DesignOfRibocomputingDevices/Fig 3 - Sample NUPACK code for the first-generation toehold switch.txt at main · AlexGreenLab/DesignOfRibocomputingDevices · GitHub

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#Sample NUPACK Code for the First-Generation Toehold Switch

material = rna1999
temperature = 37.0
trials = 10

# target structures

#the T7 terminator contains G-U wobble base pairs that can sometimes cause
#errors during NUPACK design. As a result, we assert that the terminator is
#fully single stranded. This modification will lead to an increased normalized
#ensemble defect for the designs but still allow good riboregulators to be
#generated.
structure trigger = U3 D8 U6 U3 U30 U3 U47

#We similarly assert that the linker and output gene are single-stranded
#to avoid design errors.
structure switch = U3 U12 D9 (U3 D6 U12 U3) U50

structure complex = U3 D8 U6 U3 D30 (U3 U47 + U3) U12 U18 U50

# sequence domains
domain T7_term = UAGCAUAACCCCUUGGGGCCUCUAAACGGGUCUUGAGGGGUUUUUUG
domain linker21 = AACCUGGCGGCAGCGCAAAAG

#first 29 nt of GFP
domain output_gene = AUGCGUAAAGGAGAAGAACUUUUCACUGG

domain preG = GGG
domain trig_stem = N8
domain trig_loop = N6
domain trig_spacer1 = N3
domain trig_spacer2 = N3

domain toehold = N12
domain stem1 = N9
domain stem2 = N3
domain stem3 = N6

#using a 12-nt loop domain to make full switch a multiple of 3 and in-frame in gate RNAs
domain loop  = N4 AGAGGAGA
domain stem2_star = AUG #start codon

switch.seq = preG toehold stem1 stem2 stem3 loop stem3* stem2_star stem1* linker21 output_gene
trigger.seq = preG trig_stem trig_loop trig_stem* trig_spacer1 stem3* stem2* stem1* toehold* trig_spacer2 T7_term
complex.seq = preG trig_stem trig_loop trig_stem* trig_spacer1 stem3* stem2* stem1* toehold* trig_spacer2 T7_term preG toehold stem1 stem2 stem3 loop stem3* stem2_star stem1* linker21 output_gene

# specify stop conditions for normalized ensemble defect
# Stop condition is increased to 10 to accommodate terminator
# and output gene non-ideal secondary structure

switch.stop = 10
trigger.stop = 10
complex.stop = 10

#
# prevent sequence patterns
#
prevent = AAAA, CCCC, GGGG, UUUU, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY