207
(a) Synthetic DNA Fragment Coding for Elementary Autoinhibited Protease Module
gctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaa
ggtgaaatcatgccgaacatccctcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacg
ccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgatcacaagtttgta
caaaaaagcaggctcggaaaccgtgcgtttccagtctggtggttctgggtcttctaaagctttgctgaag
ggcgtgcgcgattttaatccgatctctgcttgcgtatgcctgctggaaaactcctcggatggtcatagtg
aacgtctgtttggcattggttttggcccgtatatcattgccaaccagcatctgtttcgtcgtaacaatgg
cgaactgaccatcaaaaccatgcatggtgaattcaaagtcaaaaactctacccagctgcagatgaaaccg
gttgaaggccgtgacattatcgttatcaaaatggctaaagacttcccgccgttcccgcagaaactgaaat
tccgtcagccgaccatcaaagatcgtgtgtgcatggtgtccaccaactttcagcagaaaagcgtctcgag
cctggtgtctgaatcctctcacattgtgcataaagaagacacttctttctggcagcactggatcaccact
aaagatggccagtgtggcagcccactagtttccatcattgatggcaacattctgggcatccacagcctga
ctcataccaccaacggtagcaactacttcgtggaatttccggaaaaattcgtggcgacttatctagatgc
cgcggatggttggtgcaaaaactggaaattcaacgcggataaaatcagctggggttcctttaccctggtt
gaagatgcgccggaagatttcatgagtggtctggtgccgcgcggtgtaggtcgcgaaaccgtgcgctttc
agtctggaagcacccaccaccatcatcatcactgaggatcctctagagtcgacctgcaggcaagcttggc
actggccgtcgttttacaacg
Sequence Annotation
- Restriction sites NcoI and BamHI, and USER Enzyme recombination sites arehighlighted in black
- 50 bp homologous recombination sites with vector backbone are underlined
- TVMV protease is dotted-underlined
- TVMV cleavage sites are double-underlined
- Thrombin cleavage site is wave-underlined
- His affinity purification tag is thick-underlined
- P1-P1’ junction is highlighted in grey
(b) Primer Pair N-terminal TVMV Fragment
5’-GCTGAAGTCTTACGAGGAAGAGTTGGC-3’
T M : 70.5 ˚C
5’-ACGGTUTCGCGACCTACACCG-3’
T M : 72.1 ˚C
(c) Primer Pair C-terminal TVMVFragmentwith Randomized P1-P1’ Junction
5’-AACCGUGCGCTTTNNNNNNGGAAGCACCCACCACCATCAT-3’
T M : 70.3 ˚C
5’-CGTTGTAAAACGACGGCCAGTG-3’
T M : 70.3 ˚C
(d) USER Enzyme Dependent Recombination Site with Randomized P1-P1’ Junction
G V G R E T V R F X X G S T H H H H
5’-AACCGUGCGCTTTNNNNNNGGAAGCACCCACCACCATCAT-3’
3’-GCCACATCCAGCGCTUTGGCA-5’
Engineering Synthetic Protease Switches
(a) Synthetic DNA Fragment Coding for Elementary Autoinhibited Protease Module
gctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccaccatggaaaacgcccagaaa
ggtgaaatcatgccgaacatccctcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacg
ccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgatcacaagtttgta
caaaaaagcaggctcggaaaccgtgcgtttccagtctggtggttctgggtcttctaaagctttgctgaag
ggcgtgcgcgattttaatccgatctctgcttgcgtatgcctgctggaaaactcctcggatggtcatagtg
aacgtctgtttggcattggttttggcccgtatatcattgccaaccagcatctgtttcgtcgtaacaatgg
cgaactgaccatcaaaaccatgcatggtgaattcaaagtcaaaaactctacccagctgcagatgaaaccg
gttgaaggccgtgacattatcgttatcaaaatggctaaagacttcccgccgttcccgcagaaactgaaat
tccgtcagccgaccatcaaagatcgtgtgtgcatggtgtccaccaactttcagcagaaaagcgtctcgag
cctggtgtctgaatcctctcacattgtgcataaagaagacacttctttctggcagcactggatcaccact
aaagatggccagtgtggcagcccactagtttccatcattgatggcaacattctgggcatccacagcctga
ctcataccaccaacggtagcaactacttcgtggaatttccggaaaaattcgtggcgacttatctagatgc
cgcggatggttggtgcaaaaactggaaattcaacgcggataaaatcagctggggttcctttaccctggtt
gaagatgcgccggaagatttcatgagtggtctggtgccgcgcggtgtaggtcgcgaaaccgtgcgctttc
agtctggaagcacccaccaccatcatcatcactgaggatcctctagagtcgacctgcaggcaagcttggc
actggccgtcgttttacaacg
Sequence Annotation
- Restriction sites NcoI and BamHI, and USER Enzyme recombination sites arehighlighted in black
- 50 bp homologous recombination sites with vector backbone are underlined
- TVMV protease is dotted-underlined
- TVMV cleavage sites are double-underlined
- Thrombin cleavage site is wave-underlined
- His affinity purification tag is thick-underlined
- P1-P1’ junction is highlighted in grey
(b) Primer Pair N-terminal TVMV Fragment
5’-GCTGAAGTCTTACGAGGAAGAGTTGGC-3’
T M : 70.5 ˚C
5’-ACGGTUTCGCGACCTACACCG-3’
T M : 72.1 ˚C
(c) Primer Pair C-terminal TVMVFragmentwith Randomized P1-P1’ Junction
5’-AACCGUGCGCTTTNNNNNNGGAAGCACCCACCACCATCAT-3’
T M : 70.3 ˚C
5’-CGTTGTAAAACGACGGCCAGTG-3’
T M : 70.3 ˚C
(d) USER Enzyme Dependent Recombination Site with Randomized P1-P1’ Junction
G V G R E T V R F X X G S T H H H H
5’-AACCGUGCGCTTTNNNNNNGGAAGCACCCACCACCATCAT-3’
3’-GCCACATCCAGCGCTUTGGCA-5’
Engineering Synthetic Protease Switches