Features
Primers
CDS
misc_recomb
promoter
RBS
Circle
Line
Sequence
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100
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700
800
900
AmpR
GFP
T7 promoter
RBS
attB1
attB2
attB-GFP forward (114 .. 139)
attB-GFP reverse (805 .. 830)
Colony PCR forward (37 .. 58)
Colony PCR reverse (879 .. 907)
Sequencing forward (889 .. 908)
Sequencing reverse (50 .. 68)
pDEST-demo-GFP
915 bp
100
200
300
400
500
600
700
800
900
AmpR
RBS
GFP
attB2
T7 promoter
attB1
attB-GFP forward (114 .. 139)
attB-GFP reverse (805 .. 830)
Colony PCR forward (37 .. 58)
Colony PCR reverse (879 .. 907)
Sequencing forward (889 .. 908)
Sequencing reverse (50 .. 68)
pDEST-demo-GFP
915 bp
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
AmpR
RBS
attB1
GFP
attB2
T7 promoter
attB-GFP forward (114 .. 139)
attB-GFP reverse (805 .. 830)
Colony PCR forward (37 .. 58)
Colony PCR reverse (879 .. 907)
Sequencing forward (889 .. 908)
Sequencing reverse (50 .. 68)
pDEST-demo-GFP
915 bp
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
AmpR
T7 promoter
RBS
attB1
GFP
attB2
attB-GFP forward (114 .. 139)
attB-GFP reverse (805 .. 830)
Colony PCR forward (37 .. 58)
Colony PCR reverse (879 .. 907)
Sequencing forward (889 .. 908)
Sequencing reverse (50 .. 68)
pDEST-demo-GFP
915 bp
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
480
500
520
540
560
580
600
620
640
660
680
700
720
740
760
780
800
820
840
860
880
900
AmpR
T7 promoter
RBS
attB1
GFP
attB2
attB-GFP forward (114 .. 139)
attB-GFP reverse (805 .. 830)
Colony PCR forward (37 .. 58)
Colony PCR reverse (879 .. 907)
Sequencing forward (889 .. 908)
Sequencing reverse (50 .. 68)
pDEST-demo-GFP
915 bp
Both strands
Copy
10 20 30 40 50 60
TTGACCGCTTAAGGCTAACGTCAGTTGCATGCCTTAAGGCTTGACCAATGCGTTCAGGCT
AACTGGCGAATTCCGATTGCAGTCAACGTACGGAATTCCGAACTGGTTACGCAAGTCCGA
60
Leu Thr Ala Gly Arg Gln Leu His Ala Leu Arg Leu Asp Gln Cys Val Gln Ala
Ter Ter
AmpR
Colony PCR forward (37 .. 58)
Sequencing reverse (50 .. 68)
70 80 90 100 110 120
TAATACGACTCACTATAGGGAAGGAGATACAAGTTTGTACAAAAAAGCAGGCTATGAGTA
ATTATGCTGAGTGATATCCCTTCCTCTATGTTCAAACATGTTTTTTCGTCCGATACTCAT
120
T7 promoter
RBS
attB1
Met Ser
GFP
attB-GFP forward (114 .. 139)
Sequencing reverse (50 .. 68)
130 140 150 160 170 180
AAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTA
TTCCTCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAAT
180
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
GFP
attB-GFP forward (114 .. 139)
190 200 210 220 230 240
ATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTA
TACCCGTGTTTAAAAGACAGTCACCTCTCCCACTTCCACTACGTTGTATGCCTTTTGAAT
240
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
GFP
250 260 270 280 290 300
CCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTA
GGGAATTTAAATAAACGTGATGACCTTTTGATGGACAAGGTACCGGTTGTGAACAGTGAT
300
Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
GFP
310 320 330 340 350 360
CTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACT
GAAAGAGAATACCACAAGTTACGAAAAGTTCTATGGGTCTAGTATACTTTGCCGTACTGA
360
Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp
GFP
370 380 390 400 410 420
TTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATG
AAAAGTTCTCACGGTACGGGCTTCCAATACATGTCCTTTCTTGATATAAAAAGTTTCTAC
420
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
GFP
430 440 450 460 470 480
ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAA
TGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGGGAACAATTATCTT
480
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
GFP
490 500 510 520 530 540
TCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAAT
AGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTGTTTAACCTTA
540
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
GFP
550 560 570 580 590 600
ACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAG
TGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACCTTAGTTTC
600
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys
GFP
610 620 630 640 650 660
TTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATC
AATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCTGGTAATAG
660
Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
GFP
670 680 690 700 710 720
AACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCA
TTGTTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGACAGGT
720
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
GFP
730 740 750 760 770 780
CACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGT
GTGTTAGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAACTCA
780
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
GFP
790 800 810 820 830 840
TTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGACCCAGCTTT
AACATTGTCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATCTGGGTCGAAA
840
Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
Ter
GFP
attB2
attB-GFP reverse (805 .. 830)
850 860 870 880 890 900
CTTGTACAAAGTGGTGCATCGTAGCCTATGACGATCAGTCCATGAACTGCTAAGGTCATC
GAACATGTTTCACCACGTAGCATCGGATACTGCTAGTCAGGTACTTGACGATTCCAGTAG
900
attB2
Sequencing forward (889 .. 908)
Colony PCR reverse (879 .. 907)
910
AGGCTATCGGATCAA
TCCGATAGCCTAGTT
915
Sequencing forward (889 .. 908)
Colony PCR reverse (879 .. 907)
10 20 30
TTGACCGCTTAAGGCTAACGTCAGTTGCAT
AACTGGCGAATTCCGATTGCAGTCAACGTA
30
Leu Thr Ala Gly Arg Gln Leu His
Ter Ter
AmpR
40 50 60
GCCTTAAGGCTTGACCAATGCGTTCAGGCT
CGGAATTCCGAACTGGTTACGCAAGTCCGA
60
Ala Leu Arg Leu Asp Gln Cys Val Gln Ala
AmpR
Colony PCR forward (37 .. 58)
Sequencing reverse (50 .. 68)
70 80 90
TAATACGACTCACTATAGGGAAGGAGATAC
ATTATGCTGAGTGATATCCCTTCCTCTATG
90
T7 promoter
RBS
attB1
Sequencing reverse (50 .. 68)
100 110 120
AAGTTTGTACAAAAAAGCAGGCTATGAGTA
TTCAAACATGTTTTTTCGTCCGATACTCAT
120
attB1
Met Ser
GFP
attB-GFP forward (114 .. 139)
130 140 150
AAGGAGAAGAACTTTTCACTGGAGTTGTCC
TTCCTCTTCTTGAAAAGTGACCTCAACAGG
150
Lys Gly Glu Glu Leu Phe Thr Gly Val Val
GFP
attB-GFP forward (114 .. 139)
160 170 180
CAATTCTTGTTGAATTAGATGGTGATGTTA
GTTAAGAACAACTTAATCTACCACTACAAT
180
Pro Ile Leu Val Glu Leu Asp Gly Asp Val
GFP
190 200 210
ATGGGCACAAATTTTCTGTCAGTGGAGAGG
TACCCGTGTTTAAAAGACAGTCACCTCTCC
210
Asn Gly His Lys Phe Ser Val Ser Gly Glu
GFP
220 230 240
GTGAAGGTGATGCAACATACGGAAAACTTA
CACTTCCACTACGTTGTATGCCTTTTGAAT
240
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
GFP
250 260 270
CCCTTAAATTTATTTGCACTACTGGAAAAC
GGGAATTTAAATAAACGTGATGACCTTTTG
270
Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys
GFP
280 290 300
TACCTGTTCCATGGCCAACACTTGTCACTA
ATGGACAAGGTACCGGTTGTGAACAGTGAT
300
Leu Pro Val Pro Trp Pro Thr Leu Val Thr
GFP
310 320 330
CTTTCTCTTATGGTGTTCAATGCTTTTCAA
GAAAGAGAATACCACAAGTTACGAAAAGTT
330
Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser
GFP
340 350 360
GATACCCAGATCATATGAAACGGCATGACT
CTATGGGTCTAGTATACTTTGCCGTACTGA
360
Arg Tyr Pro Asp His Met Lys Arg His Asp
GFP
370 380 390
TTTTCAAGAGTGCCATGCCCGAAGGTTATG
AAAAGTTCTCACGGTACGGGCTTCCAATAC
390
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr
GFP
400 410 420
TACAGGAAAGAACTATATTTTTCAAAGATG
ATGTCCTTTCTTGATATAAAAAGTTTCTAC
420
Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
GFP
430 440 450
ACGGGAACTACAAGACACGTGCTGAAGTCA
TGCCCTTGATGTTCTGTGCACGACTTCAGT
450
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
GFP
460 470 480
AGTTTGAAGGTGATACCCTTGTTAATAGAA
TCAAACTTCCACTATGGGAACAATTATCTT
480
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
GFP
490 500 510
TCGAGTTAAAAGGTATTGATTTTAAAGAAG
AGCTCAATTTTCCATAACTAAAATTTCTTC
510
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu
GFP
520 530 540
ATGGAAACATTCTTGGACACAAATTGGAAT
TACCTTTGTAAGAACCTGTGTTTAACCTTA
540
Asp Gly Asn Ile Leu Gly His Lys Leu Glu
GFP
550 560 570
ACAACTATAACTCACACAATGTATACATCA
TGTTGATATTGAGTGTGTTACATATGTAGT
570
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
GFP
580 590 600
TGGCAGACAAACAAAAGAATGGAATCAAAG
ACCGTCTGTTTGTTTTCTTACCTTAGTTTC
600
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys
GFP
610 620 630
TTAACTTCAAAATTAGACACAACATTGAAG
AATTGAAGTTTTAATCTGTGTTGTAACTTC
630
Val Asn Phe Lys Ile Arg His Asn Ile Glu
GFP
640 650 660
ATGGAAGCGTTCAACTAGCAGACCATTATC
TACCTTCGCAAGTTGATCGTCTGGTAATAG
660
Asp Gly Ser Val Gln Leu Ala Asp His Tyr
GFP
670 680 690
AACAAAATACTCCAATTGGCGATGGCCCTG
TTGTTTTATGAGGTTAACCGCTACCGGGAC
690
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
GFP
700 710 720
TCCTTTTACCAGACAACCATTACCTGTCCA
AGGAAAATGGTCTGTTGGTAATGGACAGGT
720
Val Leu Leu Pro Asp Asn His Tyr Leu Ser
GFP
730 740 750
CACAATCTGCCCTTTCGAAAGATCCCAACG
GTGTTAGACGGGAAAGCTTTCTAGGGTTGC
750
Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn
GFP
760 770 780
AAAAGAGAGACCACATGGTCCTTCTTGAGT
TTTTCTCTCTGGTGTACCAGGAAGAACTCA
780
Glu Lys Arg Asp His Met Val Leu Leu Glu
GFP
790 800 810
TTGTAACAGCTGCTGGGATTACACATGGCA
AACATTGTCGACGACCCTAATGTGTACCGT
810
Phe Val Thr Ala Ala Gly Ile Thr His Gly
GFP
attB-GFP reverse (805 .. 830)
820 830 840
TGGATGAACTATACAAATAGACCCAGCTTT
ACCTACTTGATATGTTTATCTGGGTCGAAA
840
Met Asp Glu Leu Tyr Lys
Ter
GFP
attB2
attB-GFP reverse (805 .. 830)
850 860 870
CTTGTACAAAGTGGTGCATCGTAGCCTATG
GAACATGTTTCACCACGTAGCATCGGATAC
870
attB2
880 890 900
ACGATCAGTCCATGAACTGCTAAGGTCATC
TGCTAGTCAGGTACTTGACGATTCCAGTAG
900
Sequencing forward (889 .. 908)
Colony PCR reverse (879 .. 907)
910
AGGCTATCGGATCAA
TCCGATAGCCTAGTT
915
Sequencing forward (889 .. 908)
Colony PCR reverse (879 .. 907)