Addgene pX458-Dual-Guide-Donor-Cas9-H2B-mCherry Sequencing Result - Sequence Analyzer Skip to main content
Addgene

Sequence Analyzer: pX458-Dual-Guide-Donor-Cas9-H2B-mCherry Sequencing Result


Map View

FspI (9776) PvuI (9630) Amp-R (9430 .. 9449) pBRforEco (9021 .. 9039) pGEX 3' (8961 .. 8983) pRS-marker (8842 .. 8861) PsiI (8728) F1ori-F (8667 .. 8688) F1ori-R (8457 .. 8476) NotI (8148) BGH-rev (7933 .. 7950) SgrAI (7879) mCherry-R (7343 .. 7361) BamHI (7179) AfeI (7060) BsmI (6221) BsaBI * (6079) KflI (5958) PmlI (5370) pBR322ori-F (513 .. 532) XbaI (669) hU6-F (680 .. 700) LKO.1 5' (851 .. 870) NdeI (858) Acc65I (1113) KpnI (1117) BspQI - SapI (1250) MfeI (1341) AflII (1653) PaeR7I - XhoI (2239) EF1a-F (2400 .. 2420) EcoRV (4434) pX458-Dual-Guide-Donor-Cas9-H2B-mCherry 10,219 bp

Sequence View

5ʹ 3ʹ 60 ori g t a g a a a a g a t c a a a g g a t c t t c t t g a g a t c c t t t t t t t c t g c g c g t a a t c t g c t g c t t g c a t c t t t t c t a g t t t c c t a g a a g a a c t c t a g g a a a a a a a g a c g c g c a t t a g a c g a c g a a c

120 ori c a a a c a a a a a a a c c a c c g c t a c c a g c g g t g g t t t g t t t g c c g g a t c a a g a g c t a c c a a c t g t t t g t t t t t t t g g t g g c g a t g g t c g c c a c c a a a c a a a c g g c c t a g t t c t c g a t g g t t g a

180 ori c t t t t t c c g a a g g t a a c t g g c t t c a g c a g a g c g c a g a t a c c a a a t a c t g t t c t t c t a g t g g a a a a a g g c t t c c a t t g a c c g a a g t c g t c t c g c g t c t a t g g t t t a t g a c a a g a a g a t c a c

240 ori t a g c c g t a g t t a g g c c a c c a c t t c a a g a a c t c t g t a g c a c c g c c t a c a t a c c t c g c t c t g a t c g g c a t c a a t c c g g t g g t g a a g t t c t t g a g a c a t c g t g g c g g a t g t a t g g a g c g a g a c

300 ori c t a a t c c t g t t a c c a g t g g c t g c t g c c a g t g g c g a t a a g t c g t g t c t t a c c g g g t t g g a c g a t t a g g a c a a t g g t c a c c g a c g a c g g t c a c c g c t a t t c a g c a c a g a a t g g c c c a a c c t g

360 ori t c a a g a c g a t a g t t a c c g g a t a a g g c g c a g c g g t c g g g c t g a a c g g g g g g t t c g t g c a c a a g t t c t g c t a t c a a t g g c c t a t t c c g c g t c g c c a g c c c g a c t t g c c c c c c a a g c a c g t g t

420 ori c a g c c c a g c t t g g a g c g a a c g a c c t a c a c c g a a c t g a g a t a c c t a c a g c g t g a g c t a t g a g t c g g g t c g a a c c t c g c t t g c t g g a t g t g g c t t g a c t c t a t g g a t g t c g c a c t c g a t a c t

480 ori g a a a g c g c c a c g c t t c c c g a a g g g a g a a a g g c g g a c a g g t a t c c g g t a a g c g g c a g g g t c c t t t c g c g g t g c g a a g g g c t t c c c t c t t t c c g c c t g t c c a t a g g c c a t t c g c c g t c c c a g

540 ori G G G A A A C G C C T G G T A T C T T T pBR322ori-F g g a a c a g g a g a g c g c a c g a g g g a g c t t c c a g g g g g a a a c g c c t g g t a t c t t t a t a g t c c t c c t t g t c c t c t c g c g t g c t c c c t c g a a g g t c c c c c t t t g c g g a c c a t a g a a a t a t c a g g a

600 ori g t c g g g t t t c g c c a c c t c t g a c t t g a g c g t c g a t t t t t g t g a t g c t c g t c a g g g g g g c g g c a g c c c a a a g c g g t g g a g a c t g a a c t c g c a g c t a a a a a c a c t a c g a g c a g t c c c c c c g c c

660 ori a g c c t a t g g a a a a a c g c c a g c a a c g c g g c c t t t t t a c g g t t c c t g g c c t t t t g c t g g c c t t c g g a t a c c t t t t t g c g g t c g t t g c g c c g g a a a a a t g c c a a g g a c c g g a a a a c g a c c g g a

XbaI 720 U6 promoter G A G G G C C T A T T T C C C A T G A T T hU6-F t t t g c t c a t c t a g a c a t g t g a g g g c c t a t t t c c c a t g a t t c c t t c a t a t t t g c a t a t a c g a a a c g a g t a g a t c t g t a c a c t c c c g g a t a a a g g g t a c t a a g g a a g t a t a a a c g t a t a t g c

780 U6 promoter a t a c a a g g c t g t t a g a g a g a t a a t t g g a a t t a a t t t g a c t g t a a a c a c a a a g a t a t t a g t t a t g t t c c g a c a a t c t c t c t a t t a a c c t t a a t t a a a c t g a c a t t t g t g t t t c t a t a a t c a

840 U6 promoter a c a a a a t a c g t g a c g t a g a a a g t a a t a a t t t c t t g g g t a g t t t g c a g t t t t a a a a t t a t g t g t t t t a t g c a c t g c a t c t t t c a t t a t t a a a g a a c c c a t c a a a c g t c a a a a t t t t a a t a c

NdeI 900 U6 promoter G A C T A T C A T A T G C T T A C C G T LKO.1 5' t t t t a a a a t g g a c t a t c a t a t g c t t a c c g t a a c t t g a a a g t a t t t c g a t t t c t t g g c t t t a a a a t t t t a c c t g a t a g t a t a c g a a t g g c a t t g a a c t t t c a t a a a g c t a a a g a a c c g a a a

960 U6 promoter gRNA scaffold a t a t a t c t t g t g g a a a g g a c g a a a c a c c g g g t c t t c g a g a a g a c c t g t t t t a g a g c t a g a t a t a t a g a a c a c c t t t c c t g c t t t g t g g c c c a g a a g c t c t t c t g g a c a a a a t c t c g a t c t

1020 gRNA scaffold a a t a g c a a g t t a a a a t a a g g c t a g t c c g t t a t c a a c t t g a a a a a g t g g c a c c g a g t c g g t t t a t c g t t c a a t t t t a t t c c g a t c a g g c a a t a g t t g a a c t t t t t c a c c g t g g c t c a g c c a

1080 gRNA scaffold g c t t t t t t g t t t t a g a g c t a g a a a t a g c a a g t t a a a a t a a g g c t a g t c c g t t t t t a g c g c c g a a a a a a c a a a a t c t c g a t c t t t a t c g t t c a a t t t t a t t c c g a t c a g g c a a a a a t c g c g

Acc65IKpnI 1140 g t g c g c c a a t t c t g c a g a c a a a t g g c t c t a c a g g t a c c a g t a a t t c a t a c a a a a g g a c t c c a c g c g g t t a a g a c g t c t g t t t a c c g a g a t g t c c a t g g t c a t t a a g t a t g t t t t c c t g a g

1200 g c c c c t g c c t t g g g g a a t c c c a g g g a c c g t c g t t a a a c t c c c a c t a a c g t a g a a c c c a g a c g g g g a c g g a a c c c c t t a g g g t c c c t g g c a g c a a t t t g a g g g t g a t t g c a t c t t g g g t c t

BspQISapI 1260 g a t c g c t g c g t t c c c g c c c c c t c a c c c g c c c g c t c t c g t c a t c a c t g a g g t g g a g a a g a g c t a g c g a c g c a a g g g c g g g g g a g t g g g c g g g c g a g a g c a g t a g t g a c t c c a c c t c t t c t c

1320 EF-1 α promoter c a t g c g t g a g g c t c c g g t g c c c g t c a g t g g g c a g a g c g c a c a t c g c c c a c a g t c c c c g a g g t a c g c a c t c c g a g g c c a c g g g c a g t c a c c c g t c t c g c g t g t a g c g g g t g t c a g g g g c t c

MfeI 1380 EF-1 α promoter a a g t t g g g g g g a g g g g t c g g c a a t t g a a c c g g t g c c t a g a g a a g g t g g c g c g g g g t a a a c t t c a a c c c c c c t c c c c a g c c g t t a a c t t g g c c a c g g a t c t c t t c c a c c g c g c c c c a t t t g

1440 EF-1 α promoter t g g g a a a g t g a t g t c g t g t a c t g g c t c c g c c t t t t t c c c g a g g g t g g g g g a g a a c c g t a t a c c c t t t c a c t a c a g c a c a t g a c c g a g g c g g a a a a a g g g c t c c c a c c c c c t c t t g g c a t a

1500 EF-1 α promoter a t a a g t g c a g t a g t c g c c g t g a a c g t t c t t t t t c g c a a c g g g t t t g c c g c c a g a a c a c a g t a t t c a c g t c a t c a g c g g c a c t t g c a a g a a a a a g c g t t g c c c a a a c g g c g g t c t t g t g t c

1560 EF-1 α promoter EF-1 α intron A *PGQT g t a a g t g c c g t g t g t g g t t c c c g c g g g c c t g g c c t c t t t a c g g g t t a t g g c c c t t g c g t g c a t t c a c g g c a c a c a c c a a g g g c g c c c g g a c c g g a g a a a t g c c c a a t a c c g g g a a c g c a c

1620 EF-1 α promoter EF-1 α intron A GQIVEVGRAATRSEQDRAEP c c t t g a a t t a c t t c c a c g c c c c t g g c t g c a g t a c g t g a t t c t t g a t c c c g a g c t t c g g g t g g a a c t t a a t g a a g g t g c g g g g a c c g a c g t c a t g c a c t a a g a a c t a g g g c t c g a a g c c c a

AflII 1680 EF-1 α promoter EF-1 α intron A QFHTPSNSAKRKLLGKAEHK t g g a a g t g g g t g g g a g a g t t c g a g g c c t t g c g c t t a a g g a g c c c c t t c g c c t c g t g c t t g a c c t t c a c c c a c c c t c t c a a g c t c c g g a a c g c g a a t t c c t c g g g g a a g c g g a g c a c g a a c

1740 EF-1 α promoter EF-1 α intron A LQPRAQASPGGRAFRTAGER a g t t g a g g c c t g g c t t g g g c g c t g g g g c c g c c g c g t g c g a a t c t g g t g g c a c c t t c g c g c t c a a c t c c g g a c c g a a c c c g c g a c c c c g g c g g c g c a c g c t t a g a c c a c c g t g g a a g c g c g

1800 EF-1 α promoter EF-1 α intron A RDRQKRYTELWKFNKIVQQS c t g t c t c g c t g c t t t c g a t a a g t c t c t a g c c a t t t a a a a t t t t t g a t g a c c t g c t g c g a c g a c a g a g c g a c g a a a g c t a t t c a g a g a t c g g t a a a t t t t a a a a a c t a c t g g a c g a c g c t g

1860 EF-1 α promoter EF-1 α intron A AKKRALYDQLHPGLDACQYK g c t t t t t t t c t g g c a a g a t a g t c t t g t a a a t g c g g g c c a a g a t c t g c a c a c t g g t a t t t c c g a a a a a a a g a c c g t t c t a t c a g a a c a t t t a c g c c c g g t t c t a g a c g t g t g a c c a t a a a g

1920 EF-1 α promoter EF-1 α intron A PKQPRPRRRPGHTGACMNPS g g t t t t t g g g g c c g c g g g c g g c g a c g g g g c c c g t g c g t c c c a g c g c a c a t g t t c g g c g a g c c a a a a a c c c c g g c g c c c g c c g c t g c c c c g g g c a c g c a g g g t c g c g t g t a c a a g c c g c t c

1980 EF-1 α promoter EF-1 α intron A APGALAAVSFRVPTTELQGA g c g g g g c c t g c g a g c g c g g c c a c c g a g a a t c g g a c g g g g g t a g t c t c a a g c t g g c c g g c c c g c c c c g g a c g c t c g c g c c g g t g g c t c t t a g c c t g c c c c c a t c a g a g t t c g a c c g g c c g g

2040 EF-1 α promoter EF-1 α intron A QEPAQGRAATYRGARPPLAP t g c t c t g g t g c c t g g c c t c g c g c c g c c g t g t a t c g c c c c g c c c t g g g c g g c a a g g c t g g c a c g a g a c c a c g g a c c g g a g c g c g g c g g c a c a t a g c g g g g c g g g a c c c g c c g t t c c g a c c g

2100 EF-1 α promoter EF-1 α intron A GTPVLQTLPFIAAERGQQLS c c g g t c g g c a c c a g t t g c g t g a g c g g a a a g a t g g c c g c t t c c c g g c c c t g c t g c a g g g a g g g c c a g c c g t g g t c a a c g c a c t c g c c t t t c t a c c g g c g a a g g g c c g g g a c g a c g t c c c t c

2160 EF-1 α promoter EF-1 α intron A SLISSAASPLAPPHTVWVFS c t c a a a a t g g a g g a c g c g g c g c t c g g g a g a g c g g g c g g g t g a g t c a c c c a c a c a a a g g a a g a g t t t t a c c t c c t g c g c c g c g a g c c c t c t c g c c c g c c c a c t c a g t g g g t g t g t t t c c t t

2220 EF-1 α promoter EF-1 α intron A FPRETRLRRKM a a g g g c c t t t c c g t c c t c a g c c g t c g c t t c a t g t g a c t c c a c g g a g t a c c g g g c g c c g t c t t c c c g g a a a g g c a g g a g t c g g c a g c g a a g t a c a c t g a g g t g c c t c a t g g c c c g c g g c a g

PaeR7IXhoI 2280 EF-1 α promoter EF-1 α intron A c a g g c a c c t c g a t t a g t t c t c g a g c t t t t g g a g t a c g t c g t c t t t a g g t t g g g g g g a g g g g t c c g t g g a g c t a a t c a a g a g c t c g a a a a c c t c a t g c a g c a g a a a t c c a a c c c c c c t c c c

2340 EF-1 α promoter EF-1 α intron A g t t t t a t g c g a t g g a g t t t c c c c a c a c t g a g t g g g t g g a g a c t g a a g t t a g g c c a g c t t g c a a a a t a c g c t a c c t c a a a g g g g t g t g a c t c a c c c a c c t c t g a c t t c a a t c c g g t c g a a c

2400 EF-1 α promoter EF-1 α intron A T EF1a-F g c a c t t g a t g t a a t t c t c c t t g g a a t t t g c c c t t t t t g a g t t t g g a t c t t g g t t c a t t c t c g t g a a c t a c a t t a a g a g g a a c c t t a a a c g g g a a a a a c t c a a a c c t a g a a c c a a g t a a g a

2460 EF-1 α promoter Kozak sequence EF-1 α intron A C A A G C C T C A G A C A G T G G T T C EF1a-F *TDHGTG c a a g c c t c a g a c a g t g g t t c a a a g t t t t t t t c t t c c a t t t c a g g t g t c g t g a c c g g t g c c g t t c g g a g t c t g t c a c c a a g t t t c a a a a a a a g a a g g t a a a g t c c a c a g c a c t g g c c a c g g

2520 1 5 10 15 DYKDHDGDYKDHDIDYKD 3xFLAG Kozak sequence MDYKDHDGDYKDHDIDYKD GHVILVVVSVVLIMINIVFV a c c a t g g a c t a t a a g g a c c a c g a c g g a g a c t a c a a g g a t c a t g a t a t t g a t t a c a a a g a c t g g t a c c t g a t a t t c c t g g t g c t g c c t c t g a t g t t c c t a g t a c t a t a a c t a a t g t t t c t g

2580 20 DDDK MA 130 PKKKRKV (in frame with SV40 NLS) GIHGVPA 3xFLAG SV40 NLS DDDKMAPKKKRKVGIHGVPA IVILHGWLLLPLDTDVSDWC g a t g a c g a t a a g a t g g c c c c a a a g a a g a a g c g g a a g g t c g g t a t c c a c g g a g t c c c a g c a c t a c t g c t a t t c t a c c g g g g t t t c t t c t t c g c c t t c c a g c c a t a g g t g c c t c a g g g t c g t

2640 5 10 15 20 DKKYSIGLDIGTNSVGWAV Cas9 A 5 10 15 20 DKKYSIGLDIGTNSVGWAV Cas9 ADKKYSIGLDIGTNSVGWAV GVLLVADAQVDAGVRHAPGH g c c g a c a a g a a g t a c a g c a t c g g c c t g g a c a t c g g c a c c a a c t c t g t g g g c t g g g c c g t g c g g c t g t t c t t c a t g t c g t a g c c g g a c c t g t a g c c g t g g t t g a g a c a c c c g a c c c g g c a c

2700 25 30 35 40 ITDEYKVPSKKFKVLGNTDR Cas9 25 30 35 40 ITDEYKVPSKKFKVLGNTDR Cas9 ITDEYKVPSKKFKVLGNTDR DGVLVLHGALFELHQAVGVP a t c a c c g a c g a g t a c a a g g t g c c c a g c a a g a a a t t c a a g g t g c t g g g c a a c a c c g a c c g g t a g t g g c t g c t c a t g t t c c a c g g g t c g t t c t t t a a g t t c c a c g a c c c g t t g t g g c t g g c c

2760 45 50 55 60 HSIKKNLIGALLFDSGETAE Cas9 45 50 55 60 HSIKKNLIGALLFDSGETAE Cas9 HSIKKNLIGALLFDSGETAE VADLLVQDSGQQEVAAFCGL c a c a g c a t c a a g a a g a a c c t g a t c g g a g c c c t g c t g t t c g a c a g c g g c g a a a c a g c c g a g g t g t c g t a g t t c t t c t t g g a c t a g c c t c g g g a c g a c a a g c t g t c g c c g c t t t g t c g g c t c

2820 65 70 75 80 ATRLKRTARRRYTRRKNRIC Cas9 65 70 75 80 ATRLKRTARRRYTRRKNRIC Cas9 ATRLKRTARRRYTRRKNRIC GGPQLSGGSSSVGSPLVPDA g c c a c c c g g c t g a a g a g a a c c g c c a g a a g a a g a t a c a c c a g a c g g a a g a a c c g g a t c t g c c g g t g g g c c g a c t t c t c t t g g c g g t c t t c t t c t a t g t g g t c t g c c t t c t t g g c c t a g a c g

2880 85 90 95 100 YLQEIFSNEMAKVDDSFFHR Cas9 85 90 95 100 YLQEIFSNEMAKVDDSFFHR Cas9 YLQEIFSNEMAKVDDSFFHR IQLLDEAVLHGLHVVAEEVS t a t c t g c a a g a g a t c t t c a g c a a c g a g a t g g c c a a g g t g g a c g a c a g c t t c t t c c a c a g a a t a g a c g t t c t c t a g a a g t c g t t g c t c t a c c g g t t c c a c c t g c t g t c g a a g a a g g t g t c t

2940 105 110 115 120 LEESFLVEEDKKHERHPIFG Cas9 105 110 115 120 LEESFLVEEDKKHERHPIFG Cas9 LEESFLVEEDKKHERHPIFG QFLGEQHFLILLVLPVGDEA c t g g a a g a g t c c t t c c t g g t g g a a g a g g a t a a g a a g c a c g a g c g g c a c c c c a t c t t c g g c g a c c t t c t c a g g a a g g a c c a c c t t c t c c t a t t c t t c g t g c t c g c c g t g g g g t a g a a g c c g

3000 125 130 135 140 NIVDEVAYHEKYPTIYHLRK Cas9 125 130 135 140 NIVDEVAYHEKYPTIYHLRK Cas9 NIVDEVAYHEKYPTIYHLRK VDHVLHGVVLLVGGDVVQSL a a c a t c g t g g a c g a g g t g g c c t a c c a c g a g a a g t a c c c c a c c a t c t a c c a c c t g a g a a a g t t g t a g c a c c t g c t c c a c c g g a t g g t g c t c t t c a t g g g g t g g t a g a t g g t g g a c t c t t t c

3060 145 150 155 160 KLVDSTDKADLRLIYLALAH Cas9 145 150 155 160 KLVDSTDKADLRLIYLALAH Cas9 KLVDSTDKADLRLIYLALAH FQHVAGVLGVQPQDIQGQGV a a a c t g g t g g a c a g c a c c g a c a a g g c c g a c c t g c g g c t g a t c t a t c t g g c c c t g g c c c a c t t t g a c c a c c t g t c g t g g c t g t t c c g g c t g g a c g c c g a c t a g a t a g a c c g g g a c c g g g t g

3120 165 170 175 180 MIKFRGHFLIEGDLNPDNSD Cas9 165 170 175 180 MIKFRGHFLIEGDLNPDNSD Cas9 MIKFRGHFLIEGDLNPDNSD HDLEPAVEQDLAVQVGVVAV a t g a t c a a g t t c c g g g g c c a c t t c c t g a t c g a g g g c g a c c t g a a c c c c g a c a a c a g c g a c t a c t a g t t c a a g g c c c c g g t g a a g g a c t a g c t c c c g c t g g a c t t g g g g c t g t t g t c g c t g

3180 185 190 195 200 VDKLFIQLVQTYNQLFEENP Cas9 185 190 195 200 VDKLFIQLVQTYNQLFEENP Cas9 VDKLFIQLVQTYNQLFEENP HVLQEDLQHLGVVLQELFVG g t g g a c a a g c t g t t c a t c c a g c t g g t g c a g a c c t a c a a c c a g c t g t t c g a g g a a a a c c c c c a c c t g t t c g a c a a g t a g g t c g a c c a c g t c t g g a t g t t g g t c g a c a a g c t c c t t t t g g g g

3240 205 210 215 220 INASGVDAKAILSARLSKSR Cas9 205 210 215 220 INASGVDAKAILSARLSKSR Cas9 INASGVDAKAILSARLSKSR DVGAAHVGLGDQRGSQALAS a t c a a c g c c a g c g g c g t g g a c g c c a a g g c c a t c c t g t c t g c c a g a c t g a g c a a g a g c a g a t a g t t g c g g t c g c c g c a c c t g c g g t t c c g g t a g g a c a g a c g g t c t g a c t c g t t c t c g t c t

3300 225 230 235 240 RLENLIAQLPGEKKNGLFGN Cas9 225 230 235 240 RLENLIAQLPGEKKNGLFGN Cas9 RLENLIAQLPGEKKNGLFGN PQFIQDGLQGALLLIAQESV c g g c t g g a a a a t c t g a t c g c c c a g c t g c c c g g c g a g a a g a a g a a t g g c c t g t t c g g a a a c g c c g a c c t t t t a g a c t a g c g g g t c g a c g g g c c g c t c t t c t t c t t a c c g g a c a a g c c t t t g

3360 245 250 255 260 LIALSLGLTPNFKSNFDLAE Cas9 245 250 255 260 LIALSLGLTPNFKSNFDLAE Cas9 LIALSLGLTPNFKSNFDLAE QNGQAQAQGGVELAVEVQGL c t g a t t g c c c t g a g c c t g g g c c t g a c c c c c a a c t t c a a g a g c a a c t t c g a c c t g g c c g a g g a c t a a c g g g a c t c g g a c c c g g a c t g g g g g t t g a a g t t c t c g t t g a a g c t g g a c c g g c t c

3420 265 270 275 280 DAKLQLSKDTYDDDLDNLLA Cas9 265 270 275 280 DAKLQLSKDTYDDDLDNLLA Cas9 DAKLQLSKDTYDDDLDNLLA IGFQLQALVGVVVVQVVQQG g a t g c c a a a c t g c a g c t g a g c a a g g a c a c c t a c g a c g a c g a c c t g g a c a a c c t g c t g g c c c t a c g g t t t g a c g t c g a c t c g t t c c t g t g g a t g c t g c t g c t g g a c c t g t t g g a c g a c c g g

3480 285 290 295 300 QIGDQYADLFLAAKNLSDAI Cas9 285 290 295 300 QIGDQYADLFLAAKNLSDAI Cas9 QIGDQYADLFLAAKNLSDAI LDAVLVGVQKQGGLVQGVGD c a g a t c g g c g a c c a g t a c g c c g a c c t g t t t c t g g c c g c c a a g a a c c t g t c c g a c g c c a t c g t c t a g c c g c t g g t c a t g c g g c t g g a c a a a g a c c g g c g g t t c t t g g a c a g g c t g c g g t a g

3540 305 310 315 320 LLSDILRVNTEITKAPLSAS Cas9 305 310 315 320 LLSDILRVNTEITKAPLSAS Cas9 LLSDILRVNTEITKAPLSAS QQAVDQSHVGLDGLGGQAGR c t g c t g a g c g a c a t c c t g a g a g t g a a c a c c g a g a t c a c c a a g g c c c c c c t g a g c g c c t c t g a c g a c t c g c t g t a g g a c t c t c a c t t g t g g c t c t a g t g g t t c c g g g g g g a c t c g c g g a g a

3600 325 330 335 340 MIKRYDEHHQDLTLLKALVR Cas9 325 330 335 340 MIKRYDEHHQDLTLLKALVR Cas9 MIKRYDEHHQDLTLLKALVR HDLSVVLVVLVQGQQFSEHP a t g a t c a a g a g a t a c g a c g a g c a c c a c c a g g a c c t g a c c c t g c t g a a a g c t c t c g t g c g g t a c t a g t t c t c t a t g c t g c t c g t g g t g g t c c t g g a c t g g g a c g a c t t t c g a g a g c a c g c c

3660 345 350 355 360 QQLPEKYKEIFFDQSKNGYA Cas9 345 350 355 360 QQLPEKYKEIFFDQSKNGYA Cas9 QQLPEKYKEIFFDQSKNGYA LLQRLLVFLNEEVLALVAVG c a g c a g c t g c c t g a g a a g t a c a a a g a g a t t t t c t t c g a c c a g a g c a a g a a c g g c t a c g c c g t c g t c g a c g g a c t c t t c a t g t t t c t c t a a a a g a a g c t g g t c t c g t t c t t g c c g a t g c g g

3720 365 370 375 380 GYIDGGASQEEFYKFIKPIL Cas9 365 370 375 380 GYIDGGASQEEFYKFIKPIL Cas9 GYIDGGASQEEFYKFIKPIL AVNVASGALFLEVLEDLGDQ g g c t a c a t t g a c g g c g g a g c c a g c c a g g a a g a g t t c t a c a a g t t c a t c a a g c c c a t c c t g c c g a t g t a a c t g c c g c c t c g g t c g g t c c t t c t c a a g a t g t t c a a g t a g t t c g g g t a g g a c

3780 385 390 395 400 EKMDGTEELLVKLNREDLLR Cas9 385 390 395 400 EKMDGTEELLVKLNREDLLR Cas9 EKMDGTEELLVKLNREDLLR FLHVAGLFQEHLQVSLVQQP g a a a a g a t g g a c g g c a c c g a g g a a c t g c t c g t g a a g c t g a a c a g a g a g g a c c t g c t g c g g c t t t t c t a c c t g c c g t g g c t c c t t g a c g a g c a c t t c g a c t t g t c t c t c c t g g a c g a c g c c

3840 405 410 415 420 KQRTFDNGSIPHQIHLGELH Cas9 405 410 415 420 KQRTFDNGSIPHQIHLGELH Cas9 KQRTFDNGSIPHQIHLGELH LLPGEVVAADGVLDVQSLQV a a g c a g c g g a c c t t c g a c a a c g g c a g c a t c c c c c a c c a g a t c c a c c t g g g a g a g c t g c a c t t c g t c g c c t g g a a g c t g t t g c c g t c g t a g g g g g t g g t c t a g g t g g a c c c t c t c g a c g t g

3900 425 430 435 440 AILRRQEDFYPFLKDNREKI Cas9 425 430 435 440 AILRRQEDFYPFLKDNREKI Cas9 AILRRQEDFYPFLKDNREKI GNQPPLFIKVWEQLVVPFLD g c c a t t c t g c g g c g g c a g g a a g a t t t t t a c c c a t t c c t g a a g g a c a a c c g g g a a a a g a t c c g g t a a g a c g c c g c c g t c c t t c t a a a a a t g g g t a a g g a c t t c c t g t t g g c c c t t t t c t a g

3960 445 450 455 460 EKILTFRIPYYVGPLARGNS Cas9 445 450 455 460 EKILTFRIPYYVGPLARGNS Cas9 EKILTFRIPYYVGPLARGNS LLDQGEADGVVHARQGPSVA g a g a a g a t c c t g a c c t t c c g c a t c c c c t a c t a c g t g g g c c c t c t g g c c a g g g g a a a c a g c c t c t t c t a g g a c t g g a a g g c g t a g g g g a t g a t g c a c c c g g g a g a c c g g t c c c c t t t g t c g

4020 465 470 475 480 RFAWMTRKSEETITPWNFEE Cas9 465 470 475 480 RFAWMTRKSEETITPWNFEE Cas9 RFAWMTRKSEETITPWNFEE SEGPHGSLALFGDGGPVELF a g a t t c g c c t g g a t g a c c a g a a a g a g c g a g g a a a c c a t c a c c c c c t g g a a c t t c g a g g a a t c t a a g c g g a c c t a c t g g t c t t t c t c g c t c c t t t g g t a g t g g g g g a c c t t g a a g c t c c t t

4080 485 490 495 500 VVDKGASAQSFIERMTNFDK Cas9 485 490 495 500 VVDKGASAQSFIERMTNFDK Cas9 VVDKGASAQSFIERMTNFDK HHVLASGGLAEDLPHGVEIL g t g g t g g a c a a g g g c g c t t c c g c c c a g a g c t t c a t c g a g c g g a t g a c c a a c t t c g a t a a g c a c c a c c t g t t c c c g c g a a g g c g g g t c t c g a a g t a g c t c g c c t a c t g g t t g a a g c t a t t c

4140 505 510 515 520 NLPNEKVLPKHSLLYEYFTV Cas9 505 510 515 520 NLPNEKVLPKHSLLYEYFTV Cas9 NLPNEKVLPKHSLLYEYFTV VQGVLLHQGLVAQQVLVEGH a a c c t g c c c a a c g a g a a g g t g c t g c c c a a g c a c a g c c t g c t g t a c g a g t a c t t c a c c g t g t t g g a c g g g t t g c t c t t c c a c g a c g g g t t c g t g t c g g a c g a c a t g c t c a t g a a g t g g c a c

4200 525 530 535 540 YNELTKVKYVTEGMRKPAFL Cas9 525 530 535 540 YNELTKVKYVTEGMRKPAFL Cas9 YNELTKVKYVTEGMRKPAFL IVLQGFHFVHGLSHSLGGEQ t a t a a c g a g c t g a c c a a a g t g a a a t a c g t g a c c g a g g g a a t g a g a a a g c c c g c c t t c c t g a t a t t g c t c g a c t g g t t t c a c t t t a t g c a c t g g c t c c c t t a c t c t t t c g g g c g g a a g g a c

4260 545 550 555 560 SGEQKKAIVDLLFKTNRKVT Cas9 545 550 555 560 SGEQKKAIVDLLFKTNRKVT Cas9 SGEQKKAIVDLLFKTNRKVT AALLFLGDHVQQELGVPFHG a g c g g c g a g c a g a a a a a g g c c a t c g t g g a c c t g c t g t t c a a g a c c a a c c g g a a a g t g a c c t c g c c g c t c g t c t t t t t c c g g t a g c a c c t g g a c g a c a a g t t c t g g t t g g c c t t t c a c t g g

4320 565 570 575 580 VKQLKEDYFKKIECFDSVEI Cas9 565 570 575 580 VKQLKEDYFKKIECFDSVEI Cas9 VKQLKEDYFKKIECFDSVEI HLLQFLVVELFDLAEVGHFD g t g a a g c a g c t g a a a g a g g a c t a c t t c a a g a a a a t c g a g t g c t t c g a c t c c g t g g a a a t c c a c t t c g t c g a c t t t c t c c t g a t g a a g t t c t t t t a g c t c a c g a a g c t g a g g c a c c t t t a g

4380 585 590 595 600 SGVEDRFNASLGTYHDLLKI Cas9 585 590 595 600 SGVEDRFNASLGTYHDLLKI Cas9 SGVEDRFNASLGTYHDLLKI GAHFIPEVGGQACVVIQQFN t c c g g c g t g g a a g a t c g g t t c a a c g c c t c c c t g g g c a c a t a c c a c g a t c t g c t g a a a a t t a g g c c g c a c c t t c t a g c c a a g t t g c g g a g g g a c c c g t g t a t g g t g c t a g a c g a c t t t t a a

EcoRV 4440 605 610 615 620 IKDKDFLDNEENEDILEDIV Cas9 605 610 615 620 IKDKDFLDNEENEDILEDIV Cas9 IKDKDFLDNEENEDILEDIV DLVLVEQVILFVLVNQFIDH a t c a a g g a c a a g g a c t t c c t g g a c a a t g a g g a a a a c g a g g a c a t t c t g g a a g a t a t c g t g t a g t t c c t g t t c c t g a a g g a c c t g t t a c t c c t t t t g c t c c t g t a a g a c c t t c t a t a g c a c

4500 625 630 635 640 LTLTLFEDREMIEERLKTYA Cas9 625 630 635 640 LTLTLFEDREMIEERLKTYA Cas9 LTLTLFEDREMIEERLKTYA *A QGQCQKLVSLHDLFPQFGIG c t g a c c c t g a c a c t g t t t g a g g a c a g a g a g a t g a t c g a g g a a c g g c t g a a a a c c t a t g c c g a c t g g g a c t g t g a c a a a c t c c t g t c t c t c t a c t a g c t c c t t g c c g a c t t t t g g a t a c g g

4560 645 650 655 660 HLFDDKVMKQLKRRRYTGWG Cas9 645 650 655 660 HLFDDKVMKQLKRRRYTGWG Cas9 HLFDDKVMKQLKRRRYTGWG WRNSSLTIFCSFRRLYVPQP VQEVVFHHLLQLPPSVGAPA c a c c t g t t c g a c g a c a a a g t g a t g a a g c a g c t g a a g c g g c g g a g a t a c a c c g g c t g g g g c g t g g a c a a g c t g c t g t t t c a c t a c t t c g t c g a c t t c g c c g c c t c t a t g t g g c c g a c c c c g

4620 665 670 675 680 RLSRKLINGIRDKQSGKTIL Cas9 665 670 675 680 RLSRKLINGIRDKQSGKTIL Cas9 RLSRKLINGIRDKQSGKTIL LSLRFSILPMRSLCDPLVIR PQAPLQDVADPVLLGALCDQ a g g c t g a g c c g g a a g c t g a t c a a c g g c a t c c g g g a c a a g c a g t c c g g c a a g a c a a t c c t g t c c g a c t c g g c c t t c g a c t a g t t g c c g t a g g c c c t g t t c g t c a g g c c g t t c t g t t a g g a c

4680 685 690 695 700 DFLKSDGFANRNFMQLIHDD Cas9 685 690 695 700 DFLKSDGFANRNFMQLIHDD Cas9 DFLKSDGFANRNFMQLIHDD SKRFDSPKALLFKMCSIWSS IEQLGVAEGVSVEHLQDVVV g a t t t c c t g a a g t c c g a c g g c t t c g c c a a c a g a a a c t t c a t g c a g c t g a t c c a c g a c g a c c t a a a g g a c t t c a g g c t g c c g a a g c g g t t g t c t t t g a a g t a c g t c g a c t a g g t g c t g c t g

4740 705 710 715 720 SLTFKEDIQKAQVSGQGDSL Cas9 705 710 715 720 SLTFKEDIQKAQVSGQGDSL Cas9 SLTFKEDIQKAQVSGQGDSL LRVKLSSMWFAWTDPWPSLR AQGKFLVDLFGLHGALAIAQ a g c c t g a c c t t t a a a g a g g a c a t c c a g a a a g c c c a g g t g t c c g g c c a g g g c g a t a g c c t g t c g g a c t g g a a a t t t c t c c t g t a g g t c t t t c g g g t c c a c a g g c c g g t c c c g c t a t c g g a c

4800 725 730 735 740 HEHIANLAGSPAIKKGILQT Cas9 725 730 735 740 HEHIANLAGSPAIKKGILQT Cas9 HEHIANLAGSPAIKKGILQT CSCMALRAPLGAMLFPMRCV VLVNGIQGAAGGNLLADQLC c a c g a g c a c a t t g c c a a t c t g g c c g g c a g c c c c g c c a t t a a g a a g g g c a t c c t g c a g a c a g t g c t c g t g t a a c g g t t a g a c c g g c c g t c g g g g c g g t a a t t c t t c c c g t a g g a c g t c t g t

4860 745 750 755 760 VKVVDELVKVMGRHKPENIV Cas9 745 750 755 760 VKVVDELVKVMGRHKPENIV Cas9 VKVVDELVKVMGRHKPENIV TFTTSSSTFTIPRCLGSFMT HLHHVLEHFHHAPVLGLVDH g t g a a g g t g g t g g a c g a g c t c g t g a a a g t g a t g g g c c g g c a c a a g c c c g a g a a c a t c g t g c a c t t c c a c c a c c t g c t c g a g c a c t t t c a c t a c c c g g c c g t g t t c g g g c t c t t g t a g c a c

4920 765 770 775 780 IEMARENQTTQKGQKNSRER Cas9 765 770 775 780 IEMARENQTTQKGQKNSRER Cas9 IEMARENQTTQKGQKNSRER ISIALSFWVVWFPCFFLRSL DFHGSLVLGGLLSLLVAALS a t c g a a a t g g c c a g a g a g a a c c a g a c c a c c c a g a a g g g a c a g a a g a a c a g c c g c g a g a g a t a g c t t t a c c g g t c t c t c t t g g t c t g g t g g g t c t t c c c t g t c t t c t t g t c g g c g c t c t c t

4980 785 790 795 800 MKRIEEGIKELGSQILKEHP Cas9 785 790 795 800 MKRIEEGIKELGSQILKEHP Cas9 MKRIEEGIKELGSQILKEHP IFRISSPM HLPDFLADFLQAALDQFFVG a t g a a g c g g a t c g a a g a g g g c a t c a a a g a g c t g g g c a g c c a g a t c c t g a a a g a a c a c c c c t a c t t c g c c t a g c t t c t c c c g t a g t t t c t c g a c c c g t c g g t c t a g g a c t t t c t t g t g g g g

5040 805 810 815 820 VENTQLQNEKLYLYYLQNGR Cas9 805 810 815 820 VENTQLQNEKLYLYYLQNGR Cas9 VENTQLQNEKLYLYYLQNGR HFVGLQLVLLQVQVVQLIPP g t g g a a a a c a c c c a g c t g c a g a a c g a g a a g c t g t a c c t g t a c t a c c t g c a g a a t g g g c g g c a c c t t t t g t g g g t c g a c g t c t t g c t c t t c g a c a t g g a c a t g a t g g a c g t c t t a c c c g c c

5100 825 830 835 840 DMYVDQELDINRLSDYDVDH Cas9 825 830 835 840 DMYVDQELDINRLSDYDVDH Cas9 DMYVDQELDINRLSDYDVDH IHVHVLFQVDVPQGVVIHVM g a t a t g t a c g t g g a c c a g g a a c t g g a c a t c a a c c g g c t g t c c g a c t a c g a t g t g g a c c a t c t a t a c a t g c a c c t g g t c c t t g a c c t g t a g t t g g c c g a c a g g c t g a t g c t a c a c c t g g t a

5160 845 850 855 860 IVPQSFLKDDSIDNKVLTRS Cas9 845 850 855 860 IVPQSFLKDDSIDNKVLTRS Cas9 IVPQSFLKDDSIDNKVLTRS a t c g t g c c t c a g a g c t t t c t g a a g g a c g a c t c c a t c g a c a a c a a g g t g c t g a c c a g a a g c t a g c a c g g a g t c t c g a a a g a c t t c c t g c t g a g g t a g c t g t t g t t c c a c g a c t g g t c t t c g

5220 865 870 875 880 DKNRGKSDNVPSEEVVKKMK Cas9 865 870 875 880 DKNRGKSDNVPSEEVVKKMK Cas9 DKNRGKSDNVPSEEVVKKMK g a c a a g a a c c g g g g c a a g a g c g a c a a c g t g c c c t c c g a a g a g g t c g t g a a g a a g a t g a a g c t g t t c t t g g c c c c g t t c t c g c t g t t g c a c g g g a g g c t t c t c c a g c a c t t c t t c t a c t t c

5280 885 890 895 900 NYWRQLLNAKLITQRKFDNL Cas9 885 890 895 900 NYWRQLLNAKLITQRKFDNL Cas9 NYWRQLLNAKLITQRKFDNL a a c t a c t g g c g g c a g c t g c t g a a c g c c a a g c t g a t t a c c c a g a g a a a g t t c g a c a a t c t g t t g a t g a c c g c c g t c g a c g a c t t g c g g t t c g a c t a a t g g g t c t c t t t c a a g c t g t t a g a c

5340 905 910 915 920 TKAERGGLSELDKAGFIKRQ Cas9 905 910 915 920 TKAERGGLSELDKAGFIKRQ Cas9 TKAERGGLSELDKAGFIKRQ a c c a a g g c c g a g a g a g g c g g c c t g a g c g a a c t g g a t a a g g c c g g c t t c a t c a a g a g a c a g t g g t t c c g g c t c t c t c c g c c g g a c t c g c t t g a c c t a t t c c g g c c g a a g t a g t t c t c t g t c

PmlI 5400 925 930 935 940 LVETRQITKHVAQILDSRMN Cas9 925 930 935 940 LVETRQITKHVAQILDSRMN Cas9 LVETRQITKHVAQILDSRMN c t g g t g g a a a c c c g g c a g a t c a c a a a g c a c g t g g c a c a g a t c c t g g a c t c c c g g a t g a a c g a c c a c c t t t g g g c c g t c t a g t g t t t c g t g c a c c g t g t c t a g g a c c t g a g g g c c t a c t t g

5460 945 950 955 960 TKYDENDKLIREVKVITLKS Cas9 945 950 955 960 TKYDENDKLIREVKVITLKS Cas9 TKYDENDKLIREVKVITLKS a c t a a g t a c g a c g a g a a t g a c a a g c t g a t c c g g g a a g t g a a a g t g a t c a c c c t g a a g t c c t g a t t c a t g c t g c t c t t a c t g t t c g a c t a g g c c c t t c a c t t t c a c t a g t g g g a c t t c a g g

5520 965 970 975 980 KLVSDFRKDFQFYKVREINN Cas9 965 970 975 980 KLVSDFRKDFQFYKVREINN Cas9 KLVSDFRKDFQFYKVREINN a a g c t g g t g t c c g a t t t c c g g a a g g a t t t c c a g t t t t a c a a a g t g c g c g a g a t c a a c a a c t t c g a c c a c a g g c t a a a g g c c t t c c t a a a g g t c a a a a t g t t t c a c g c g c t c t a g t t g t t g

5580 985 990 995 1000 YHHAHDAYLNAVVGTALIKK Cas9 985 990 995 1000 YHHAHDAYLNAVVGTALIKK Cas9 YHHAHDAYLNAVVGTALIKK t a c c a c c a c g c c c a c g a c g c c t a c c t g a a c g c c g t c g t g g g a a c c g c c c t g a t c a a a a a g a t g g t g g t g c g g g t g c t g c g g a t g g a c t t g c g g c a g c a c c c t t g g c g g g a c t a g t t t t t c

5640 1005 1010 1015 1020 YPKLESEFVYGDYKVYDVRK Cas9 1005 1010 1015 1020 YPKLESEFVYGDYKVYDVRK Cas9 YPKLESEFVYGDYKVYDVRK t a c c c t a a g c t g g a a a g c g a g t t c g t g t a c g g c g a c t a c a a g g t g t a c g a c g t g c g g a a g a t g g g a t t c g a c c t t t c g c t c a a g c a c a t g c c g c t g a t g t t c c a c a t g c t g c a c g c c t t c

5700 1025 1030 1035 1040 MIAKSEQEIGKATAKYFFYS Cas9 1025 1030 1035 1040 MIAKSEQEIGKATAKYFFYS Cas9 MIAKSEQEIGKATAKYFFYS a t g a t c g c c a a g a g c g a g c a g g a a a t c g g c a a g g c t a c c g c c a a g t a c t t c t t c t a c a g c t a c t a g c g g t t c t c g c t c g t c c t t t a g c c g t t c c g a t g g c g g t t c a t g a a g a a g a t g t c g

5760 1045 1050 1055 1060 NIMNFFKTEITLANGEIRKR Cas9 1045 1050 1055 1060 NIMNFFKTEITLANGEIRKR Cas9 NIMNFFKTEITLANGEIRKR a a c a t c a t g a a c t t t t t c a a g a c c g a g a t t a c c c t g g c c a a c g g c g a g a t c c g g a a g c g g t t g t a g t a c t t g a a a a a g t t c t g g c t c t a a t g g g a c c g g t t g c c g c t c t a g g c c t t c g c c

5820 1065 1070 1075 1080 PLIETNGETGEIVWDKGRDF Cas9 1065 1070 1075 1080 PLIETNGETGEIVWDKGRDF Cas9 PLIETNGETGEIVWDKGRDF c c t c t g a t c g a g a c a a a c g g c g a a a c c g g g g a g a t c g t g t g g g a t a a g g g c c g g g a t t t t g g a g a c t a g c t c t g t t t g c c g c t t t g g c c c c t c t a g c a c a c c c t a t t c c c g g c c c t a a a a

5880 1085 1090 1095 1100 ATVRKVLSMPQVNIVKKTEV Cas9 1085 1090 1095 1100 ATVRKVLSMPQVNIVKKTEV Cas9 ATVRKVLSMPQVNIVKKTEV g c c a c c g t g c g g a a a g t g c t g a g c a t g c c c c a a g t g a a t a t c g t g a a a a a g a c c g a g g t g c g g t g g c a c g c c t t t c a c g a c t c g t a c g g g g t t c a c t t a t a g c a c t t t t t c t g g c t c c a c

5940 1105 1110 1115 1120 QTGGFSKESILPKRNSDKLI Cas9 1105 1110 1115 1120 QTGGFSKESILPKRNSDKLI Cas9 QTGGFSKESILPKRNSDKLI c a g a c a g g c g g c t t c a g c a a a g a g t c t a t c c t g c c c a a g a g g a a c a g c g a t a a g c t g a t c g t c t g t c c g c c g a a g t c g t t t c t c a g a t a g g a c g g g t t c t c c t t g t c g c t a t t c g a c t a g

KflI 6000 1125 1130 1135 1140 ARKKDWDPKKYGGFDSPTVA Cas9 1125 1130 1135 1140 ARKKDWDPKKYGGFDSPTVA Cas9 ARKKDWDPKKYGGFDSPTVA g c c a g a a a g a a g g a c t g g g a c c c t a a g a a g t a c g g c g g c t t c g a c a g c c c c a c c g t g g c c c g g t c t t t c t t c c t g a c c c t g g g a t t c t t c a t g c c g c c g a a g c t g t c g g g g t g g c a c c g g

6060 1145 1150 1155 1160 YSVLVVAKVEKGKSKKLKSV Cas9 1145 1150 1155 1160 YSVLVVAKVEKGKSKKLKSV Cas9 YSVLVVAKVEKGKSKKLKSV t a t t c t g t g c t g g t g g t g g c c a a a g t g g a a a a g g g c a a g t c c a a g a a a c t g a a g a g t g t g a t a a g a c a c g a c c a c c a c c g g t t t c a c c t t t t c c c g t t c a g g t t c t t t g a c t t c t c a c a c

BsaBI* 6120 1165 1170 1175 1180 KELLGITIMERSSFEKNPID Cas9 1165 1170 1175 1180 KELLGITIMERSSFEKNPID Cas9 KELLGITIMERSSFEKNPID a a a g a g c t g c t g g g g a t c a c c a t c a t g g a a a g a a g c a g c t t c g a g a a g a a t c c c a t c g a c t t t c t c g a c g a c c c c t a g t g g t a g t a c c t t t c t t c g t c g a a g c t c t t c t t a g g g t a g c t g

6180 1185 1190 1195 1200 FLEAKGYKEVKKDLIIKLPK Cas9 1185 1190 1195 1200 FLEAKGYKEVKKDLIIKLPK Cas9 FLEAKGYKEVKKDLIIKLPK t t t c t g g a a g c c a a g g g c t a c a a a g a a g t g a a a a a g g a c c t g a t c a t c a a g c t g c c t a a g a a a g a c c t t c g g t t c c c g a t g t t t c t t c a c t t t t t c c t g g a c t a g t a g t t c g a c g g a t t c

BsmI 6240 1205 1210 1215 1220 YSLFELENGRKRMLASAGEL Cas9 1205 1210 1215 1220 YSLFELENGRKRMLASAGEL Cas9 YSLFELENGRKRMLASAGEL t a c t c c c t g t t c g a g c t g g a a a a c g g c c g g a a g a g a a t g c t g g c c t c t g c c g g c g a a c t g a t g a g g g a c a a g c t c g a c c t t t t g c c g g c c t t c t c t t a c g a c c g g a g a c g g c c g c t t g a c

6300 1225 1230 1235 1240 QKGNELALPSKYVNFLYLAS Cas9 1225 1230 1235 1240 QKGNELALPSKYVNFLYLAS Cas9 QKGNELALPSKYVNFLYLAS c a g a a g g g a a a c g a a c t g g c c c t g c c c t c c a a a t a t g t g a a c t t c c t g t a c c t g g c c a g c g t c t t c c c t t t g c t t g a c c g g g a c g g g a g g t t t a t a c a c t t g a a g g a c a t g g a c c g g t c g

6360 1245 1250 1255 1260 HYEKLKGSPEDNEQKQLFVE Cas9 1245 1250 1255 1260 HYEKLKGSPEDNEQKQLFVE Cas9 HYEKLKGSPEDNEQKQLFVE c a c t a t g a g a a g c t g a a g g g c t c c c c c g a g g a t a a t g a g c a g a a a c a g c t g t t t g t g g a a g t g a t a c t c t t c g a c t t c c c g a g g g g g c t c c t a t t a c t c g t c t t t g t c g a c a a a c a c c t t

6420 1265 1270 1275 1280 QHKHYLDEIIEQISEFSKRV Cas9 1265 1270 1275 1280 QHKHYLDEIIEQISEFSKRV Cas9 QHKHYLDEIIEQISEFSKRV c a g c a c a a g c a c t a c c t g g a c g a g a t c a t c g a g c a g a t c a g c g a g t t c t c c a a g a g a g t g g t c g t g t t c g t g a t g g a c c t g c t c t a g t a g c t c g t c t a g t c g c t c a a g a g g t t c t c t c a c

6480 1285 1290 1295 1300 ILADANLDKVLSAYNKHRDK Cas9 1285 1290 1295 1300 ILADANLDKVLSAYNKHRDK Cas9 ILADANLDKVLSAYNKHRDK a t c c t g g c c g a c g c t a a t c t g g a c a a a g t g c t g t c c g c c t a c a a c a a g c a c c g g g a t a a g t a g g a c c g g c t g c g a t t a g a c c t g t t t c a c g a c a g g c g g a t g t t g t t c g t g g c c c t a t t c

6540 1305 1310 1315 1320 PIREQAENIIHLFTLTNLGA Cas9 1305 1310 1315 1320 PIREQAENIIHLFTLTNLGA Cas9 PIREQAENIIHLFTLTNLGA c c c a t c a g a g a g c a g g c c g a g a a t a t c a t c c a c c t g t t t a c c c t g a c c a a t c t g g g a g c c g g g t a g t c t c t c g t c c g g c t c t t a t a g t a g g t g g a c a a a t g g g a c t g g t t a g a c c c t c g g

6600 1325 1330 1335 1340 PAAFKYFDTTIDRKRYTSTK Cas9 1325 1330 1335 1340 PAAFKYFDTTIDRKRYTSTK Cas9 PAAFKYFDTTIDRKRYTSTK c c t g c c g c c t t c a a g t a c t t t g a c a c c a c c a t c g a c c g g a a g a g g t a c a c c a g c a c c a a a g g a c g g c g g a a g t t c a t g a a a c t g t g g t g g t a g c t g g c c t t c t c c a t g t g g t c g t g g t t t

6660 1345 1350 1355 1360 EVLDATLIHQSITGLYETRI Cas9 1345 1350 1355 1360 EVLDATLIHQSITGLYETRI Cas9 EVLDATLIHQSITGLYETRI g a g g t g c t g g a c g c c a c c c t g a t c c a c c a g a g c a t c a c c g g c c t g t a c g a g a c a c g g a t c c t c c a c g a c c t g c g g t g g g a c t a g g t g g t c t c g t a g t g g c c g g a c a t g c t c t g t g c c t a g

6720 1365 DLSQLGGD (in frame with Cas9) KRPAATKKAGQA Cas9 1365 DLSQLGGD 1 5 10 KRPAATKKAGQA Cas9 nucleoplasmin NLS DLSQLGGDKRPAATKKAGQA g a c c t g t c t c a g c t g g g a g g c g a c a a a a g g c c g g c g g c c a c g a a a a a g g c c g g c c a g g c a c t g g a c a g a g t c g a c c c t c c g c t g t t t t c c g g c c g c c g g t g c t t t t t c c g g c c g g t c c g t

6780 (in frame with Cas9) KKKKEFGSGEGRGSLLTCGD 15 KKKK EFGSG 140 145 EGRGSLLTCGD nucleoplasmin NLS T2A KKKKEFGSGEGRGSLLTCGD a a a a a g a a a a a g g a a t t c g g c a g t g g a g a g g g c a g a g g a a g t c t g c t a a c a t g c g g t g a c t t t t t c t t t t t c c t t a a g c c g t c a c c t c t c c c g t c t c c t t c a g a c g a t t g t a c g c c a c t g

6840 (in frame with Cas9) VEENPGPMPEPSKSAPAPKK 150 155 VEENPGP 1 5 10 MPEPSKSAPAPKK T2A H2B VEENPGPMPEPSKSAPAPKK g t c g a g g a g a a t c c t g g c c c a a t g c c t g a a c c c t c t a a g t c t g c t c c a g c c c c t a a a a a g c a g c t c c t c t t a g g a c c g g g t t a c g g a c t t g g g a g a t t c a g a c g a g g t c g g g g a t t t t t c

6900 (in frame with Cas9) GSKKAITKAQKKDGKKRKRS 15 20 25 30 GSKKAITKAQKKDGKKRKRS H2B GSKKAITKAQKKDGKKRKRS g g t t c t a a g a a g g c t a t c a c t a a g g c g c a g a a g a a g g a t g g t a a g a a g c g t a a g c g c a g c c c a a g a t t c t t c c g a t a g t g a t t c c g c g t c t t c t t c c t a c c a t t c t t c g c a t t c g c g t c g

6960 (in frame with Cas9) RKESYSIYVYKVLKQVHPDT 35 40 45 50 RKESYSIYVYKVLKQVHPDT H2B RKESYSIYVYKVLKQVHPDT c g c a a g g a g a g c t a t t c t a t c t a t g t g t a c a a g g t t c t g a a g c a g g t c c a c c c c g a c a c c g c g t t c c t c t c g a t a a g a t a g a t a c a c a t g t t c c a a g a c t t c g t c c a g g t g g g g c t g t g g

7020 (in frame with Cas9) GISSKAMGIMNSFVNDIFER 55 60 65 70 GISSKAMGIMNSFVNDIFER H2B GISSKAMGIMNSFVNDIFER g g c a t c t c a t c c a a g g c c a t g g g g a t c a t g a a c t c c t t c g t c a a c g a c a t c t t c g a g c g c c c g t a g a g t a g g t t c c g g t a c c c c t a g t a c t t g a g g a a g c a g t t g c t g t a g a a g c t c g c g

AfeI 7080 (in frame with Cas9) IAGEASRLAHYNKRSTITSR 75 80 85 90 IAGEASRLAHYNKRSTITSR H2B IAGEASRLAHYNKRSTITSR a t c g c g g g c g a g g c t t c t c g c c t g g c t c a c t a c a a t a a g c g c t c g a c c a t c a c c t c c a g g t a g c g c c c g c t c c g a a g a g c g g a c c g a g t g a t g t t a t t c g c g a g c t g g t a g t g g a g g t c c

7140 (in frame with Cas9) EIQTAVRLLLPGELAKHAVS 95 100 105 110 EIQTAVRLLLPGELAKHAVS H2B EIQTAVRLLLPGELAKHAVS g a g a t t c a g a c g g c t g t g c g c c t g c t g c t g c c t g g g g a g c t g g c t a a g c a t g c t g t g t c c c t c t a a g t c t g c c g a c a c g c g g a c g a c g a c g g a c c c c t c g a c c g a t t c g t a c g a c a c a g g

BamHI 7200 (in frame with Cas9) EGTKAVTKYTSSKDPPVATM 115 120 125 EGTKAVTKYTSSK (in frame with H2B) DPPVAT 1 M H2B mCherry Kozak sequence EGTKAVTKYTSSKDPPVATM g a g g g c a c t a a g g c a g t t a c c a a g t a c a c t a g c t c t a a g g a t c c a c c g g t c g c c a c c a t g c t c c c g t g a t t c c g t c a a t g g t t c a t g t g a t c g a g a t t c c t a g g t g g c c a g c g g t g g t a c

7260 (in frame with Cas9) VSKGEEDNMAIIKEFMRFKV 5 10 15 20 VSKGEEDNMAIIKEFMRFKV mCherry Kozak sequence VSKGEEDNMAIIKEFMRFKV g t g a g c a a g g g c g a g g a g g a t a a c a t g g c c a t c a t c a a g g a g t t c a t g c g c t t c a a g g t g c a c t c g t t c c c g c t c c t c c t a t t g t a c c g g t a g t a g t t c c t c a a g t a c g c g a a g t t c c a c

7320 (in frame with Cas9) HMEGSVNGHEFEIEGEGEGR 25 30 35 40 HMEGSVNGHEFEIEGEGEGR mCherry HMEGSVNGHEFEIEGEGEGR c a c a t g g a g g g c t c c g t g a a c g g c c a c g a g t t c g a g a t c g a g g g c g a g g g c g a g g g c c g c g t g t a c c t c c c g a g g c a c t t g c c g g t g c t c a a g c t c t a g c t c c c g c t c c c g c t c c c g g c g

7380 (in frame with Cas9) PYEGTQTAKLKVTKGGPLPF 45 50 55 60 PYEGTQTAKLKVTKGGPLPF mCherry G G T T C G A C T T C C A C T G G T T mCherry-R PYEGTQTAKLKVTKGGPLPF c c c t a c g a g g g c a c c c a g a c c g c c a a g c t g a a g g t g a c c a a g g g t g g c c c c c t g c c c t t c g g g a t g c t c c c g t g g g t c t g g c g g t t c g a c t t c c a c t g g t t c c c a c c g g g g g a c g g g a a g

7440 (in frame with Cas9) AWDILSPQFMYGSKAYVKHP 65 70 75 80 AWDILSPQFMYGSKAYVKHP mCherry AWDILSPQFMYGSKAYVKHP g c c t g g g a c a t c c t g t c c c c t c a g t t c a t g t a c g g c t c c a a g g c c t a c g t g a a g c a c c c c c g g a c c c t g t a g g a c a g g g g a g t c a a g t a c a t g c c g a g g t t c c g g a t g c a c t t c g t g g g g

7500 (in frame with Cas9) ADIPDYLKLSFPEGFKWERV 85 90 95 100 ADIPDYLKLSFPEGFKWERV mCherry ADIPDYLKLSFPEGFKWERV g c c g a c a t c c c c g a c t a c t t g a a g c t g t c c t t c c c c g a g g g c t t c a a g t g g g a g c g c g t g c g g c t g t a g g g g c t g a t g a a c t t c g a c a g g a a g g g g c t c c c g a a g t t c a c c c t c g c g c a c

7560 (in frame with Cas9) MNFEDGGVVTVTQDSSLQDG 105 110 115 120 MNFEDGGVVTVTQDSSLQDG mCherry MNFEDGGVVTVTQDSSLQDG a t g a a c t t c g a g g a c g g c g g c g t g g t g a c c g t g a c c c a g g a c t c c t c c c t g c a g g a c g g c t a c t t g a a g c t c c t g c c g c c g c a c c a c t g g c a c t g g g t c c t g a g g a g g g a c g t c c t g c c g

7620 (in frame with Cas9) EFIYKVKLRGTNFPSDGPVM 125 130 135 140 EFIYKVKLRGTNFPSDGPVM mCherry EFIYKVKLRGTNFPSDGPVM g a g t t c a t c t a c a a g g t g a a g c t g c g c g g c a c c a a c t t c c c c t c c g a c g g c c c c g t a a t g c t c a a g t a g a t g t t c c a c t t c g a c g c g c c g t g g t t g a a g g g g a g g c t g c c g g g g c a t t a c

7680 (in frame with Cas9) QKKTMGWEASSERMYPEDGA 145 150 155 160 QKKTMGWEASSERMYPEDGA mCherry QKKTMGWEASSERMYPEDGA c a g a a g a a a a c c a t g g g c t g g g a g g c c t c c t c c g a g c g g a t g t a c c c c g a g g a c g g c g c c g t c t t c t t t t g g t a c c c g a c c c t c c g g a g g a g g c t c g c c t a c a t g g g g c t c c t g c c g c g g

7740 (in frame with Cas9) LKGEIKQRLKLKDGGHYDAE 165 170 175 180 LKGEIKQRLKLKDGGHYDAE mCherry LKGEIKQRLKLKDGGHYDAE c t g a a g g g c g a g a t c a a g c a g a g g c t g a a g c t g a a g g a c g g c g g c c a c t a c g a c g c t g a g g a c t t c c c g c t c t a g t t c g t c t c c g a c t t c g a c t t c c t g c c g c c g g t g a t g c t g c g a c t c

7800 (in frame with Cas9) VKTTYKAKKPVQLPGAYNVN 185 190 195 200 VKTTYKAKKPVQLPGAYNVN mCherry VKTTYKAKKPVQLPGAYNVN g t c a a g a c c a c c t a c a a g g c c a a g a a g c c c g t g c a g c t g c c c g g c g c c t a c a a c g t c a a c c a g t t c t g g t g g a t g t t c c g g t t c t t c g g g c a c g t c g a c g g g c c g c g g a t g t t g c a g t t g

7860 (in frame with Cas9) IKLDITSHNEDYTIVEQYER 205 210 215 220 IKLDITSHNEDYTIVEQYER mCherry IKLDITSHNEDYTIVEQYER a t c a a g t t g g a c a t c a c c t c c c a c a a c g a g g a c t a c a c c a t c g t g g a a c a g t a c g a a c g c t a g t t c a a c c t g t a g t g g a g g g t g t t g c t c c t g a t g t g g t a g c a c c t t g t c a t g c t t g c g

SgrAI 7920 (in frame with Cas9) AEGRHSTGGMDELYKEF* 225 230 235 AEGRHSTGGMDELYK EF* mCherry AEGRHSTGGMDELYKEF* g c c g a g g g c c g c c a c t c c a c c g g c g g c a t g g a c g a g c t g t a c a a g g a a t t c t a a c t a g a g c g g c t c c c g g c g g t g a g g t g g c c g c c g t a c c t g c t c g a c a t g t t c c t t a a g a t t g a t c t c

7980 bGH poly(A) signal G G A G C T G A C A C G G A A G A T BGH-rev c t c g c t g a t c a g c c t c g a c t g t g c c t t c t a g t t g c c a g c c a t c t g t t g t t t g c c c c t c c c g a g c g a c t a g t c g g a g c t g a c a c g g a a g a t c a a c g g t c g g t a g a c a a c a a a c g g g g a g g g

8040 bGH poly(A) signal c c g t g c c t t c c t t g a c c c t g g a a g g t g c c a c t c c c a c t g t c c t t t c c t a a t a a a a t g a g g g g c a c g g a a g g a a c t g g g a c c t t c c a c g g t g a g g g t g a c a g g a a a g g a t t a t t t t a c t c c

8100 bGH poly(A) signal a a a t t g c a t c g c a t t g t c t g a g t a g g t g t c a t t c t a t t c t g g g g g g t g g g g t g g g g c a g g t t t a a c g t a g c g t a a c a g a c t c a t c c a c a g t a a g a t a a g a c c c c c c a c c c c a c c c c g t c c

NotI 8160 bGH poly(A) signal AAV2 ITR AAV2 ITR a c a g c a a g g g g g a g g a t t g g g a a g a g a a t a g c a g g c a t g c t g g g g a g c g g c c g c a g g a a c t g t c g t t c c c c c t c c t a a c c c t t c t c t t a t c g t c c g t a c g a c c c c t c g c c g g c g t c c t t g

8220 AAV2 ITR AAV2 ITR c c c t a g t g a t g g a g t t g g c c a c t c c c t c t c t g c g c g c t c g c t c g c t c a c t g a g g c c g g g c g g g a t c a c t a c c t c a a c c g g t g a g g g a g a g a c g c g c g a g c g a g c g a g t g a c t c c g g c c c g

8280 AAV2 ITR AAV2 ITR g a c c a a a g g t c g c c c g a c g c c c g g g c t t t g c c c g g g c g g c c t c a g t g a g c g a g c g a g c g c c t g g t t t c c a g c g g g c t g c g g g c c c g a a a c g g g c c c g c c g g a g t c a c t c g c t c g c t c g c g

8340 AAV2 ITR AAV2 ITR g c a g c t g c c t g c a g g g g c g c c t g a t g c g g t a t t t t c t c c t t a c g c a t c t g t g c g g t a t t t c g t c g a c g g a c g t c c c c g c g g a c t a c g c c a t a a a a g a g g a a t g c g t a g a c a c g c c a t a a a

8400 f1 ori c a c a c c g c a t a c g t c a a a g c a a c c a t a g t a c g c g c c c t g t a g c g g c g c a t t a a g c g c g g c g t g t g g c g t a t g c a g t t t c g t t g g t a t c a t g c g c g g g a c a t c g c c g c g t a a t t c g c g c c g

8460 f1 ori G A G G F1ori-R g g g t g t g g t g g t t a c g c g c a g c g t g a c c g c t a c a c t t g c c a g c g c c t t a g c g c c c g c t c c c c c a c a c c a c c a a t g c g c g t c g c a c t g g c g a t g t g a a c g g t c g c g g a a t c g c g g g c g a g g

8520 f1 ori A A A G C G A A A G A A G G G A F1ori-R t t t c g c t t t c t t c c c t t c c t t t c t c g c c a c g t t c g c c g g c t t t c c c c g t c a a g c t c t a a a a a a g c g a a a g a a g g g a a g g a a a g a g c g g t g c a a g c g g c c g a a a g g g g c a g t t c g a g a t t t

8580 f1 ori t c g g g g g c t c c c t t t a g g g t t c c g a t t t a g t g c t t t a c g g c a c c t c g a c c c c a a a a a a c t a g c c c c c g a g g g a a a t c c c a a g g c t a a a t c a c g a a a t g c c g t g g a g c t g g g g t t t t t t g a

8640 f1 ori t g a t t t g g g t g a t g g t t c a c g t a g t g g g c c a t c g c c c t g a t a g a c g g t t t t t c g c c c t t t a c t a a a c c c a c t a c c a a g t g c a t c a c c c g g t a g c g g g a c t a t c t g c c a a a a a g c g g g a a a

8700 f1 ori G T G G A C T C T T G T T C C A A A C T G G F1ori-F g a c g t t g g a g t c c a c g t t c t t t a a t a g t g g a c t c t t g t t c c a a a c t g g a a c a a c a c t c a a c t g c a a c c t c a g g t g c a a g a a a t t a t c a c c t g a g a a c a a g g t t t g a c c t t g t t g t g a g t t

PsiI 8760 f1 ori c t c t a t c t c g g g c t a t t c t t t t g a t t t a t a a g g g a t t t t g c c g a t t t c g g t c t a t t g g t t g a g a t a g a g c c c g a t a a g a a a a c t a a a t a t t c c c t a a a a c g g c t a a a g c c a g a t a a c c a a

8820 f1 ori a a a a a a t g a g c t g a t t t a a c a a a a a t t t a a c g c g a a t t t t a a c a a a a t a t t a a c g t t t a c t t t t t t a c t c g a c t a a a t t g t t t t t a a a t t g c g c t t a a a a t t g t t t t a t a a t t g c a a a t g

8880 f1 ori A T G T T A G A C G A G A C T A C G G C pRS-marker a a t t t t a t g g t g c a c t c t c a g t a c a a t c t g c t c t g a t g c c g c a t a g t t a a g c c a g c c c c g t t a a a a t a c c a c g t g a g a g t c a t g t t a g a c g a g a c t a c g g c g t a t c a a t t c g g t c g g g g c

8940 a c a c c c g c c a a c a c c c g c t g a c g c g c c c t g a c g g g c t t g t c t g c t c c c g g c a t c c g c t t a t g t g g g c g g t t g t g g g c g a c t g c g c g g g a c t g c c c g a a c a g a c g a g g g c c g t a g g c g a a t

9000 C C G G G A G C T G C A T G T G T C A G A G G pGEX 3' c a g a c a a g c t g t g a c c g t c t c c g g g a g c t g c a t g t g t c a g a g g t t t t c a c c g t c a t c a c c g t c t g t t c g a c a c t g g c a g a g g c c c t c g a c g t a c a c a g t c t c c a a a a g t g g c a g t a g t g g

9060 C G G A G C A C T A T G C G G A T A A pBRforEco g a a a c g c g c g a g a c g a a a g g g c c t c g t g a t a c g c c t a t t t t t a t a g g t t a a t g t c a t g a t c t t t g c g c g c t c t g c t t t c c c g g a g c a c t a t g c g g a t a a a a a t a t c c a a t t a c a g t a c t a

9120 AmpR promoter a a t a a t g g t t t c t t a g a c g t c a g g t g g c a c t t t t c g g g g a a a t g t g c g c g g a a c c c c t a t t t a t t a c c a a a g a a t c t g c a g t c c a c c g t g a a a a g c c c c t t t a c a c g c g c c t t g g g g a t a

9180 AmpR promoter t t g t t t a t t t t t c t a a a t a c a t t c a a a t a t g t a t c c g c t c a t g a g a c a a t a a c c c t g a t a a a c a a a t a a a a a g a t t t a t g t a a g t t t a t a c a t a g g c g a g t a c t c t g t t a t t g g g a c t a t

9240 1 5 10 signal sequence MSIQHFRVAL AmpR promoter AmpR MSIQHFRVAL a a t g c t t c a a t a a t a t t g a a a a a g g a a g a g t a t g a g t a t t c a a c a t t t c c g t g t c g c c c t t t a c g a a g t t a t t a t a a c t t t t t c c t t c t c a t a c t c a t a a g t t g t a a a g g c a c a g c g g g a

9300 15 20 signal sequence IPFFAAFCLPVFA 25 30 HPETLVK AmpR IPFFAAFCLPVFAHPETLVK t a t t c c c t t t t t t g c g g c a t t t t g c c t t c c t g t t t t t g c t c a c c c a g a a a c g c t g g t g a a a t a a g g g a a a a a a c g c c g t a a a a c g g a a g g a c a a a a a c g a g t g g g t c t t t g c g a c c a c t t

9360 35 40 45 50 VKDAEDQLGARVGYIELDLN AmpR VKDAEDQLGARVGYIELDLN a g t a a a a g a t g c t g a a g a t c a g t t g g g t g c a c g a g t g g g t t a c a t c g a a c t g g a t c t c a a t c a t t t t c t a c g a c t t c t a g t c a a c c c a c g t g c t c a c c c a a t g t a g c t t g a c c t a g a g t t

9420 55 60 65 70 SGKILESFRPEERFPMMSTF AmpR SGKILESFRPEERFPMMSTF c a g c g g t a a g a t c c t t g a g a g t t t t c g c c c c g a a g a a c g t t t t c c a a t g a t g a g c a c t t t g t c g c c a t t c t a g g a a c t c t c a a a a g c g g g g c t t c t t g c a a a a g g t t a c t a c t c g t g a a a

9480 75 80 85 90 KVLLCGAVLSRIDAGQEQLG AmpR C G A T A C A C C G C G C C A T A A T A Amp-R KVLLCGAVLSRIDAGQEQLG t a a a g t t c t g c t a t g t g g c g c g g t a t t a t c c c g t a t t g a c g c c g g g c a a g a g c a a c t c g g a t t t c a a g a c g a t a c a c c g c g c c a t a a t a g g g c a t a a c t g c g g c c c g t t c t c g t t g a g c c

9540 95 100 105 110 RRIHYSQNDLVEYSPVTEKH AmpR RRIHYSQNDLVEYSPVTEKH t c g c c g c a t a c a c t a t t c t c a g a a t g a c t t g g t t g a g t a c t c a c c a g t c a c a g a a a a g c a a g c g g c g t a t g t g a t a a g a g t c t t a c t g a a c c a a c t c a t g a g t g g t c a g t g t c t t t t c g t

9600 115 120 125 130 LTDGMTVRELCSAAITMSDN AmpR LTDGMTVRELCSAAITMSDN t c t t a c g g a t g g c a t g a c a g t a a g a g a a t t a t g c a g t g c t g c c a t a a c c a t g a g t g a t a a a g a a t g c c t a c c g t a c t g t c a t t c t c t t a a t a c g t c a c g a c g g t a t t g g t a c t c a c t a t t

PvuI 9660 135 140 145 150 TAANLLLTTIGGPKELTAFL AmpR TAANLLLTTIGGPKELTAFL c a c t g c g g c c a a c t t a c t t c t g a c a a c g a t c g g a g g a c c g a a g g a g c t a a c c g c t t t t t t g t g a c g c c g g t t g a a t g a a g a c t g t t g c t a g c c t c c t g g c t t c c t c g a t t g g c g a a a a a a

9720 155 160 165 170 HNMGDHVTRLDRWEPELNEA AmpR HNMGDHVTRLDRWEPELNEA g c a c a a c a t g g g g g a t c a t g t a a c t c g c c t t g a t c g t t g g g a a c c g g a g c t g a a t g a a g c c g t g t t g t a c c c c c t a g t a c a t t g a g c g g a a c t a g c a a c c c t t g g c c t c g a c t t a c t t c g

FspI 9780 175 180 185 190 IPNDERDTTMPVAMATTLRK AmpR IPNDERDTTMPVAMATTLRK c a t a c c a a a c g a c g a g c g t g a c a c c a c g a t g c c t g t a g c a a t g g c a a c a a c g t t g c g c a a g t a t g g t t t g c t g c t c g c a c t g t g g t g c t a c g g a c a t c g t t a c c g t t g t t g c a a c g c g t t

9840 195 200 205 210 LLTGELLTLASRQQLIDWME AmpR LLTGELLTLASRQQLIDWME a c t a t t a a c t g g c g a a c t a c t t a c t c t a g c t t c c c g g c a a c a a t t a a t a g a c t g g a t g g a t g a t a a t t g a c c g c t t g a t g a a t g a g a t c g a a g g g c c g t t g t t a a t t a t c t g a c c t a c c t

9900 215 220 225 230 ADKVAGPLLRSALPAGWFIA AmpR ADKVAGPLLRSALPAGWFIA g g c g g a t a a a g t t g c a g g a c c a c t t c t g c g c t c g g c c c t t c c g g c t g g c t g g t t t a t t g c c c g c c t a t t t c a a c g t c c t g g t g a a g a c g c g a g c c g g g a a g g c c g a c c g a c c a a a t a a c g

9960 235 240 245 250 DKSGAGERGSRGIIAALGPD AmpR DKSGAGERGSRGIIAALGPD t g a t a a a t c t g g a g c c g g t g a g c g t g g a a g c c g c g g t a t c a t t g c a g c a c t g g g g c c a g a a c t a t t t a g a c c t c g g c c a c t c g c a c c t t c g g c g c c a t a g t a a c g t c g t g a c c c c g g t c t

10,020 255 260 265 270 GKPSRIVVIYTTGSQATMDE AmpR GKPSRIVVIYTTGSQATMDE t g g t a a g c c c t c c c g t a t c g t a g t t a t c t a c a c g a c g g g g a g t c a g g c a a c t a t g g a t g a a c c a t t c g g g a g g g c a t a g c a t c a a t a g a t g t g c t g c c c c t c a g t c c g t t g a t a c c t a c t

10,080 275 280 285 RNRQIAEIGASLIKHW* AmpR RNRQIAEIGASLIKHW* a c g a a a t a g a c a g a t c g c t g a g a t a g g t g c c t c a c t g a t t a a g c a t t g g t a a c t g t c a g a t g c t t t a t c t g t c t a g c g a c t c t a t c c a c g g a g t g a c t a a t t c g t a a c c a t t g a c a g t c t

10,140 c c a a g t t t a c t c a t a t a t a c t t t a g a t t g a t t t a a a a c t t c a t t t t t a a t t t a a a a g g a t g g t t c a a a t g a g t a t a t a t g a a a t c t a a c t a a a t t t t g a a g t a a a a a t t a a a t t t t c c t a

10,200 c t a g g t g a a g a t c c t t t t t g a t a a t c t c a t g a c c a a a a t c c c t t a a c g t g a g t t t t c g t t g a t c c a c t t c t a g g a a a a a c t a t t a g a g t a c t g g t t t t a g g g a a t t g c a c t c a a a a g c a a

3ʹ 5ʹ 10,219 c c a c t g a g c g t c a g a c c c c g g t g a c t c g c a g t c t g g g g

Restriction Enzymes

Instructions: By default, all cutters are shown. Filter on number of cut sites or search by enzyme name.

Filter

Features

Primers

BLAST

BLAST (Basic Local Alignment Search Tool) finds regions of similarity between biological sequences. Click on the buttons below to submit a BLAST search to NCBI. The results will appear in a new window. See your recent BLAST results on NCBI's website.

  • Nucleotide-Nucleotide BLAST (BLASTN)

  • Translated Nucleotide-Protein BLAST (BLASTX)

  • Sequence alignment using BLAST (BLAST2)

Sequence Analyzer Guide

Map

Displays a graphical map based on nucleotide sequence data labeled with restriction enzymes, plasmid features, ORFs (theoretical open reading frames) and primers. Hovering over data labels will display additional information (e.g. cut site)

To select a portion of sequence, click one location on the plasmid and then a second location to display the sequence between the two locations.

Sequence

Displays both strands of base paired nucleotide sequences with annotated enzymes, plasmid features, ORFs (theoretical open reading frames) and primers. Hovering over data labels will display additional information (e.g. cut site).

To select a portion of sequence, click one location on the sequence and then a second location to display the sequence between the two locations.

Enzymes

List of restriction enzymes that can cut a given nucleotide sequence. Table lists enzyme name and the sequence location of the cut.

Features

List of common features detected in a given nucleotide sequence. Table lists feature name, location, size, color used to indicate its position on the map, and direction (if relevant).

Primers

List of commonly used primers detected in a given nucleotide sequence. Table lists primer name, sequence, length, binding site location, and direction.

BLAST

Use Basic Local Alignment Search Tool (BLAST) via the NCBI website to determine similarity between a given sequence and nucleotide (BLASTN) or protein (BLASTX) sequence databases. Additionally, align a custom nucleotide sequence against a given sequence using BLAST2.

File Downloads

GenBank

File contains the nucleotide sequence and annotated features in GenBank flat file format. Open the file with a text editor or plasmid mapping software to view the sequence.

SnapGene

File contains the nucleotide sequence and enhanced annotations from SnapGene Server. Open the file with SnapGene software or the free Viewer to view the plasmid map, sequence, and perform additional sequence analysis.