Supplementary
Fig.
:
Sequence
alignment
of
the
original
nAG
mRNA
in
salamanders
and
the
newly
designed
nAG
(suitable
for
higher
vertebrates).
5'UTR
CACGAGTGGAGCACTTCCCCAGCACGCAAGACAAGTGCAGGTGGGAGACCAAGC
CATCGCTCAAC…………………………………………...........Original
XHO1 (restriction site)
5'UTR
CTCGAGCACGAGTGGAGCACTTCCCCAGCACGCAAGACAAGTGCAGGTGGGAGACCAAGC
CATCGCTCAAC……………………………..………….………Optimized
1
* ATG GTG AAA GGT TAC CTG GCA GCT CTT CTG CTC CTA GCG CTT TCT TCA TTC
1
† ATG GTG AAA GGG TAT TTG GCG GCG TTG CTT CTC CTT GCG CTG AGC TCA TTC
1
#
52
* AGC CTA GCC AAA GAG AGC GCC AAG AGA CCG GAA GTG AAG AAG GTC CAG ACT
52
† TCC CTC GCC AAA GAG TCG GCA AAG AGG CCC GAG GTG AAA AAG GTA CAG ACC
18
#
103
* CTT TCG AGG GGG TGG GGC GAC AGT CTC GAA TGG GCT CAG ACG TAT GAG GAA
103
† CTC TCG CGC GGT TGG GGA GAT AGC CTG GAA TGG GCG CAA ACG TAT GAA GAG
35
#
154
* AGC CTG TCC AAA TCC AGG AGC AGC AAC AAA CCA CTG CTC GTT ATC AAC CAC
154
† TCA TTG TCC AAG TCG AGA TCA TCG AAC AAA CCC CTG CTG GTG ATC AAT CAC
52
#
M
S
L
S
V
L
S
L
K
A
R
S
G
K
G
K
Y
E
W
S
L
S
G
R
A
A
D
S
A
K
S
S
L
R
L
N
L
P
E
K
L
E
W
P
L
V
A
L
A
K
Q
L
L
K
T
V
S
V
Y
I
S
Q
E
N
F
T
E
H
205
* AGA GAT GAC TGT CCA CAC TCT CAA GCT TTG AAG AAA GCA TTT GCT GAG CAC
205
† CGC GAT GAC TGC CCT CAT TCG CAA GCT TTG AAG AAA GCA TTC GCG GAG CAC
68
#
256
* AAA GGC ATC CAG AAA CTC GCA GAG AAG TTC ATT CTT CTT AAC GTT GTT CAT
256
† AAG GGG ATC CAG AAG TTG GCC GAG AAG TTT ATC TTG CTC AAC GTG GTA CAC
R
D
D
C
P
H
S
Q
A
L
K
K
A
F
A
E
H
85
# K
G
I
Q
K
L
A
E
K
F
I
L
L
N
V
V
H
307
* GAT CCA ACT GAC AAG AAC CTT GTA CTT GAT GGC ATG TAT GTA CCC AAG CTT
307
† GAT CCC ACC GAT AAG AAT CTG GTC TTG GAT GGG ATG TAC GTA CCA AAA CTC
102
#
358
* GTT TTC GTA GAT CCA TCT ATG GTA GTG AGA GCT GAT CTT CCT GGA AAA TAC
358
† GTC TTT GTG GAC CCT AGC ATG GTC GTC AGG GCC GAC CTC CCG GGA AAG TAC
119
#
409
* TCC AAT CAT CGG TAC ACC TAT GAG CCT GCA GAC ATT GAT CTG TTG TAT GGT
409
† TCG AAT CAT CGG TAC ACT TAC GAA CCC GCG GAC ATT GAC CTT CTC TAT GGC
136
#
460
* AAC ATG CAG AAA GCA CTC AAA CTT CTG AAA ACT GAA CTG …… V5 Peptide……
460
† AAT ATG CAG AAA GCA CTT AAG TTG CTG
153
#
D
V
S
N
P
F
N
M
T
V
H
Q
D
D
R
K
K
P
Y
A
N
S
T
L
L
M
Y
K
V
V
E
L
L
V
P
L
D
R
A
K
G
A
D
M
D
I
Y
L
D
V
P
P
L
K
G
K
L
Y
L
Y
G
AAA ACG GAG CTT GGA AAA CCG ATT
T
E
L
G
K
P
I
……………………………… V5 Peptide………………………………………
511
† CCG AAC CCA CTC CTT GGT CTG GAC TCC ACA TGA GCGGCCGC
170
#
P
N
P
L
L
G
L
D
S
T
*
Not1 (restriction site)
3'UTR
TGAGCGAAGAATGCCTAGACAAGTGACCCCCGCATCCTGTTTCCGCATGAGACTGC
ACAACCAGAAAGTTGACTTCAGTTGATTTGAAATTCATGAAGACACTGTAAAAGCA
TAACTGGGATTATGATTCATCTGGCTGTAAACACTTCCTGGCATTTTGACGTTTGAC
TGTGCTAGATTTTTTTAAAATGTATTCTTTATGCTTCATCTGTAAGCAACACATTTTT
AAATAAATCCATTTTTGGGTATTTATTATT……………………………..………..original
*
Original
sequence
†
Optimized
sequence
#
Amino
Acids
sequence
Sequence
alignment
of
the
original
PROD
1
mRNA
in
salamanders
and
the
newly
designed
PROD
1
(suitable
for
higher
vertebrates).
 GGCACTGGGGCGCACACCTCGCGCTGATTTTACCTGGACTCGAAGCGTTGAGGGTTTCGTCAGCTACAAG
AC
 AAGCTGGCTAGCGCCGCCACC
 ATG
ATG
CTT
CTA
CCA
CTC
TCC
TTG
TTT
CTG
GTG
GCA
TGC
CTG
CAC
TCA
ACT
ACA
GCG
TTA
 ATG
TAG
CTG
CTC
CCA
CTG
TCC
TTG
TTC
CTC
GTC
GCC
TGT
CTG
CAC
TCC
ACT
ACC
GCG
CTC
• M
M
L
L
P
L
S
L
F
L
V
A
C
L
H
S
T
T
A
L
 AAA
TGC
TTC
ACC
AGA
AAC
GGA
GAC
GAC
AGG
ACT
GTG
ACC
ACC
TGC
GCC
GAG
GAA
CAG
ACT
 AAG
TGC
TTC
ACA
AGA
AAC
GGC
GAT
GAT
AGG
ACC
GTG
ACC
ACT
TGT
GCC
GAG
GAA
CAG
ACT
• K
C
F
T
R
N
G
D
D
R
T
V
T
T
C
A
E
E
Q
T
 CGA
TGC
CTC
TTC
GTA
CAA
CTG
CCA
TAT
TCT
GAG
ATA
CAA
GAA
TGC
AAG
ACG
GTG
CAA
CAG
 CGG
TGT
CTG
TTC
GTG
CAA
CTG
CCG
TAC
TCG
GAG
ATC
CAG
GAG
TGC
AAA
ACC
GTG
CAG
CAG
• R
C
L
F
V
Q
L
P
Y
S
E
I
Q
E
C
K
T
V
Q
Q
 TGT
GCT
GAG
GTG
TTA
GAG
GAA
GTC
ACT
GCC
ATT
GGA
TAT
CCA
GCA
AAG
TGC
TGC
TGC
GAG
 TGC
GCA
GAA
GTG
CTG
GAA
GAA
GTG
ACC
GCC
ATT
GGA
TAC
CCC
GCT
AAG
TGC
TGC
TGC
GAG
• C
A
E
V
L
E
E
V
T
A
I
G
Y
P
A
K
C
C
C
E
 GAT
CTC
TGC
AAC
CGG
AGT
GAG
CAA
GAT
TTT
GAG
ACC
ACC
ACC
CAG
ACC
ACA
ACA
CTA
GCA
 GAT
CTT
TGC
AAC
CGC
AGC
GAG
CAG
GAC
TTT
GAA
ACC
ACG
ACC
CAG
ACC
ACC
ACT
CTG
GCC
• D
L
C
N
R
S
E
Q
D
F
E
T
T
T
Q
T
T
T
L
A
 TTC
TTG
GAT
GGA
CCA
CAG
 TTC
CTG
GAC
GGG
CCT
CAA
FLAG‐tag
peptides
• F
L
D
G
P
Q
3’UTR
 TGA
CAC
CAA
AAC
GGC
CCA
GAC
ACT
GCA
TTC
CCA
GCA
TCC
TAA
TTG
AAG
TGG
GGC
ATA
ACT
GTG
AAC
ACA
TTC
TGT
GCC
TTT
TTT
GTT
TTT
CCT
TCA
GCT
CTT
CCC
TAG
AAT
TTG
GGA
ACG
TTT
TCC
CTC
CTT
CCT
TTA
TAA
CAC
TTT
TTC
TTA
TGA
CTG
GTG
 GAC
TAC
AAG
GAC
GAC
GAC
GAC
AAG
GGT
TGA
CAC
CAA
AAC
GGC
CCA
GAC
ACT
GCA
TTC
 Original salamander sequence.
 Optimized sequence.
• Amino acids sequence.