATTACAATATACTAGTAGCGGCCGCTGCAG
These were ordered as dsDNA from Twist Biosciences within a Kanamycin-resistance Twist plasmid vector with a ColE1 origin:
Activatory:
Homo sym 8 code used to define 1* sequence. (RBS sequence from Toehold paper. First 10 mKate2 codons used as domain gene):
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure apt = U14
structure Tact = .. ...... (((((((((((((( (((((((((((((( ...... ........ .......... .......... .......... +))))))))))))))+))))))))))))))
structure Toff = .. (((((( ((............ ............)) )))))) ........ .......... .......... ..........
# define sequence domains
domain end1 = N2
domain stem1 = N6
domain bs= N14
domain stem2= AGA GGA
domain end2= GA N6
domain gene= ATGGTGAGCGAGCTGATTAAGGAGAACATG
# thread domains onto strands
strand switch1 = end1 stem1 bs bs stem2 end2 gene
strand apt1 = bs*
# thread strands onto target structures
Tact.seq = switch1 apt1 apt1
Toff.seq = switch1
apt.seq = apt1
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
Positive control (both Tact and Toff transcriptionally active):
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 10
# target structure defined in dot-parens-plus notation
structure apt = U14
structure Tact = .. ...... .............. .............. ...... ........ .......... .......... ..........
structure Toff = .. ...... .............. .............. ...... ........ .......... .......... ..........
# define sequence domains
domain end1 = N2
domain stem1 = N6
domain bs= ACAAGAATAGGAGT
domain ns= N14
domain stem2= AGA GGA
domain end2= GA N6
domain gene= ATGGTGAGCGAGCTGATTAAGGAGAACATG
# thread domains onto strands
strand switch1 = end1 stem1 ns ns stem2 end2 gene
strand apt1 = bs*
# thread strands onto target structures
Tact.seq = switch1
Toff.seq = switch1
apt.seq = apt1
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
Homo sym Long:
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 10
# target structure defined in dot-parens-plus notation
structure apt = U20
structure Tact = .. ................. (((((((((((((((((((( (((((((((((((((((((( ................. ........................... + ))))))))))))))))))))+))))))))))))))))))))
structure Toff = .. ((((((((((((((((( ((((................ ................)))) ))))))))))))))))) ...........................
# define sequence domains
domain end = N2
domain stem1 = N17
domain bs= N20
domain stem2= AGA GGA GAN NNN NN ATG
domain gene= GTGAGCGAGCTGATTAAGGAGAACATG
# thread domains onto strands
strand switch1 = end stem1 bs bs stem2 gene
strand apt1 = bs*
# thread strands onto target structures
Tact.seq = switch1 apt1 apt1
Toff.seq = switch1
apt.seq = apt1
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
Homo Sym 12 using 1* sequence defined above. Modified from homo sym 8. Similar process used for homo sym 18.
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure apt = U14
structure Toff = (((( (((( (((( ............ ............ )))) )))) )))) .... .......... .......... ..........
structure Tact = .......... (((((((((((((( (((((((((((((( .......... .... .......... .......... .......... + )))))))))))))) + ))))))))))))))
# define sequence domains
domain stem1 = N10
domain bs = ACAAGAATAGGAGT
domain stem2 = AGA GGA GA NN
domain spacer = N4
domain gene = ATGGTGAGCGAGCTGATTAAGGAGAACATG
# thread domains onto strands
strand switch1 = stem1 bs bs stem2 spacer gene
strand apt1 = bs*
# thread strands onto target structures
Tact.seq = switch1 apt1 apt1
Toff.seq = switch1
apt.seq = apt1
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
Hetero sym 8 using previously defined 1* sequence and undefined 2*:
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure aptA = U14
structure aptB = U14
structure Tact = .. ...... (((((((((((((( (((((((((((((( ...... ........ .......... .......... .......... +))))))))))))))+))))))))))))))
structure Toff = .. (((((( ((............ ............)) )))))) ........ .......... .......... ..........
# define sequence domains
domain end1 = N2
domain stem1 = N6
domain bsA= ACAAGAATAGGAGT
domain bsB= N14
domain stem2= AGA GGA
domain end2= GA N6
domain gene= ATGGTGAGCGAGCTGATTAAGGAGAACATG
# thread domains onto strands
strand switch1 = end1 stem1 bsA bsB stem2 end2 gene
strand apt1 = bsA*
strand apt2 = bsB*
# thread strands onto target structures
Tact.seq = switch1 apt2 apt1
Toff.seq = switch1
aptA.seq = apt1
aptB.seq = apt2
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
Repressive:
aabb20 by splitting the domains a and b within the trigger. Later changed to loops of 6 and 50 with same domain sequences:
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure tact = U100
structure toff = ......((((((((((( ((((((((((( .......... .......... ((((((((((( ((((((((((( .......... .......... ..........+)))))))))))+)))))))))))+)))))))))))+)))))))))))
structure apta=U11
structure aptb=U11
​
# define sequence domains
domain a = N11
domain b = GGA GAN NNA GA
domain end = N6
domain end2= ATGGTGAGCGAGCTGATTAAGGAGAACATG
domain loop= N20
​
# thread domains onto strands
strand switch1 = end a a loop b b end2
strand apt1 = a*
strand apt2 = b*
​
# thread strands onto target structures
tact.seq = switch1
toff.seq = switch1 apt2 apt2 apt1 apt1
apta.seq= apt1
aptb.seq= apt2
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
acdb20 using domains a and b specified from above:
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure tact = U130
structure toff = ......((((((((((( ((((((((((( .......... .......... .......... .......... .......... ((((((((((( ((((((((((( .......... .......... ..........+)))))))))))+)))))))))))+)))))))))))+)))))))))))
structure apta=U11
structure aptb=U11
structure aptc=U11
structure aptd=U11
​
# define sequence domains
domain a = CAAGACAAAGA
domain b = GGAGACAUAGA
domain c = N11
domain d = GGAGA N6
domain end = N6
domain end2= ATGGTGAGCGAGCTGATTAAGGAGAACATG
domain loop= AACAATCATAAACATAACACTAACATCACTACAACTATCAATAACTAACA
​
# thread domains onto strands
strand switch1 = end c a loop b d end2
strand apt1 = a*
strand apt2 = b*
strand apt3 =c*
strand apt4= d*
​
# thread strands onto target structures
tact.seq = switch1
toff.seq = switch1 apt4 apt2 apt1 apt3
apta.seq= apt1
aptb.seq= apt2
aptc.seq= apt3
aptd.seq= apt4
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
AABB design to get domain sequences, then manually changed to make AB and AAABBB, using Salis RBS calculator and an E. coli codon table:
# parameters for NUPACK design
material = rna
temperature = 37
dangles = none
trials = 5
# target structure defined in dot-parens-plus notation
structure tact = U130
structure toff = ......((((((((((( ((((((((((( .......... .......... .......... .......... .......... ((((((((((( ((((((((((( .......... .......... ..........+)))))))))))+)))))))))))+)))))))))))+)))))))))))
structure apta=U11
structure aptb=U11
# define sequence domains
domain A = N11
domain B = CGGCUC AUG UC
domain end = N6
domain end2= GTG AGC GAG CTG ATT AAG GAG AAC ATG CAC
domain rbs = AGA GGA GA
domain loop= N42
# thread domains onto strands
strand switch1 = end A A loop rbs B B end2
strand apt1 = A*
strand apt2 = B*
# thread strands onto target structures
tact.seq = switch1
toff.seq = switch1 apt2 apt2 apt1 apt1
apta.seq= apt1
aptb.seq= apt2
# prevent sequence patterns
prevent = AAAA, CCCC, GGGG, TTTT, CCGG, GGCC, CGCG, GCGC, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY
ssDNA sequences used for in vitro testing
Ordered as ssDNA from a combination of IDT and Sigma Aldrich.