Supplementary Data
Light regulation on growth, development and secondary metabolism of marine-derived
filamentous fungi
Menghao Cai · Zhe Fang · Chuanpeng Niu · Xiangshan Zhou · Yuanxing Zhang
The affiliation and address of all the authors:
State Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, China
Corresponding author: Xiangshan Zhou
Tel./fax: +86-21-64253025; E-mail address: xszhou@ecust.edu.cn
1. Degenerate PCR Procedure
The primers for light-responding genes of A. glaucus HB 1-19 (AgFphA, AglreB, AgveA) are
listed in Supplementary Table 1. The PCR mixtures are shown in Supplementary Table 2 and the
results are described in Supplementary Fig. 1. Genomic DNA for PCR was extracted with the
Plant Genomic DNA Extraction Kit (TIANGEN, BIOTECH Co. Ltd, China). Premix Taq (EX
TaqTM Version) was bought from TIANGEN BIOTECH Co. Ltd, China. The PCR procedure is,
95°C 4 min; 94°C 30 sec; 35°C-69°C 1 min; 72°C 1 min/kb (35 cycles); 72°C 7 min.
Table 1 Degenerate primers used for cloning AgfphA, AglreB and AgveA
Gene
Primer
Sequence
AgfphA
WFPH2Fa
5’-TGYGADGAYGARCCIATICA-3’
WFPH2Rb
5’-TGDATIGGIGACATIGCICG-3’
WC2Fa
5’-CTCGAYTCVTTHYTSGARCA-3’
WC2Rb
5’-TCVGGBCCYTTYCTCCAYTC-3’
VeAFa
5’-GAYCGYCGHCCDGTYGATCC-3’
VeARb
5’-CCDGGGAAYTTCTTRGCRYT-3’
AglreB
AgveA
a Forward
primer; b Reverse primer.
Table 2 Degenerate PCR reaction system
PCR mixture
50 μL
15 μL
Premix Taq (2×)
25 μL
7.5 μL
Genome DNA
100 ng-200 ng
30 ng-50 ng
Primer F
1.5 μL (100 μM)
0.5 μL (100 μM)
Primer R
1.5 μL (100 μM)
0.5 μL (100 μM)
ddH2O
Add to 50 μL
Add to 15 μL
Fig. 2 Amplification genes of AgfphA (A), AglreB (B) and AgveA (C) by degenerate PCR. (A),
(B) and (C) Lane 1-16: different anneal temperatures with 35.0, 36.1, 38.1, 40.9, 44.2, 47.0,
49.0, 50.0, 51.0, 52.1, 54.2, 56.9, 60.3, 63.0, 65.0, 66.0°C; M1: marker III, M2: 100 bp DNA
marker.
2. Genome Walking Procedure
The walking PCR system and PCR program are listed in Table 3 and Table 4.
Table 3 Walking PCR system
Mixture
25 μL
50 μL
First
Genomic DNA
50 ng
dNTP Mixture
4 μL
10× LA PCR buffer II (Mg2+ plus)
2.5 μL
Takara LA Taq
0.25 μL
AP1 Primer#
0.5 μL
SP1 Primer*
0.5 μL
ddH2O
Add to 25 μL
Second
Template DNA(from 1st PCR solution)
1 μL
dNTP Mixture
4 μL
10× LA PCR buffer II (Mg2+ plus)
2.5 μL
Takara LA Taq
0.25 μL
AP1 Primer
0.5 μL
SP2 Primer*
0.5 μL
ddH2O
Add to 25 μL
Third
Template DNA(from 2nd PCR solution)
1 μL
2 μL
dNTP Mixture
4 μL
8 μL
10× LA PCR buffer II (Mg2+ plus)
2.5 μL
5 μL
Takara LA Taq
0.25 μL
0.5 μL
AP1 Primer
0.5 μL
1 μL
SP3 Primer*
0.5 μL
1 μL
ddH2O
Add to 25 μL
Add to 50 μL
#AP1
is from the Genome Walking Kit (TAKARA); *SP1, SP2, SP3 for each walking were
designed by the software Primer Premier 5.0.
Table 4 Thermal protocols for walking PCR
1st PCR
2nd PCR
3rd PCR
Step
Temperature
Time
Step
Temperature
Time
Step
Temperature
Time
1
94 °C
1 min
1
94 °C
30 sec
1
94 °C
30 sec
2
98 °C
1 min
2
65 °C
1 min
2
65 °C
1 min
3
94 °C
30 sec
3
72 °C
2 min
3
72 °C
2 min
4
65 °C
1 min
4
94 °C
30 sec
4
94 °C
30 sec
5
72 °C
2 min
5
65 °C
1 min
5
65 °C
1 min
6
go to 3
5 times
6
72 °C
2 min
6
72 °C
2 min
7
94 °C
30 sec
7
94 °C
30 sec
7
94 °C
30 sec
8
25 °C
3 min
8
44 °C
1 min
8
44 °C
1 min
9
72 °C
2 min
9
72 °C
2 min
9
72 °C
2 min
10
94 °C
30 sec
10
go to 1
15 times
10
go to 1
15 times
11
65 °C
1 min
11
72 °C
10 min
11
72 °C
10 min
12
72 °C
2 min
12
end
12
end
13
94 °C
30 sec
14
65 °C
1 min
15
72 °C
2 min
16
94 °C
30 sec
17
44 °C
1 min
18
72 °C
2 min
19
go to 10
15 times
20
72 °C
21
end
10 min
3. Gene sequences
Agfph A (3732 bp)
ATGACGTCTCCGAAGAAAGCTGGTGTTAACGAGGCACCGCCAGATCAAAGCCCAAAT
CGTCTCACACCCCCCACCCAACCAACACTCCAGCGCGAAAACAGCTTCGATGGCACA
GATATCGTCTATCCTATACGCTCTGTCGTCTCGGTTGATCCAACTTCACAGAATTCTGAG
CCAGTCCCATCTCATCCGGCCTCGCCTATCAGGGATGGCGTTCGTCGGTACTCTATAATC
GATGCGAACACTTGGGACCAGATGCAGTCTCGAGCCAGGACGGATTCTGTGAACGATC
CCCCCAAGCCGATCAATGCGACAGAAAACCCCGACCTTGACGAACCGGAATACCAAC
GTCGCAGTTCCGAGGCGACTGAAGAGAGTCTCCCGGTTATCAATCCGGAACATCCACA
AAATCCAGAAATCCTCGAAGGAACCAGTCTTGTTACCTCACGATTCGAGCATGTGATTA
CAGAGCGCGGCCATGCTGTCGCCACGGGACCGGGTGCCCCATTCCAGCCTTGCGAGG
ACGAACCGATCCATATCCCTGGTGCAGTACAGAGCTTTGGTGTGTTAGTGGTTATCCGT
GAGGAAGCGGGCGACCAGTACCTCGTCCGCATCGCTAGCGAGAATTCCAAGGATCTTC
TTGGGTATTCCCCGAACGAGTTATTCGCCCTTCAAAATTTCTGTGATATCCTCGGAGAC
GACCAAGCTGATGACCTTTTGGACCACATTGATTTTATTCGCGACGATTCGTTCGACCC
CAGTGTGGACGGCCCCGAAGTATTCCTCTTGTCCATCAAACAGCCCTCGCGACGCTCT
CGCAAGTTCTGGTGTGCCATCCATCGCTGTCCAGCCGATCCCAGTCTGATAATTTGTGA
ATTTGAATTGGAGGATGACCAGATCAATCCACTGAATGTTTCTGGTCGCGGATCGCCAG
CATCGCCCAGCGCGACTCTGGGGTTGGCACCGACCCCAGAGCAGCTAGCAGCTAGCA
CCAGCAGCATGAGCCAGCCCTTGCGCACACTGCGCAATGCTCGTCGGCGGAGGGGAG
AGGCGGCCGCCATGGAAGTGTTCAGCATCCTCACCCAGATCCAAGATCAGCTGGCCCG
CACAGAGACGCTTGAGTCTTTACTGAACACAGCGACCGGACTGGTGAAAGAGTTGAC
CGGCTTCCATCGGGTTCTGATTTATCAATTTGATCTGAATTGGAACGGCACCGTCGTGG
CCGAACTGGTTGACCCCAGAGCTTCCGTCGATCTATATAAAGGTCTCCATTTCCCCGCC
ATGGACATTCCCAGACAGGCTCGTGATCTGTACCGGGTTAACAAGGTTCGACTACTATA
TGACCGTGATCAGACAACGTCGCGACTCGTCTGTCGGACAGCCAAAGATCTGGAGAC
ACCGCTGGACATGACCCATGCCTATCTGCGTGCGATGTCTCCAATTCACATAAAGTATCT
GGCAAATATGGAAATCCGCGCATCTATGTCAATCAGTATCAACGGGTTCAATGAGCTTT
GGGGTTTGATATCGTGCCACTCGTATGGAAAGTCTGGCATGAGAGTGTCATTCCCCATC
GGAAAGATGTGCCGGTTGGTTGGAGATAGCGTCTCGCGAAACATCGAGCGTCTGTCCT
ATTCGGCTCGGCTTCAGGCACGTAAATTAATCAATACCGCCCCAACCGATGCAAACCCT
TCCGGATACATCATCGCCTCGTCGGACGATCTACTCAAATTATTCGGTGCTGACTACGGT
GCTCCGTGTATTCGAGACGAGACGAAACTATTGGGCAAACTCACTCACTCTCAGGAGA
TCCTGGCCTTGTTGGAGTATCTGAAGTTGCGCCAGATCAACTCCGTCCTTGCCTCGCAT
CATGTCACCAAAGACTTCCCTGACTTGCGCTATCCTCCAGGGTTCAAGCATATTTCAGG
TTTGCTTTATGTACCTCTATCTACAGGCGGAACGGACTTCATGGTCTTTTTCCGTCGCCG
CCGCTTGACAGAAATCAAATGGGCGGGTAATCCATACGACAAGAAAGTCGTGGATGGG
TACCTACAGCCACGAAAGAGTTTCCAGACCTGGTGCGAAACTATTCTCGATCAATCCC
GGGAATGGACCGAGACCGATGTGGAAACAGCGGCGGTGCTGTGTTTGGTGTACGGAA
AGTTCATCAAAGTGTGGCGACAGAAGGAAGCGGCCATGGAGAACTCGCAGCTCACGC
AGCTCCTGTTGGCCAATTCGGCCCACGAAGTTCGGACCCCTTTGAATGCTATTATTAAC
TACCTCGAAATTGCAATGGAAGGTTCATTGGACACCGAGACCCGAGACAACCTGACCA
AGTCTTACTCTGCTTCCAAATCGCTGATCTACGTCATCAACGACTTGCTCGATCTGACC
AACATCGAGAAGGGGCAGAATCTTATCAAGGATGAGTCTTTCGACCTGCCTCAGACGA
TCAAACAGGCCGCAGAGATGTTCGAAGGTGAAGCCAAACGCAAGGGAATCTCTTTCA
ATGTCCACTCTCACCCCGGACTCCCGACTGCAGTCGTAGGAGATCAGCGGCGCATTCG
TCAAGTGATCTCTAATCTCATTTCGAATGCCATGCAGCATACCTCGACCGGGGGCATTA
CTGTCGAGGTCTGGCGCGCGCCATCCCAGCCCGACAATAGCGATGTGATGATTGAAAT
GGTGGTCGTGGACACCGGTTCGGGGATGTCTCAAGACACCCTCGAGGCATTATTCCAG
CAGCTCGAGCAGGTGGGGTCAATGGATGACGAAGAAGGCCAAGATCACCTCCCTTCC
CTGGAAGGGCAAGAGAAACGGGTGCTTGGCTTGGGACTCGCGTTGGTGGCTCGGATC
GTCCGCAACCTACAAGGACAGCTGGGCGTGCGGTCCAAAGAGGGCAAAGGCAGTCGT
TTCAAGATCTCGTTGCAGTTCCCTGTTCCAGAGGGTACAACTTCGACAGCTGATAGCGA
TCCTCGTCCTCCCAGTCGGATGGCGGAGACGACGGCTCCGTTCCAGCACCAGCAAGA
GATCATGCTGGTGGATAGGAACCGCTCACTGGATCGCTCACGCCGTCAAAGCGCCGAG
AGCAACCGCAGCGGACAGAGCGGTAGCAGCGCGCAGAGTAACCGGAGTGAAGCCGA
TCGCCTGATCAGCGCCATGCAAGAGCCGCTGTTGGTCCGTAGCGCCAGCGAGGGCGGC
GCTACACGAACTCAGCAGCTGGATGCGAATGATCCCTTGATTCCTAAACCGCGAAAGA
CTACTAGCAGCTCTGTCGCGGCAACACCCACCACGGAGTTTGCTCCTGGTCCTGATATT
CGCACGACCGTCCCCCCTGTTTCGAACATGCCGCCGGGCCAGACAACTGTGAAGGATT
TTGGAGTGCCATTGGGTGCTATTCGAGTACCTGAACAGGATGTTGAGAGGCCGCCATC
ACCTCCTAAGCTGGTGGATGGGGAGCCTGCGCCGGCACTAGGTTCGCCTGAGAAGGTA
CCCGACAAAGCCCCGCAACAAAAGGCTGCTGTTGCGGACAGATTCAACGTGCTGGTG
GCAGAGGATGATCCCATCAACAGCAAGATTATCCAAAAGCGGTTGGAGAAACTGGGTC
ATAAGGTGCATCTGACTCCCAATGGGGAGGCATGTGCTGGTGCATATCGTGCGGATTGT
AAGAACTTCGACATCGTTCTTATGGACATTCAGGTATGCTTTGCCCCGTCACAGCTCAC
TACCTAG
AglreA (2559 bp)
ATGAGCGGGTTCGATGACTTCTATCACCCGCAGTACTATTCCCTGCCGGCTTCAAATCG
AGACCACGATCACCCGCAAACCGTTCAAGATCCAGATGGCGATCCCGTGGTGACATCG
TCTCTCATGGTCTATTCCCAATCAGGCGCGTATGATTTCGGTCACTATCAGAATGTGTAC
GATCAGTCGCAAGAGCCCAGTGTGTTCCGGGACGGCATTCCAAGTACAAGCAGCAGC
ATCAACACAGGACATCCGACACATGCGAATCAGATGCAGACGCAGAGCCAGCAATCTT
CCCACCATGTGGGCTATCCAGCACAGGGATCGAATCTGGAAGGTCGCCTGTCAAATGT
TAACATCTCTTATGATCCCTGGTCCGGCGCAGAGACTTCAATGCTCATGAACCCACATG
CGAGTAGTGCATTTCCATACGACTTCGCCAATGCGTATTCAATGCCTCCGCAATTAACTT
CACAGCACATGTGTCTGAACCCTCAGTCGACCTATTCCACCCTTCAGACGCAAACACC
GGCTCAAAACACATATTGGGGAATGGACGGCTATCGTGCTCCACAACAGGACGAACCC
GTATCACAAGGACAGTATCAAAATCGTCAAGCATATCCTCAGACATATGCTCCTCAGCG
GACGTTGCAATCGAGGGCTATTCAGCCAAAAAACCAACCATTCCAGGGTGGTTTTCCG
TCCAAAGTAAAATCCGAGTCGCACTTTGAGGCCTCACCTTATACTAATATCTATTCAACA
AGTGGCTTTGATATTCTGGGTGTTCTGGCTCGCGTAATATCGAGACCAGACCCGAAAAT
CAACATCGGTGCCGTCGATCTATCCTGTGCATTTGTGCTCTGCGATATGACCCAAGCTG
ATCACCCCATCGTGTATATGTCAGAAGCCTTTGGGCGGTTAACAGGATACTCCGAAGAG
GAGATCATTGGCCGGAACTGTCGTTTTCTCCAAGGTCCCGATGGAAACGTAGAACCTG
GGGCACAGCGAAAATTCGCCGACTCTAACACGGTTTACCGGATGCGAACAACCATTGA
AGAACGAAACGAGACCCAAGTCAGTGTGATCAATTACAGGAAAGGGGGGCAGCCATT
TATGAATTTGGTTACAATGATTCCTATACGTTGGGATACGAACGAGTATCGATTTTATGTT
GGTTTCCAGGTCGATTTGGTGGAGAAGCCGGATGCTGTCACCAAGAAGAATTCAAATG
GGACCTACATGATCAATTACCAACGCAGTCAGTTACCCAACTACGTGGTGCCTCAGCCT
GACATCCGCCAAAGCCATCCTGATCTGGTGGTGCAATTTGACCATGATCAAGTGTCTGC
CATTTTGCGCTCAATTAGCGCTTCTCGACCAGAATATAAACACTACCTCGATCGAATCCT
TGTCGAGAACGCAGATGACGTGATCCACGTTTTATCTTACGATGGGGTGTTCCTCTACC
TTTCCCCGTCATGCCAAAAAATTCTGGAATATGAGTCCGTCGAGTTAGTCAATAAGACA
TTATCATCAATCTGCCATCCCAGTGATATTGGCACCGTCACTAGGGACCTTCGGACCAG
CATCACCACAGCCCCGGTGAGCGTGGTGTATCGAATTCGCAAAAAGCACAGCGGTTAT
GCTTGGTTCGAGAGCCATGGTGCATGGCATGTGGAGCAAGGTCGAGGACGAACGTTC
CTTGTTTTGGTCGGAAGAGAACGTTTGGTGTATCATCTCGGACAAGTCGCCAGGTTAG
GGAGTGAGGCCCTTGGCGAGACAGATGTTTGGGCGAAGATATCTGCGTCAGGGATCAT
TCTGTATATCTCCACAAAAGCACGACCGGTTCTGGGCCGAACACAGGATGACCTAGTC
GGGACGAGTTTCAAAGACTTGTTAGGCGCGGAAAGCCACTCAGAAGTGCAACAGGCT
TTGCAGAACTCCCGCAATGGTCAACGAACAATGTTTAACCATCAAGTCCGTCATAAGA
AGGGTCATATGGTTCAGGCGCAGACGGTTCTATTTCCTGGTGACACAAAAGAAGGCAC
CAAACCTTCATTTCTTGTCGCCCAGATACGGGTTCTGAAATCATCGCAGTCGTCTAGCG
AGGAGCCTATGCCAACAGAGACGACAACGTTAATACCAGCTAGGAATCGACCGAGGTC
AAGCACAGACCAACAACAAAACCTTGTCAACGCCTCAAGTGCAAACCACTTACCCCC
AGACAACCAAATCACATCCCCCACAGAACAAATCACAATCTTCCCCGAACTCATCCCA
ACCCGCGGCTCAAGCTGGCAATTCGAGCTCCGCGAGCTCGAAAAACGAAACCGCGGC
CTCTCCGACGAACTTCAGCGACTACTCACCCGGAGAAAGAAGCGCAAGCGGAAGCAA
ACGTCAATGCCAGTGGGCAAGAGCTGCGTCATATGCCACACAAAGAACACGCCGGAG
TGGAGACGGGGGCCGAGCGGGAATCGGGATCTGTGTAATAGTTGTGGGTTACGGTGGG
CGAAGCAGGTGAGGAGTGCGGCGCAGTTGGAGAGATCGGAATCTGGTTGA
AglreB (1245 bp)
ATGGAGCCTGCTTCTATAAGACATGGCCAGGCATATGGCTTGCAAATCGACGGAGGCAA
TTCCTATACAATACAAGCTCAGCGCATGCGACAAGCACAGTCCCTCACCGGCGACTTC
GAGGCGCAAACCGTGCAACAATCCGGTGCTAGAAGCTGGCCACAACGCGCAATAGCT
GAGATGAAGGACATGTTTCTGCTTCTAAGCGCAGATGGCACGATAACATACGCCTCGTC
CTTGTGCCAGTCGATTACGGGATACACCACGAAACAATTGGAGGGAAAGCCGCTGTCC
TACTTCACACACCAAGACGATCGCGCAATATTTTCCGAGGAACTAGACCAATGTGTTGC
GACGGGATGTGCTCTACATCTGCATTTCCGGTTCAGCCGCGTGGGTGATTCGCACTGCA
TTCTGGAAGCCTCCGGGCATCCCCACATGTCGAATCAGGGCGGGAACAACGCAGATGG
AAATAGCAAACCCTACGACGGAGTCTTTATCATATGCCGGCCATATCCGACCAAAAGCG
CTCAGTTCCTCGATTCGTTTTTCGAGCACAAGATCGAGAATATCCGATTGATGCAACAG
GTCACACGACTCAAGGAGGAAGAGGAGGAGGAATCAAAAGCGAACAAAGCACCATA
TGCAAGATCGGACCATTCAAGCAGCAGCAACAAGACATATTTCAACCAACCTCCACGT
TTCATCGCATCAAACCCAGCTGGTTTCACCGGTACCATTATCCCCGATTTCATTACATCA
GCAGAAGAATATGAATCATCAGATACCCTTGAGCCCGAGCAACCCAAACCATTTGCCG
CCGAGAATCTATCTCTCATTGAAGGGATCGAAGTCCTAACTGGTTTGCACTACGGAGAA
GGAGAGAGATCTCAGGGTATTAGCACTGGTATCCGCCGTGGTCGCCTCATCCAATGCGA
CACCGATTTTACTAAGATAGACCAGCAGGTCCGCAGTATGGAGGAGAGCGATAGGAGG
AAACGTCTGAGAGGAGAATATCGATGTAGCGACTGTGGGACTTCAGACTCGCCCGAGT
GGCGAAAGGGTCCTAAGGGACCTAAGACTTTGTGTAATGCTTGTGGATGTAAGTTGGT
CACCTTCCAATGGACATTTCGGTTATATGCTAACGTCTCCAGTACGATGGGCCAAAGAG
AAGCGCCAGACACCACCAACCTAGGTTTCTGTTCTGGACGCACTGCTCGTCGCTCTTT
CTTGACTCCAGTTGTATAG
AgveA (1734 bp)
ATGGCGACGCGAATGCCCCTCATGCCTCCGGAGAATGAGACGGAAGCCCATTTCAGCC
GGATTACCCGTGAGGGTAAGAGGATATCGTACAAGCTCAGTGTCATGCAGCAACCTGA
GCGTGCCAGAGCCTGCGGTGCAGGTGCCAAATGTATGCCATTACCCCTTGGATATATGG
AAAGAAAGGGAAGGCTAACTGAAACAGCGTCTGCCGACCGTCGACCGGTTGATCCTC
CGCCCGTGGTTGAACTGCGCATCTTCGAATCCGACCCCAATGACGACCTGCACAAGAC
CGACATCACCTTTGCCTACAACGCCAGTTTCTTCTTGTACGCAACATTGCAAACGGCGC
GCCCCATTGCGCATGGGCGAGTCGCTGGTGCTCCGACTACCCCCGTGTTGACCGGTGT
GCCCGTCGCGGGAATCGCCTATTTGGACCGTCCGTCTCAGGCTGGCTACTTCATCTTTC
CCGATCTGTCGGTCCGTCATGAGGGTCGATACCGCTTGACCTTCCACCTGTACGAAGA
AACCAAGGACCTCAAGGATGCCGACAGAGATCCCCCGATACCGGACACCACCGTTCA
ACCGCTTAAACCGGGCGCTAGCACTCCTGACGCTTTCAACTTCCGTCTTTTGGTCAACT
CTGTCCCCTTTACCGTGTACAGCGCCAAGAAGTTCCCGGGTCTGGCCGCCAGTACCTC
CCTGAGTCGAATTATCGCTGAACAAGGCTGCCGTGTTCGCATTCGCCGTGATGTCCGCA
TGAGACGCCGCACCGACAAGCGCAACGACGACTACGATGAATACTCTGCGGCTCCCC
GGCAAGCAGACAACAGGTACTCCACGCCAGATGCCTATACCGCTGCTGCCGCTGCTGC
TGCGGCAGCAGCCACCCCCATCGAACGCCCACGATCTGCCAGCACCAGCACGATCGA
ACCCGCTTTGCCATACGCTCCTGATGCGCAGCGTCGACCTTCTGGATCCGACTACGGAT
ACCCTTGTGCGCAGCCCTATCAAAGAGCCATTCCTGTCGCGCCGCAGGCATCACCGTAT
CCTTCGCACCTTTCTTTCGGCTCTGGTCAGCCTCAGTACCATGCTCCCCCGCTTCCACC
CACTCCCCAAGCGACCGCACCGGTAATCCCATACTCGCCTCATCTGTCATACTCCCATA
CTAGAAATCCATCCACCGAATACGAACCCACTGGGTACCCATATCCTCAACCGCGACCA
CCCACCGAGCGGACTTCCAGCTACCCCGGACCTCTACCGCCTCTCCGTCCTCTTGAGC
CACCAAAGGGCTTCACACAACCAGCGGGTGAACCCCGTTCCTCGGATCCCAACGCTTA
TCACCAATCTCAAGCCCTGCCTCCAGCGCCACGATCTCATACACCCTCAACAGGTATTG
CACCTTCCCTTCCGCCTCTCTCGGCCCTGTCTAGCGGCAGTGAATACGCCCACCACATG
CCCAGCAGTATGGTTCTGAACCCGAGCCACGAGATGACTCCGGGCAAACGGTTGTACG
ATGCCCCCGGACCGAAGTTGAGCAAACGGTCGCATGATGAATCATTTGGGCCCGATGA
CCGCCCGGTGCAAAACGGCATGCGACCAGATACAGAATTGTATCCGAGCATGCAACGG
AAGCTTTCTGAGGCCAGTCGCGCCTACTTTGGTGATACCCGCGATGAGATGGCATACAA
GCGAGCCAACGGCCGCATGGCCATGAAGATTCCGCCGACTTTTTCTAGTTAA