Part sequences for cgRNA, trigger, and control gRNA

MATERIALS AND METHODS

A.4 Part sequences for cgRNA, trigger, and control gRNA

Splinted switch mechanism in vitro

Name Sequence Legend

IVSS_tD 5⁰-CTTCTTACTTCTCCAATCATAATTTCTCCC-3⁰ trigger XD

Table A.2: Splinted switch sequences for in vitro studies (Chapter 2). Nucleotides shaded orange are constrained by the target gene. Nucleotides shaded gray are con- strained by Cas9. Nucleotides shaded blue are designed as described in Chapter 2.

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

i-15_cg 5⁰-TATCATCCATCAACCCATCTAATTCAACAAGAAT

TGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGG TGCTTTTTTT-3⁰

5⁰extension: 15

i-25_cg 5⁰-ACTATAGACTTATCATCCATCAACCCATCTAATTC

AACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTT-3⁰

5⁰extension: 25

i-35_cg 5⁰-GCTACTCATTACTATAGACTTATCATCCATCAAC

CCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

5⁰extension: 35

ii-15a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCT

ATTCGAGAAAGTTAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT-3⁰

Handle loop: 15a

ii-15b_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATC

CCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT TTTTT-3⁰

Handle loop: 15b

ii-25a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCT

ATTCGAGAAAGTTTCAGATCCCTAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT-3⁰

Handle loop: 25a

ii-25b_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAAA

GTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT-3⁰

Handle loop: 25b

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

ii-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCT

ATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCA AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA GTGGCACCGAGTCGGTGCTTTTTTT-3⁰

Handle loop: 35

iii-15a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TAAACCTTACCACAATAAGTGGCACCGAGTCGGTGCT TTTTTT-3⁰

Terminator loop 1: 15a

iii-15b_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCT TTTTTT-3⁰

Terminator loop 1: 15b

iii-25a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TAAACCTTACCACAATTTCACCATTCAAGTGGCACCG AGTCGGTGCTTTTTTT-3⁰

Terminator loop 1: 25a

iii-25b_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TACAATTTCACCATTCACATAAGCACAAGTGGCACCG AGTCGGTGCTTTTTTT-3⁰

Terminator loop 1: 25b

iii-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TAAACCTTACCACAATTTCACCATTCACATAAGCACA AGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

Terminator loop 1: 35

iv-15_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGCAAGAACGTAACAATCGGTGCT TTTTTT-3⁰

Terminator loop 2: 15

iv-25_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACA GAACGGTGCTTTTTTT-3⁰

Terminator loop 2: 25

iv-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACA GAAGATAGAAATACGGTGCTTTTTTT-3⁰

Terminator loop 2: 35

i-15_ii-15a_cg 5⁰-TATCATCCATCAACCCATCTAATTCAACAAGAAT

TGTTTTAGAGCTAGCTATTCGAGAAAGTTAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGCTTTTTTT-3⁰

5⁰extension: 15 /

Handle loop: 15a

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

i-25_ii-25a_cg 5⁰-ACTATAGACTTATCATCCATCAACCCATCTAATTC

AACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGT TTCAGATCCCTAGCAAGTTAAAATAAGGCTAGTCCGT TATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTT-3⁰

5⁰extension: 25 /

Handle loop: 25a

i-35_ii-35_cg 5⁰-GCTACTCATTACTATAGACTTATCATCCATCAAC

CCATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTA TTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCAA GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTT-3⁰

5⁰ extension 35 /

Handle loop: 35

i-15_iii-15a_cg 5⁰-TATCATCCATCAACCCATCTAATTCAACAAGAAT

TGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTAAACCTTACCACAATAAGTGG CACCGAGTCGGTGCTTTTTTT-3⁰

5⁰extension: 15 /

Term loop 1: 15a

i-25_iii-25a_cg 5⁰-ACTATAGACTTATCATCCATCAACCCATCTAATTC

AACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCGTTATCAACTTAAACCTTACCAC AATTTCACCATTCAAGTGGCACCGAGTCGGTGCTTTT TTT-3⁰

5⁰extension: 25 /

Term loop 1: 25a

i-35_iii-35_cg 5⁰-GCTACTCATTACTATAGACTTATCATCCATCAAC

CCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTA AACCTTACCACAATTTCACCATTCACATAAGCACAAG TGGCACCGAGTCGGTGCTTTTTTT-3⁰

5⁰extension: 35 /

Term loop 1: 35

i-15_iv-15_cg 5⁰-TATCATCCATCAACCCATCTAATTCAACAAGAAT

TGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAA CGTAACAATCGGTGCTTTTTTT-3⁰

5⁰extension: 15 /

Term loop 2: 15

i-25_iv-25_cg 5⁰-ACTATAGACTTATCATCCATCAACCCATCTAATTC

AACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGCAAGAACGTAACAATGAGAACAGAACGGTGCTTT TTTT-3⁰

5⁰extension: 25 /

Term loop 2: 25

i-35_iv-35_cg 5⁰-GCTACTCATTACTATAGACTTATCATCCATCAAC

CCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAA TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAG AAGATAGAAATACGGTGCTTTTTTT-3⁰

5⁰extension: 35 /

Term loop 2: 35

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

ii-15b_iii-15a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATC

CCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTAAACCTTACCACAATAAGTGGCACCG AGTCGGTGCTTTTTTT-3⁰

Handle loop: 15b / Term loop 1:

15a

ii-25b_iii-25a_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAA

AGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAA TAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAA TTTCACCATTCAAGTGGCACCGAGTCGGTGCTTTTTT T-3⁰

Handle loop: 25b / Term loop 1:

25a

ii-35_iii-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCT

ATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCA AGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCT TACCACAATTTCACCATTCACATAAGCACAAGTGGCA CCGAGTCGGTGCTTTTTTT-3⁰

Handle loop: 35 / Term loop 1: 35

ii-15b_iv-15_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATC

CCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAA CAATCGGTGCTTTTTTT-3⁰

Handle loop: 15b / Term loop 2: 15

ii-25b_iv-25_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAAA

GTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGC AAGAACGTAACAATGAGAACAGAACGGTGCTTTTTT T-3⁰

Handle loop: 25b / Term loop 2: 25

ii-35_iv-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCT

ATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCA AGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA GTGGCACCGCAAGAACGTAACAATGAGAACAGAAGAT AGAAATACGGTGCTTTTTTT-3⁰

Handle loop: 35 / Term loop 2: 35

iii-15b_iv-15_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TCATTCACATAAGCACAAGTGGCACCGCAAGAACGTA ACAATCGGTGCTTTTTTT-3⁰

Term loop 1: 15b / Term loop 2: 15

iii-25b_iv-25_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTACAATTTCACCATTCACATAAGCACAAGTGGCACC GCAAGAACGTAACAATGAGAACAGAACGGTGCTTTTT TT-3⁰

Term loop 1: 25b / Term loop 2: 25

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

iii-35_iv-35_cg 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGA

AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TAAACCTTACCACAATTTCACCATTCACATAAGCACA AGTGGCACCGCAAGAACGTAACAATGAGAACAGAAGA TAGAAATACGGTGCTTTTTTT-3⁰

Term loop 1: 35 / Term loop 2: 35

i-15_t 5⁰-GGTTGATGGATGATA-3⁰ 5⁰ extension: 15

+Trigger

i-25_t 5⁰-GGTTGATGGATGATAAGTCTATAGT-3⁰ 5⁰ extension: 25

+Trigger

i-35_t 5⁰-GGTTGATGGATGATAAGTCTATAGTAATGAGTAG

C-3⁰

5⁰ extension: 35

+Trigger

ii-15a_t 5⁰-ACTTTCTCGAATAGC-3⁰ Handle loop: 15a

+Trigger

ii-15b_t 5⁰-CACGGAACACGGGAT-3⁰ Handle loop: 15b

+Trigger

ii-25a_t 5⁰-GGGATCTGAAACTTTCTCGAATAGC-3⁰ Handle loop: 25a

+Trigger

ii-25b_t 5⁰-CACGGAACACGGGATCTGAAACTTT-3⁰ Handle loop: 25b

+Trigger

ii-35_t 5⁰-CACGGAACACGGGATCTGAAACTTTCTCGAATAG

C-3⁰

Handle loop: 35 +Trigger

iii-15a_t 5⁰-ATTGTGGTAAGGTTT-3⁰ Terminator loop

1: 15a +Trigger

iii-15b_t 5⁰-GTGCTTATGTGAATG-3⁰ Terminator loop

1: 15b +Trigger

iii-25a_t 5⁰-GAATGGTGAAATTGTGGTAAGGTTT-3⁰ Terminator loop

1: 25a +Trigger

iii-25b_t 5⁰-GTGCTTATGTGAATGGTGAAATTGT-3⁰ Terminator loop

1: 25b +Trigger

iii-35_t 5⁰-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTT

T-3⁰

Terminator loop 1: 35 +Trigger

iv-15_t 5⁰-ATTGTTACGTTCTTG-3⁰ Terminator loop

2: 15 +Trigger

iv-25_t 5⁰-TTCTGTTCTCATTGTTACGTTCTTG-3⁰ Terminator loop

2: 25 +Trigger

iv-35_t 5⁰-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTT

G-3⁰

Terminator loop 2: 35 +Trigger

i-15_ii-15a_t 5⁰-ACTTTCTCGAATAGCGGTTGATGGATGATA-3⁰ 5⁰extension: 15 /

Handle loop: 15a +Trigger

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

i-25_ii-25a_t 5⁰-GGGATCTGAAACTTTCTCGAATAGCGGTTGATGGA

TGATAAGTCTATAGT-3⁰

5⁰extension: 25 /

Handle loop: 25a +Trigger

i-35_ii-35_t 5⁰-CACGGAACACGGGATCTGAAACTTTCTCGAATAGC

GGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-3⁰

5⁰extension: 35 /

Handle loop: 35 +Trigger

i-15_iii-15a_t 5⁰-ATTGTGGTAAGGTTTGGTTGATGGATGATA-3⁰ 5⁰extension: 15 /

Term loop 1: 15a +Trigger

i-25_iii-25a_t 5⁰-GAATGGTGAAATTGTGGTAAGGTTTGGTTGATGGA

TGATAAGTCTATAGT-3⁰

5⁰extension: 25 /

Term loop 1: 25a +Trigger

i-35_iii-35_t 5⁰-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTT

GGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-3⁰

5⁰extension: 35 /

Term loop 1: 35 +Trigger

i-15_iv-15_t 5⁰-ATTGTTACGTTCTTGGGTTGATGGATGATA-3⁰ 5⁰extension: 15 /

Term loop 2: 15 +Trigger

i-25_iv-25_t 5⁰-TTCTGTTCTCATTGTTACGTTCTTGGGTTGATGGA

TGATAAGTCTATAGT-3⁰

5⁰extension: 25 /

Term loop 2: 25 +Trigger

i-35_iv-35_t 5⁰-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTG

GGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-3⁰

5⁰extension: 35 /

Term loop 2: 35 +Trigger

ii-15b_iii-15a_t 5⁰-ATTGTGGTAAGGTTTCACGGAACACGGGAT-3⁰ Handle loop: 15b

/ Term loop 1:

15a +Trigger

ii-25b_iii-25a_t 5⁰-GAATGGTGAAATTGTGGTAAGGTTTCACGGAACAC

GGGATCTGAAACTTT-3⁰

Handle loop: 25b / Term loop 1:

25a +Trigger

ii-35_iii-35_t 5⁰-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTT

CACGGAACACGGGATCTGAAACTTTCTCGAATAGC-3⁰

Handle loop: 35 / Term loop 1: 35 +Trigger

ii-15b_iv-15_t 5⁰-ATTGTTACGTTCTTGCACGGAACACGGGAT-3⁰ Handle loop: 15b

/ Term loop 2: 15 +Trigger

ii-25b_iv-25_t 5⁰-TTCTGTTCTCATTGTTACGTTCTTGCACGGAACAC

GGGATCTGAAACTTT-3⁰

Handle loop: 25b / Term loop 2: 25 +Trigger

Insert studies inE. coli

Part name Candidate cgRNA/trigger sequence Legend

ii-35_iv-35_t 5⁰-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTG

CACGGAACACGGGATCTGAAACTTTCTCGAATAGC-3⁰

Handle loop: 35 / Term loop 2: 35 +Trigger

iii-15b_iv-15_t 5⁰-ATTGTTACGTTCTTGGTGCTTATGTGAATG-3⁰ Term loop 1: 15b

/ Term loop 2: 15 +Trigger

iii-25b_iv-25_t 5⁰-TTCTGTTCTCATTGTTACGTTCTTGGTGCTTATGT

GAATGGTGAAATTGT-3⁰

Term loop 1: 25b / Term loop 2: 25 +Trigger

iii-35_iv-35_t 5⁰-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTG

GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTT-3⁰

Term loop 1: 35 / Term loop 2: 35 +Trigger

Table A.4: Candidate cgRNA/trigger sequences for insert studies inE. coli(Chapter 3). Nucleotides shaded orange are constrained by the target gene. Nucleotides shaded gray are constrained by dCas9. Nucleotides shaded blue are designed as described in Chapter 3.

Splinted switch mechanism inE. coli

Name Sequence Legend

SplS_cgA 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTACACCTTACGCC

GGTTCAATTCCAAGTCCCTTCCAGTAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTAACACCCTTTACAAACCTTCCTCTTCCTTTAC CCTAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

cgRNA cgRNA A

SplS_cgB 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGTAATCGAATC

ATAGTAAATTTCCCATCGTCATAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTCATACGGGTCTGAAGTAGTTCATTCTTATACA GTCAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

cgRNA B

SplS_cgC 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGTCGTTACCTT

ATCAATATCAACCTCCGCATACACTAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGCACATAGGACCCAACATGCCAACAGAGAAGA GTTAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

cgRNA C

SplS_cgD 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAACATACAAAC

CACTACCGACAACTTAAACACTTTTAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTTCACTACATCATATCATACGCGGAATTAAACA ATCAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

cgRNA D

SplS_tA 5⁰-AGGGTAAAGGAAGAGGAAGGTTTGTAAAGGGTGTTCTGGAAGG

GACTTGGAATTGAACCGGCGTAAGGTG-3⁰

Trigger

Trigger XA

SplS_tB 5⁰-GACTGTATAAGAATGAACTACTTCAGACCCGTATGTTATGACG

ATGGGAAATTTACTATGATTCGATTAC-3⁰

Trigger XB

SplS_tC 5⁰-AACTCTTCTCTGTTGGCATGTTGGGTCCTATGTGCGTGTATGC

GGAGGTTGATATTGATAAGGTAACGAC-3⁰

Trigger XC

SplS_tD 5⁰-GATTGTTTAATTCCGCGTATGATATGATGTAGTGAAAAGTGTT

TAAGTTGTCGGTAGTGGTTTGTATGTT-3⁰

Trigger XD

Table A.5: Splinted switch sequences for studies inE. coli(Chapter 4). Nucleotides shaded orange are constrained by the target gene. Nucleotides shaded gray are con- strained by dCas9. Nucleotides shaded blue are designed as described in Chapter 4.

Toehold switch mechanism inE. coli

Name Sequence Legend

ToeS_cgA 5⁰-ATGTTCGTTGTATTAAGACCGCTAAACTGAAAGTTACACGCC

CAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTT-3⁰

cgRNA cgRNA A

ToeS_cgB 5⁰-GTATATGAAATTGAAAGACCGCTAAACTGAAAGTTACACGCC

CAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTT-3⁰

cgRNA B

ToeS_cgC 5⁰-AAGGTGATAGTAAAGAGACCGCTAAACTGAAAGTTACACGCC

CAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTT-3⁰

cgRNA C

ToeS_cgD 5⁰-TATACTTATACTTGGAGACCGCTAAACTGAAAGTTACACGCC

CAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTT-3⁰

cgRNA D

ToeS_tA 5⁰-AACTTTCAGTTTAGCGGTCTTAATACAACGAACAT-3⁰ Trigger

Trigger XA

ToeS_tB 5⁰-AACTTTCAGTTTAGCGGTCTTTCAATTTCATATAC-3⁰ Trigger XB

ToeS_tC 5⁰-AACTTTCAGTTTAGCGGTCTCTTTACTATCACCTT-3⁰ Trigger XC

ToeS_tD 5⁰-AACTTTCAGTTTAGCGGTCTCCAAGTATAAGTATA-3⁰ Trigger XD

Table A.6: Toehold switch sequences for studies inE. coli(Chapter 5). Nucleotides shaded orange are constrained by the target gene. Nucleotides shaded gray are con- strained by dCas9. Nucleotides shaded blue are designed as described in Chapter 5.

Control gRNA sequences

Name Sequence

CT_g 5⁰-CACATCCCATCCACCGCCACGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT T-3⁰

mRFP_g 5⁰-AACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT T-3⁰

sfGFP_g 5⁰-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAAT

AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT T-3⁰ NT_g 5⁰-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC

TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-3⁰

Table A.7: Control gRNA sequences. For the standard gRNAs, nucleotides shaded orange are constrained by the target gene (mRFP or sfGFP for bacterial studies) or target dsDNA sequence (CT gRNA binding site or sfGFP binding site for in vitro studies). Nucleotides shaded gray are constrained by Cas protein. The no-target gRNA contains no target-binding region. For bacterial studies, the autofluorescence control strain was transformed with the no-target gRNA control plasmid (sequence NT_g).

Transcriptional promoter and terminator sequences

Name Type Sequence

T7_E T7 promoter 5⁰-CCTCTAATACGACTCACTATA-3⁰

BBa_J23100 Constitutive promoter 5⁰-TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAG

C-3⁰

BBa_J23108 Constitutive promoter 5⁰-CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAG

C-3⁰

BBa_J23114 Constitutive promoter 5⁰-TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAG

C-3⁰

BBa_R0011 lacI-regulated promoter 5⁰-AATTGTGAGCGGATAACAATTGACATTGTGAGCGG

ATAACAAGATACTGAGCACA-3⁰

BBa_B0015 Terminator 5⁰-CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA

GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGG TGGGCCTTTCTGCGTTTATA-3⁰

BBa_B0050 Terminator 5⁰-AAAAAAAGGATCTCAAGAAGATCCTTTGATTT

T-3⁰

BBa_B1006 Terminator 5⁰-AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTT

TTTT-3⁰

BBa_B1010 Terminator 5⁰-CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTC

GCCGC-3⁰

Table A.8: Transcriptional promoter and terminator sequences.

A.5Plasmids ConstitutivelyactivesplintedswitchinE.coli NotethattheplasmidsusedfortheinsertstudiesofChapter3(notshown)usethesameplasmidlayoutasthecgRNA-onlyandcgRNA+cognatetriggerplasmids ofthesplintedswitchstudiesofChapter4(below). NamePartsLegend pdCas9-bacteriaAddgeneplasmid#44249 pdCas9+lacISeeFigureA.5,FigureA.6 pag-NTColE1ori;amp-R;BBa_J23108-NT_g-BBa_B1006No-targetgRNA;Autofluorescence Autofluorescence pg-sfGFPColE1ori;amp-R;BBa_J23108-sfGFP_g-BBa_B1006StandardgRNA pSplS-cgA-nTColE1ori;amp-R;BBa_J23108-SplS_cgA-BBa_B1006cgRNA cgRNAA pSplS-cgA-tAColE1ori;amp-R;BBa_J23100-SplS_tA-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgA-BBa_B1006 SeeFigureA.1,FigureA.2cgRNA+trigger cgRNAA,triggerXA pSplS-cgA-tBColE1ori;amp-R;BBa_J23100-SplS_tB-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgA-BBa_B1006cgRNAA,triggerXB pSplS-cgA-tCColE1ori;amp-R;BBa_J23100-SplS_tC-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgA-BBa_B1006cgRNAA,triggerXC pSplS-cgB-nTColE1ori;amp-R;BBa_J23108-SplS_cgB-BBa_B1006cgRNAB pSplS-cgB-tAColE1ori;amp-R;BBa_J23100-SplS_tA-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgB-BBa_B1006cgRNAB,triggerXA pSplS-cgB-tBColE1ori;amp-R;BBa_J23100-SplS_tB-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgB-BBa_B1006cgRNAB,triggerXB pSplS-cgB-tCColE1ori;amp-R;BBa_J23100-SplS_tC-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgB-BBa_B1006cgRNAB,triggerXC pSplS-cgC-nTColE1ori;amp-R;BBa_J23108-SplS_cgC-BBa_B1006cgRNAC pSplS-cgC-tAColE1ori;amp-R;BBa_J23100-SplS_tA-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgC-BBa_B1006cgRNAC,triggerXA pSplS-cgC-tBColE1ori;amp-R;BBa_J23100-SplS_tB-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgC-BBa_B1006cgRNAC,triggerXB pSplS-cgC-tCColE1ori;amp-R;BBa_J23100-SplS_tC-BBa_B1006-BBa_B0015;BBa_J23108-SplS_cgC-BBa_B1006cgRNAC,triggerXC pSplS-cgD-nTColE1ori;amp-R;BBa_J23108-SplS_cgD-BBa_B1006cgRNAD TableA.9:PlasmidsusedwithsplintedswitchcgRNAsinE.coli.

BBa_B1006de novo sequence e

de novo sequence dsfGFP target-specific spacerBBa_J23108ScarBBa_B0015BBa_B1006de novo sequence d*de novo sequence e*BBa_J23100BglIIEcoRI BamHI ColE1

AmpR pSplS-cgA-tA 3049 bp

Standard gRNA sequence

AmpR_promoter FigureA.1:ExampleplasmidmapforsplintedswitchinE.coli.Plasmid:pSplS-cgA-tAforcgRNAA+triggerXA.

1 AATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGTGGTAGGAGATCTGCATAAGGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGGGTAAAGGAAGAGGAAGGTTTGTAAAGGGTGTTCTGGAAGGGACTTGGAATTGAACCGGCGTAAG 200 >>>>>> >>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> EcoRI BglII BBa_J23100 de novo sequence e* de novo sequence d* 201 GTGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTCTACACCCTTCTAACAACTCCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGATCTGAA 400 >>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> de novo sequence d* BBa_B0015 Scar >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> BBa_B1006 401 ACATGACGAGCTGGTGAGCATAAGGAGCTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCATCTAATTCAACAAGAATTGTTTTAGAGCTACACCTTACGCCGGTTCAATTCCAAGTCCCTTCCAGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAACACCCTTTACAAACCTTCCTCTTCCTTTACCCT 600 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> BBa_J23108 sfGFP target-specific spacer de novo sequence d Standard gRNA sequence de novo sequence e >>>>>>>>>>>> Standard gRNA sequence 601 AAGTGGCACCGAGTCGGTGCTTTTTTTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGCGCTATCAGTTGACACAGTGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGA 800 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Standard gRNA sequence BBa_B1006 801 TGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAA 1000 1001 AGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCCTTACTCGAGTCTAGACTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT 1200 >>>>>> BamHI 1201 GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC 1400 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ColE1 1401 TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA 1600 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ColE1 1601 CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT 1800 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ColE1 1801 TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGT 2000 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ColE1 2001 AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG 2200 287

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< AmpR 2201 CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG 2400

225

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< AmpR 2401 GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA 2600 159

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< AmpR 2601 TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT 2800 92

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< AmpR 2801 TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT 3000 25

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<< AmpR AmpR_promoter 3001 GCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA 3049 FigureA.2:ExampleannotatedplasmidsequenceforsplintedswitchinE.coli.Plasmid:pSplS-cgA-tAforcgRNAA+triggerXA.