How to design oligos for de novo gene synthesis of superfolder GFP
Soluble SuperFolder GFP (sfGFP) has
1, Cycle3 mutations: F99S, M153T, and V163A;
2, Enhanced GFP mutations: F64L and S65T;
3, Six additional mutations: S30R, Y39N, N105T, Y145F, I171V, A206V;
4, Q80R mutation
>95% are monomer
G1-10
ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGT 60
GATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCTACAATCGGA 120
AAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT 180
GTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAAGG 240
CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTC 300
AAAGATGACGGGAAATACAAGACGCGTGCTGTAGTCAAGTTTGAAGGTGATACCCTTGTT 360
AATCGTATCGAGTTAAAGGGTACTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA 420
CTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGA 480
ATCAAAGCTAACTTCACAGTTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGAC 540
CATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC 600
CTGTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCAC
ATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACA
G11
AAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACATAA
==========
joined
ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGT 60
GATGTTAATGGGCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTGATGCTACAATCGGA 120
AAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTT 180
GTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAAGG 240
CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTC 300
AAAGATGACGGGAAATACAAGACGCGTGCTGTAGTCAAGTTTGAAGGTGATACCCTTGTT 360
AATCGTATCGAGTTAAAGGGTACTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAA 420
CTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGA 480
ATCAAAGCTAACTTCACAGTTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGAC 540
CATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC 600
CTGTCGACACAAACTGTCCTTTCGAAAGATCCCAACGAA
AAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACATAA
====================
IDT optimized
ATGAGCAAAGGCGAGGAACTCTTTACGGGCGTGGTCCCGATTCTGGTTGAATTAGATGGC 60
GACGTCAACGGTCACAAGTTTTCGGTACGCGGAGAAGGTGAAGGTGATGCAACCATTGGG 120
AAACTGACTTTGAAGTTTATTTGCACTACTGGCAAGCTGCCGGTGCCCTGGCCGACCCTG 180
GTAACCACACTGACGTATGGCGTCCAGTGCTTTTCCCGTTACCCGGACCATATGAAACGC 240
CATGACTTCTTCAAATCTGCGATGCCAGAGGGTTACGTACAAGAACGCACGATCTCGTTC 300
AAAGATGATGGTAAATATAAAACGCGCGCAGTGGTAAAGTTCGAAGGTGATACCTTAGTC 360
AACCGCATCGAACTGAAAGGCACCGACTTCAAAGAAGATGGCAACATTCTGGGTCATAAA 420
CTCGAATATAACTTCAACTCTCATAACGTCTACATCACAGCGGATAAACAGAAAAACGGC 480
ATTAAAGCCAATTTTACGGTTCGTCATAACGTGGAAGACGGGTCAGTGCAACTCGCGGAT 540
CATTATCAGCAGAATACTCCTATTGGTGATGGCCCGGTGCTTCTGCCGGATAATCATTAT 600
TTATCTACGCAGACCGTACTGAGCAAAGATCCTAATGAAAAACGTGATCATATGGTGCTC 660
CTCGAATTTGTTACCGCGGCGGGCATTACC
Non-cutter
18 BamHI GGATCC
26 BglII AGATCT
70 EcoRI GAATTC
71 EcoRV GATATC
76 HindIII AAGCTT
80 KpnI GGTACC
89 NcoI CCATGG
91 NheI GCTAGC
93 NotI GCGGCCGC
103 PmeI GTTTAAAC
104 PmlI CACGTG
110 PstI CTGCAG
115 SalI GTCGAC
121 SfiI GGCCNNNNNGGCC
135 XbaI TCTAGA
137 XhoI CTCGAG
Translate
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTL
VTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLV
NRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLAD
HYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKRDHMVLLEFVTAAGIT
G11
KRDHMVLLEFVTAAGITGT*
Amino acid sequence
GFP 11 WT KRDHMVLLEFVTAAGITGT
GFP 11 M1 (L221H) KRDHMVLHEFVTAAGITGT
GFP 11 M2 (L221H, F223S, T225N) KRDHMVLHESVNAAGGT
GFP 11 M3 (L221H, F223Y, T225N) RDHMVLHEYVNAAGIT
Mutations:
N39I, T105K, E111V, I128T, K166T, I167V and S205T
Blast vs superfolder GFP
Score = 463 bits (1191), Expect = 1e-135, Method: Compositional matrix adjust.
Identities = 223/230 (96%), Positives = 225/230 (97%), Gaps = 0/230 (0%)
ssfGFP 1 MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTL 60
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDAT-GKLTLKFICTTGKLPVPWPTL
sfGFP 1 MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTL 60
Query 61 VTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLV 120
VTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG-YKTRA-VKFEGDTLV
Sbjct 61 VTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLV 120
Query 121 NRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLAD 180
NRIELKG-DFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANF–RHNVEDGSVQLAD
Sbjct 121 NRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLAD 180
Query 181 HYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKRDHMVLLEFVTAAGIT 230
HYQQNTPIGDGPVLLPDNHYLSTQ-VLSKDPNEKRDHMVLLEFVTAAGIT
Sbjct 181 HYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGIT 230
BamH1 Kozak
ATTAAG GAT CCG GCC ACC
ATGAGCAAAGGCGAGGAACTCTTTACGGGCGTGGTCCCGATTCTGGTTGAATTAGATGGC 60
GACGTCAACGGTCACAAGTTTTCGGTACGCGGAGAAGGTGAAGGTGATGCAACCATTGGG 120
AAACTGACTTTGAAGTTTATTTGCACTACTGGCAAGCTGCCGGTGCCCTGGCCGACCCTG 180
GTAACCACACTGACGTATGGCGTCCAGTGCTTTTCCCGTTACCCGGACCATATGAAACGC 240
CATGACTTCTTCAAATCTGCGATGCCAGAGGGTTACGTACAAGAACGCACGATCTCGTTC 300
AAAGATGATGGTAAATATAAAACGCGCGCAGTGGTAAAGTTCGAAGGTGATACCTTAGTC 360
AACCGCATCGAACTGAAAGGCACCGACTTCAAAGAAGATGGCAACATTCTGGGTCATAAA 420
CTCGAATATAACTTCAACTCTCATAACGTCTACATCACAGCGGATAAACAGAAAAACGGC 480
ATTAAAGCCAATTTTACGGTTCGTCATAACGTGGAAGACGGGTCAGTGCAACTCGCGGAT 540
CATTATCAGCAGAATACTCCTATTGGTGATGGCCCGGTGCTTCTGCCGGATAATCATTAT 600
TTATCTACGCAGACCGTACTGAGCAAAGATCCTAATGAAAAACGTGATCATATGGTGCTC 660
CTCGAATTTGTTACCGCGGCGGGCATTACC
stop XbaI XhoI
TAA TCTAGA CTCGAG atcga
DNAworks http://helixweb.nih.gov/dnaworks/
FINAL SUMMARY FOR 11 SOLUTIONS
——————————————————————————–
# Tm Len | Score TmRange Short Long #Olig #Repeat #Misprime
1 62 50 | 11.709 3.1 12 50 24 12 4
2 62 51 | 7.755 2.5 14 51 24 12 0
3 62 52 | 10.733 4.3 16 52 24 12 3
4 62 53 | 11.712 3.1 15 53 22 12 4
5 62 54 | 9.728 2.1 16 54 22 12 2
6 62 55 | 9.736 2.8 15 55 22 12 2
7 62 56 | 11.709 2.9 16 56 22 12 4
8 62 57 | 7.753 3.5 16 57 20 12 0
9 62 58 | 8.736 1.9 15 58 20 12 1
10 62 59 | 7.747 1.8 16 59 20 12 0
11 62 60 | 9.728 2.1 14 60 18 12 2
PARAMETERS FOR TRIAL 10
—————————————————————-
Total Size Of Gene ……… 728 nt
Protein Residues ……….. 0
Mutatable Residues ……… 0
Fixed Nucleotides ………. 728 nt
Oligo Size …………….. 59 nt
Annealing Temp …………. 62 +/- 1*C
Oligo Concentration …….. 1.00E-7 M
Sodium Concentration ……. 5.00E-2 M
Mg2+ Concentration ……… 2.00E-3 M
Codon Frequency Threshold .. 10%
Repeat Threshold ……….. 8 nt
Mispriming Threshold ……. 8/18 (6 exact) nt
——————————————————————————–
The DNA sequence # 10 is:
—————————————————————-
1 ATTAAGGATCCGGCCACCATGAGCAAAGGCGAGGAACTCTTTACGGGCGTGGTCCCGATT
61 CTGGTTGAATTAGATGGCGACGTCAACGGTCACAAGTTTTCGGTACGCGGAGAAGGTGAA
121 GGTGATGCAACCATTGGGAAACTGACTTTGAAGTTTATTTGCACTACTGGCAAGCTGCCG
181 GTGCCCTGGCCGACCCTGGTAACCACACTGACGTATGGCGTCCAGTGCTTTTCCCGTTAC
241 CCGGACCATATGAAACGCCATGACTTCTTCAAATCTGCGATGCCAGAGGGTTACGTACAA
301 GAACGCACGATCTCGTTCAAAGATGATGGTAAATATAAAACGCGCGCAGTGGTAAAGTTC
361 GAAGGTGATACCTTAGTCAACCGCATCGAACTGAAAGGCACCGACTTCAAAGAAGATGGC
421 AACATTCTGGGTCATAAACTCGAATATAACTTCAACTCTCATAACGTCTACATCACAGCG
481 GATAAACAGAAAAACGGCATTAAAGCCAATTTTACGGTTCGTCATAACGTGGAAGACGGG
541 TCAGTGCAACTCGCGGATCATTATCAGCAGAATACTCCTATTGGTGATGGCCCGGTGCTT
601 CTGCCGGATAATCATTATTTATCTACGCAGACCGTACTGAGCAAAGATCCTAATGAAAAA
661 CGTGATCATATGGTGCTCCTCGAATTTGTTACCGCGGCGGGCATTACCTAATCTAGACTC
721 GAGATCGA
—————————————————————-
The oligonucleotide assembly is:
—————————————————————-
1 10 20 30 40 50 60
| | | | | | |
1 —> 3 —>
1 ATTAAGGATCCGGCCACCA gggcgtggtcccgatt
********* < repeat
AATTCCTAGGCCGGTGGTACTCGTTTCCGCTCCTTGAGAAATGCCCGCACCAGGGCTAA
<— 2
| | | | | | |
61 ctggttgaattagatggcgacgtcaacggtcacaagttttcgg
********* < repeat
gttgccagtgttcaaaagccatgcgcctcttccactt
| | | | | | |
5 —>
121 GTGATGCAACCATTGGGAAACTGACTTTGAAGTTTATTTGCACTACTGGCAAGCTGCCG
****** ********** < repeat
ccactacgttggtaaccctttg GATGACCGTTCGACGGC
<— 4
| | | | | | |
7 —>
181 cacactgacgtatggcgtccagtgcttttcccgttac
CACGGGACCGGCTGGGACCATTGGTGTGACTGCATACCGCAG
<— 6
| | | | | | |
9 —>
241 ccggaccatatgaaacgccatg GCCAGAGGGTTACGTACAA
********* < repeat
cctggtatactttgcggtactgaagaagtttagacgctacggtctcccaatgcatgtt
<—
| | | | | | |
11 —>
301 GAACGCACGATCTCGTTCAAAGATGATGGTAAATATAAAA agtggtaaagttc
**************** < repeat
*********** < AT rich
c GAGCAAGTTTCTACTACCATTTATATTTTGCGCGCGTCACCATTTCAAG
8
| | | | | | |
361 gaaggtgataccttagtcaaccgcatcgaactgaaaggcaccgact
********* ********** < repeat
CTTCCACTAT cttgactttccgtggctgaagtttcttctaccg
<— 10
| | | | | | |
13 —>
421 CATTCTGGGTCATAAACTCGAATATAACTTCAACTCTCATAACGTCTACATCACAGCG
******** ********* < repeat
ttgtaagacccagtatttgagcttat TATTGCAGATGTAGTGTCGC
<— 12
| | | | | | |
15 —>
481 G gcattaaagccaattttacggttcgtcataacgtggaagacggg
******** ********* < repeat
CTATTTGTCTTTTTGCCGTAATTTCGGTTAAAATGCCAA cc
<— 14
| | | | | | |
17 —>
541 tcagtgcaactcgcg TGGTGATGGCCCGGTGCTT
******** < repeat
agtcacgttgagcgcctagtaatagtcgtcttatgaggataaccactaccgggccac
<— 16
| | | | | | |
19 -
601 CTGCCGGATAATCATTATTTATCTACGCAGACCGTACTGA aaaa
******** ********* ****** < repeat
********* < AT rich
ATAGATGCGTCTGGCATGACTCGTTTCTAGGATTACTTTTT
| | | | | | |
–>
661 cgtgatcatatggtgctcctcgaatttgttaccgcggcgggcattacctaatcta
** ********* * < repeat
*********** < GC rich
GCACTAGTATACCACGAG ccgcccgtaatggattagatctgag
<— 18
| | | | | | |
721
******* < repeat
ctctagct
<— 20
—————————————————————-
The total codon usage score ……….. 0.000
The total length score ……………. 0.000
The total melting temperature score … 0.000
The total repeat score ……………. 5.549
The total pattern score …………… 0.000
The total mispriming score ………… 0.000
The total AT content score ………… 1.319
The total GC content score ………… 0.879
The total fixed gap score …………. N/A
The OVERALL score …… 7.747
Tm Range # of Overlaps
————————————-
<59 0
59 0
60 0
61 11
62 8
63 0
64 0
65 0
66 0
>=67 0
————————————-
Tm Range = 1.8
Ovrlap Len Range # of Oligos
————————————-
<17 2
17 2
18 1
19 2
20 4
21 3
22 1
23 2
24 1
25 0
26 0
>=27 0
————————————-
Lowest Overlap = 16
Length Range # of Oligos
————————————-
<47 2
47-48 0
49-50 0
51-52 0
53-54 0
55-56 0
57-58 0
59-60 18
61-62 0
63-64 0
65-66 0
>=67 0
————————————-
Longest = 59
There are 12 repeats greater than 8 nt:
—————————————————————-
DR Pos1 = 20 Pos2 = 638 Size = 9 Seq1 = TGAGCAAAG
Seq2 = TGAGCAAAG
DR Pos1 = 112 Pos2 = 118 Size = 8 Seq1 = GAAGGTGA
Seq2 = GAAGGTGA
DR Pos1 = 112 Pos2 = 361 Size = 8 Seq1 = GAAGGTGA
Seq2 = GAAGGTGA
DR Pos1 = 118 Pos2 = 361 Size = 9 Seq1 = GAAGGTGAT
Seq2 = GAAGGTGAT
IR Pos1 = 146 Pos2 = 404 Size = 9 Seq1 = CTTTGAAGT
Seq2 = ACTTCAAAG
IR Pos1 = 148 Pos2 = 448 Size = 8 Seq1 = TTGAAGTT
Seq2 = AACTTCAA
IR Pos1 = 245 Pos2 = 666 Size = 9 Seq1 = ACCATATGA
Seq2 = TCATATGGT
IR Pos1 = 308 Pos2 = 720 Size = 8 Seq1 = CGATCTCG
Seq2 = CGAGATCG
DR Pos1 = 316 Pos2 = 406 Size = 8 Seq1 = TTCAAAGA
Seq2 = TTCAAAGA
DR Pos1 = 459 Pos2 = 522 Size = 9 Seq1 = TCATAACGT
Seq2 = TCATAACGT
DR Pos1 = 489 Pos2 = 655 Size = 8 Seq1 = GAAAAACG
Seq2 = GAAAAACG
DR Pos1 = 557 Pos2 = 611 Size = 8 Seq1 = ATCATTAT
Seq2 = ATCATTAT
—————————————————————-
Sequence Patterns Screened (As Supplied By User)
—————————————————————-
None found
—————————————————————-
20 oligonucleotides need to be synthesized
—————————————————————-
1 ATTAAGGATCCGGCCACCA 19
2 AATCGGGACCACGCCCGTAAAGAGTTCCTCGCCTTTGCTCATGGTGGCCGGATCCTTAA 59
3 GGGCGTGGTCCCGATTCTGGTTGAATTAGATGGCGACGTCAACGGTCACAAGTTTTCGG 59
4 GTTTCCCAATGGTTGCATCACCTTCACCTTCTCCGCGTACCGAAAACTTGTGACCGTTG 59
5 GTGATGCAACCATTGGGAAACTGACTTTGAAGTTTATTTGCACTACTGGCAAGCTGCCG 59
6 GACGCCATACGTCAGTGTGGTTACCAGGGTCGGCCAGGGCACCGGCAGCTTGCCAGTAG 59
7 CACACTGACGTATGGCGTCCAGTGCTTTTCCCGTTACCCGGACCATATGAAACGCCATG 59
8 CTTGTACGTAACCCTCTGGCATCGCAGATTTGAAGAAGTCATGGCGTTTCATATGGTCC 59
9 GCCAGAGGGTTACGTACAAGAACGCACGATCTCGTTCAAAGATGATGGTAAATATAAAA 59
10 TATCACCTTCGAACTTTACCACTGCGCGCGTTTTATATTTACCATCATCTTTGAACGAG 59
11 AGTGGTAAAGTTCGAAGGTGATACCTTAGTCAACCGCATCGAACTGAAAGGCACCGACT 59
12 TATTCGAGTTTATGACCCAGAATGTTGCCATCTTCTTTGAAGTCGGTGCCTTTCAGTTC 59
13 CATTCTGGGTCATAAACTCGAATATAACTTCAACTCTCATAACGTCTACATCACAGCGG 59
14 AACCGTAAAATTGGCTTTAATGCCGTTTTTCTGTTTATCCGCTGTGATGTAGACGTTAT 59
15 GCATTAAAGCCAATTTTACGGTTCGTCATAACGTGGAAGACGGGTCAGTGCAACTCGCG 59
16 CACCGGGCCATCACCAATAGGAGTATTCTGCTGATAATGATCCGCGAGTTGCACTGACC 59
17 TGGTGATGGCCCGGTGCTTCTGCCGGATAATCATTATTTATCTACGCAGACCGTACTGA 59
18 GAGCACCATATGATCACGTTTTTCATTAGGATCTTTGCTCAGTACGGTCTGCGTAGATA 59
19 AAAACGTGATCATATGGTGCTCCTCGAATTTGTTACCGCGGCGGGCATTACCTAATCTA 59
20 TCGATCTCGAGTCTAGATTAGGTAATGCCCGCC 33
