Addgene pXCas9H840A - Sequence Analyzer Skip to main content
Addgene

Sequence Analyzer: pXCas9H840A


Map View

AflIII - PciI (8501) pBR322ori-F (8346 .. 8365) SacII (7548) FspI (7390) PvuI (7244) Amp-R (7044 .. 7063) pBRforEco (6635 .. 6653) pGEX 3' (6575 .. 6597) pRS-marker (6456 .. 6475) PsiI (6342) F1ori-F (6281 .. 6302) F1ori-R (6071 .. 6090) PluTI (5914) SfoI (5912) NarI (5911) KasI (5910) SbfI (5907) NotI (5762) BGH-rev (5547 .. 5564) EcoRI (5523) FseI (5500) nucleoplasmin NLS BsmI (5008) BsaBI * (4866) KflI (4745) hU6-F (1 .. 21) LKO.1 5' (172 .. 191) XbaI (428) Acc65I (434) KpnI (438) SnaBI (701) chicken β-actin promoter BseRI (1112) PaqCI (1217) AgeI (1239) SV40 NLS BglII (1618) PspOMI (2723) ApaI (2727) PasI (3137) EcoRV (3221) PmlI (4157) pXCas9H840A 8506 bp

Sequence View

60 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 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 t a c a a g g c t g t t a g a g a g 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 t a t g t t c c g a c a a t c t c t c

120 U6 promoter 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 a c a a a a t a c g t g a c g t a g a 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 t g t t t t a t g c a c t g c a t c t

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

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

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

360 gRNA scaffold 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 g c t t t t t t g t t t t a g a g c t 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 c g a a a a a a c a a a a t c t c g a

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

XbaIAcc65IKpnI 480 CMV enhancer a a a t g g c t c t a g a g g t a c c c g t t a c a t a a c t t a c g g t a a a t g g c c c g c c t g g c t g a c c g c t t t a c c g a g a t c t c c a t g g g c a a t g t a t t g a a t g c c a t t t a c c g g g c g g a c c g a c t g g c g

540 CMV enhancer c c a a c g a c c c c c g c c c a t t g a c g t c a a t a g t a a c g c c a a t a g g g a c t t t c c a t t g a c g t c g g t t g c t g g g g g c g g g t a a c t g c a g t t a t c a t t g c g g t t a t c c c t g a a a g g t a a c t g c a g

600 CMV enhancer a a t g g g t g g a g t a t t t a c g g t a a a c t g c c c a c t t g g c a g t a c a t c a a g t g t a t c a t a t g c t t a c c c a c c t c a t a a a t g c c a t t t g a c g g g t g a a c c g t c a t g t a g t t c a c a t a g t a t a c g

660 CMV enhancer c a a g t a c g c c c c c t a t t g a c g t c a a t g a c g g t a a a t g g c c c g c c t g g c a t t g t g c c c a g t g t t c a t g c g g g g g a t a a c t g c a g t t a c t g c c a t t t a c c g g g c g g a c c g t a a c a c g g g t c a

SnaBI 720 CMV enhancer a c a t g a c c t t a t g g g a c t t t c c t a c t t g g c a g t a c a t c t a c g t a t t a g t c a t c g c t a t t a t g t a c t g g a a t a c c c t g a a a g g a t g a a c c g t c a t g t a g a t g c a t a a t c a g t a g c g a t a a t

780 CMV enhancer chicken β- actin promoter c c a t g g t c g a g g t g a g c c c c a c g t t c t g c t t c a c t c t c c c c a t c t c c c c c c c c t c c c c a c g g t a c c a g c t c c a c t c g g g g t g c a a g a c g a a g t g a g a g g g g t a g a g g g g g g g g a g g g g t g

840 chicken β- actin promoter c c c c a a t t t t g t a t t t a t t t a t t t t t t a a t t a t t t t g t g c a g c g a t g g g g g c g g g g g g g g g g g g t t a a a a c a t a a a t a a a t a a a a a a t t a a t a a a a c a c g t c g c t a c c c c c g c c c c c c c c

900 chicken β- actin promoter g g g g g g g g c g c g c g c c a g g c g g g g c g g g g c g g g g c g a g g g g c g g g g c g g g g c g a g g c g g a c c c c c c c c g c g c g c g g t c c g c c c c g c c c c g c c c c g c t c c c c g c c c c g c c c c g c t c c g c c t

960 chicken β- actin promoter g a g g t g c g g c g g c a g c c a a t c a g a g c g g c g c g c t c c g a a a g t t t c c t t t t a t g g c g a g g c c t c c a c g c c g c c g t c g g t t a g t c t c g c c g c g c g a g g c t t t c a a a g g a a a a t a c c g c t c c g

1020 chicken β- actin promoter hybrid intron g g c g g c g g c g g c g g c c c t a t a a a a a g c g a a g c g c g c g g c g g g c g g g a g t c g c t g c g a c g c c c g c c g c c g c c g c c g g g a t a t t t t t c g c t t c g c g c g c c g c c c g c c c t c a g c g a c g c t g c g

1080 hybrid intron t g c c t t c g c c c c g t g c c c c g c t c c g c c g c c g c c t c g c g c c g c c c g c c c c g g c t c t g a c t g a c g g a a g c g g g g c a c g g g g c g a g g c g g c g g c g g a g c g c g g c g g g c g g g g c c g a g a c t g a c

BseRI 1140 hybrid intron a c c g c g t t a c t c c c a c a g g t g a g c g g g c g g g a c g g c c c t t c t c c t c c g g g c t g t a a t t a g t g g c g c a a t g a g g g t g t c c a c t c g c c c g c c c t g c c g g g a a g a g g a g g c c c g a c a t t a a t c

1200 hybrid intron c t g a g c a a g a g g t a a g g g t t t a a g g g a t g g t t g g t t g g t g g g g t a t t a a t g t t t a a t t a c g a c t c g t t c t c c a t t c c c a a a t t c c c t a c c a a c c a a c c a c c c c a t a a t t a c a a a t t a a t g

AgeIPaqCI 1260 1 DY hybrid intron 3xFLAG Kozak sequence MDY c t g g a g c a c c t g c c t g a a a t c a c t t t t t t t c a g g t t g g a c c g g t g c c a c c a t g g a c t a t a g a c c t c g t g g a c g g a c t t t a g t g a a a a a a a g t c c a a c c t g g c c a c g g t g g t a c c t g a t a t

1320 5 10 15 20 KDHDGDYKDHDIDYKDDDDK 3xFLAG KDHDGDYKDHDIDYKDDDDK 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 g a t g a c g a t a a g a 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 c t a c t g c t a t t c t

1380 MA 130 PKKKRKV (in frame with SV40 NLS) GIHGVPAA DKK SV40 NLS Cas9(H840A) DKK Cas9 MAPKKKRKVGIHGVPAADKK 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 g c c g a c a a g a a g 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 c g g c t g t t c t t c a

1440 5 10 15 20 YSIGLDIGTNSVGWAVITDE Cas9 5 10 15 20 YSIGLDIGTNSVGWAVITDE Cas9(H840A) YSIGLDIGTNSVGWAVITDE 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 a t c a c c g a c g a g t 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 t a g t g g c t g c t c a

1500 25 30 35 40 YKVPSKKFKVLGNTDRHSIK Cas9 25 30 35 40 YKVPSKKFKVLGNTDRHSIK Cas9(H840A) YKVPSKKFKVLGNTDRHSIK 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 c a c a g c a t c a a g 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 g t g t c g t a g t t c t

1560 45 50 55 60 KNLIGALLFDSGETAEATRL Cas9 45 50 55 60 KNLIGALLFDSGETAEATRL Cas9(H840A) KNLIGALLFDSGETAEATRL 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 c c a c c c g g c t g a 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 c g g t g g g c c g a c t

BglII 1620 65 70 75 80 KRTARRRYTRRKNRICYLQE Cas9 65 70 75 80 KRTARRRYTRRKNRICYLQE Cas9(H840A) KRTARRRYTRRKNRICYLQE 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 t a t c t g c a a g a g a 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 a t a g a c g t t c t c t

1680 85 90 95 100 IFSNEMAKVDDSFFHRLEES Cas9 85 90 95 100 IFSNEMAKVDDSFFHRLEES Cas9(H840A) IFSNEMAKVDDSFFHRLEES 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 c t g g a a g a g t c 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 g a c c t t c t c a g g a

1740 105 110 115 120 FLVEEDKKHERHPIFGNIVD Cas9 105 110 115 120 FLVEEDKKHERHPIFGNIVD Cas9(H840A) FLVEEDKKHERHPIFGNIVD 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 a a c a t c g t g g a c g 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 t t g t a g c a c c t g c

1800 125 130 135 140 EVAYHEKYPTIYHLRKKLVD Cas9 125 130 135 140 EVAYHEKYPTIYHLRKKLVD Cas9(H840A) EVAYHEKYPTIYHLRKKLVD 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 a a a c t g g t g g a c a 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 t t t g a c c a c c t g t

1860 145 150 155 160 STDKADLRLIYLALAHMIKF Cas9 145 150 155 160 STDKADLRLIYLALAHMIKF Cas9(H840A) STDKADLRLIYLALAHMIKF 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 a t g a t c a a g t t c c 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 t a c t a g t t c a a g g

1920 165 170 175 180 RGHFLIEGDLNPDNSDVDKL Cas9 165 170 175 180 RGHFLIEGDLNPDNSDVDKL Cas9(H840A) RGHFLIEGDLNPDNSDVDKL 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 g t g g a c a a g c t g t 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 c a c c t g t t c g a c a

1980 185 190 195 200 FIQLVQTYNQLFEENPINAS Cas9 185 190 195 200 FIQLVQTYNQLFEENPINAS Cas9(H840A) FIQLVQTYNQLFEENPINAS 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 a t c a a c g c c a g c g 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 t a g t t g c g g t c g c

2040 205 210 215 220 GVDAKAILSARLSKSRRLEN Cas9 205 210 215 220 GVDAKAILSARLSKSRRLEN Cas9(H840A) GVDAKAILSARLSKSRRLEN 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 c g g c t g g a a a a t 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 g c c g a c c t t t t a g

2100 225 230 235 240 LIAQLPGEKKNGLFGNLIAL Cas9 225 230 235 240 LIAQLPGEKKNGLFGNLIAL Cas9(H840A) LIAQLPGEKKNGLFGNLIAL 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 c t g a t t g c c c t g a 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 g a c t a a c g g g a c t

2160 245 250 255 260 SLGLTPNFKSNFDLAEDAKL Cas9 245 250 255 260 SLGLTPNFKSNFDLAEDAKL Cas9(H840A) SLGLTPNFKSNFDLAEDAKL 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 t g c c a a a c t g c 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 c t a c g g t t t g a c g

2220 265 270 275 280 QLSKDTYDDDLDNLLAQIGD Cas9 265 270 275 280 QLSKDTYDDDLDNLLAQIGD Cas9(H840A) QLSKDTYDDDLDNLLAQIGD 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 a g a t c g g c g a c c 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 g t c t a g c c g c t g g

2280 285 290 295 300 QYADLFLAAKNLSDAILLSD Cas9 285 290 295 300 QYADLFLAAKNLSDAILLSD Cas9(H840A) QYADLFLAAKNLSDAILLSD 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 c t g c t g a g c g a c a 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 g a c g a c t c g c t g t

2340 305 310 315 320 ILRVNTEITKAPLSASMIKR Cas9 305 310 315 320 ILRVNTEITKAPLSASMIKR Cas9(H840A) ILRVNTEITKAPLSASMIKR 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 a t g a t c a a g a g a 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 t a c t a g t t c t c t a

2400 325 330 335 340 YDEHHQDLTLLKALVRQQLP Cas9 325 330 335 340 YDEHHQDLTLLKALVRQQLP Cas9(H840A) YDEHHQDLTLLKALVRQQLP 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 c a g c a g c t g c c t g 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 g t c g t c g a c g g a c

2460 345 350 355 360 EKYKEIFFDQSKNGYAGYID Cas9 345 350 355 360 EKYKEIFFDQSKNGYAGYID Cas9(H840A) EKYKEIFFDQSKNGYAGYID 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 g c t a c a t t g a c g 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 c c g a t g t a a c t g c

2520 365 370 375 380 GGASQEEFYKFIKPILEKMD Cas9 365 370 375 380 GGASQEEFYKFIKPILEKMD Cas9(H840A) GGASQEEFYKFIKPILEKMD 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 g a a a a g a t g g a c g 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 c t t t t c t a c c t g c

2580 385 390 395 400 GTEELLVKLNREDLLRKQRT Cas9 385 390 395 400 GTEELLVKLNREDLLRKQRT Cas9(H840A) GTEELLVKLNREDLLRKQRT 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 a a g c a g c g g a c c t 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 t t c g t c g c c t g g a

2640 405 410 415 420 FDNGSIPHQIHLGELHAILR Cas9 405 410 415 420 FDNGSIPHQIHLGELHAILR Cas9(H840A) FDNGSIPHQIHLGELHAILR 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 g c c a t t c t g c g g c 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 c g g t a a g a c g c c g

2700 425 430 435 440 RQEDFYPFLKDNREKIEKIL Cas9 425 430 435 440 RQEDFYPFLKDNREKIEKIL Cas9(H840A) RQEDFYPFLKDNREKIEKIL 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 g a g a a g a t c c t g a 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 c t c t t c t a g g a c t

PspOMIApaI 2760 445 450 455 460 TFRIPYYVGPLARGNSRFAW Cas9 445 450 455 460 TFRIPYYVGPLARGNSRFAW Cas9(H840A) TFRIPYYVGPLARGNSRFAW 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 a g a t t c g c c t g g a 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 t c t a a g c g g a c c t

2820 465 470 475 480 MTRKSEETITPWNFEEVVDK Cas9 465 470 475 480 MTRKSEETITPWNFEEVVDK Cas9(H840A) MTRKSEETITPWNFEEVVDK 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 g t g g t g g a c a a g g 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 c a c c a c c t g t t c c

2880 485 490 495 500 GASAQSFIERMTNFDKNLPN Cas9 485 490 495 500 GASAQSFIERMTNFDKNLPN Cas9(H840A) GASAQSFIERMTNFDKNLPN 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 a a c c t g c c c a a c g 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 t t g g a c g g g t t g c

2940 505 510 515 520 EKVLPKHSLLYEYFTVYNEL Cas9 505 510 515 520 EKVLPKHSLLYEYFTVYNEL Cas9(H840A) EKVLPKHSLLYEYFTVYNEL 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 a t a a c g a g c t g a 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 a t a t t g c t c g a c t

3000 525 530 535 540 TKVKYVTEGMRKPAFLSGEQ Cas9 525 530 535 540 TKVKYVTEGMRKPAFLSGEQ Cas9(H840A) TKVKYVTEGMRKPAFLSGEQ 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 g c g g c g a g c a g a 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 t c g c c g c t c g t c t

3060 545 550 555 560 KKAIVDLLFKTNRKVTVKQL Cas9 545 550 555 560 KKAIVDLLFKTNRKVTVKQL Cas9(H840A) KKAIVDLLFKTNRKVTVKQL 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 g t g a a g c a g c t g a 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 c a c t t c g t c g a c t

3120 565 570 575 580 KEDYFKKIECFDSVEISGVE Cas9 565 570 575 580 KEDYFKKIECFDSVEISGVE Cas9(H840A) KEDYFKKIECFDSVEISGVE 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 t c c g g c g t g g a a g 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 a g g c c g c a c c t t c

PasI 3180 585 590 595 600 DRFNASLGTYHDLLKIIKDK Cas9 585 590 595 600 DRFNASLGTYHDLLKIIKDK Cas9(H840A) DRFNASLGTYHDLLKIIKDK 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 t c a a g g a c a a g g 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 t a g t t c c t g t t c c

EcoRV 3240 605 610 615 620 DFLDNEENEDILEDIVLTLT Cas9 605 610 615 620 DFLDNEENEDILEDIVLTLT Cas9(H840A) DFLDNEENEDILEDIVLTLT 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 c t g a c c c t g a c a 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 g a c t g g g a c t g t g

3300 625 630 635 640 LFEDREMIEERLKTYAHLFD Cas9 625 630 635 640 LFEDREMIEERLKTYAHLFD Cas9(H840A) LFEDREMIEERLKTYAHLFD *AWRNSS 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 c a c c t g t t c g a c 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 g t g g a c a a g c t g c

3360 645 650 655 660 DKVMKQLKRRRYTGWGRLSR Cas9 645 650 655 660 DKVMKQLKRRRYTGWGRLSR Cas9(H840A) DKVMKQLKRRRYTGWGRLSR LTIFCSFRRLYVPQPLSLRF 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 a g g c t g a g c c g g a 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 t c c g a c t c g g c c t

3420 665 670 675 680 KLINGIRDKQSGKTILDFLK Cas9 665 670 675 680 KLINGIRDKQSGKTILDFLK Cas9(H840A) KLINGIRDKQSGKTILDFLK SILPMRSLCDPLVIRSKRFD 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 g a t t t c c t g a a g 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 c t a a a g g a c t t c a

3480 685 690 695 700 SDGFANRNFMQLIHDDSLTF Cas9 685 690 695 700 SDGFANRNFMQLIHDDSLTF Cas9(H840A) SDGFANRNFMQLIHDDSLTF SPKALLFKMCSIWSSLRVKL 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 a g c c t g a c c t t t 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 t c g g a c t g g a a a t

3540 705 710 715 720 KEDIQKAQVSGQGDSLHEHI Cas9 705 710 715 720 KEDIQKAQVSGQGDSLHEHI Cas9(H840A) KEDIQKAQVSGQGDSLHEHI SSMWFAWTDPWPSLRCSCMA 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 c a c g a g c a c a t t g 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 g t g c t c g t g t a a c

3600 725 730 735 740 ANLAGSPAIKKGILQTVKVV Cas9 725 730 735 740 ANLAGSPAIKKGILQTVKVV Cas9(H840A) ANLAGSPAIKKGILQTVKVV LRAPLGAMLFPMRCVTFTTS 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 a a g g t g g t g g 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 c a c t t c c a c c a c c

3660 745 750 755 760 DELVKVMGRHKPENIVIEMA Cas9 745 750 755 760 DELVKVMGRHKPENIVIEMA Cas9(H840A) DELVKVMGRHKPENIVIEMA SSTFTIPRCLGSFMTISIAL 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 a t c g a a a t g g c c a 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 t a g c t t t a c c g g t

3720 765 770 775 780 RENQTTQKGQKNSRERMKRI Cas9 765 770 775 780 RENQTTQKGQKNSRERMKRI Cas9(H840A) RENQTTQKGQKNSRERMKRI SFWVVWFPCFFLRSLIFRIS 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 a t g a a g c g g a t c g 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 t a c t t c g c c t a g c

3780 785 790 795 800 EEGIKELGSQILKEHPVENT Cas9 785 790 795 800 EEGIKELGSQILKEHPVENT Cas9(H840A) EEGIKELGSQILKEHPVENT SPM 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 g t g g a a a a c a c c 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 c a c c t t t t g t g g g

3840 805 810 815 820 QLQNEKLYLYYLQNGRDMYV Cas9 805 810 815 820 QLQNEKLYLYYLQNGRDMYV Cas9(H840A) QLQNEKLYLYYLQNGRDMYV 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 g a t a t g t a c g t 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 c t a t a c a t g c a c c

3900 825 830 835 840 DQELDINRLSDYDVDAIVPQ Cas9 825 830 835 DQELDINRLSDYDVD 840 A IVPQ Cas9(H840A) DQELDINRLSDYDVDAIVPQ 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 g c t a t c g t g c c t c a g a 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 c g a t a g c a c g g a g t c t

3960 845 850 855 860 SFLKDDSIDNKVLTRSDKNR Cas9 845 850 855 860 SFLKDDSIDNKVLTRSDKNR Cas9(H840A) SFLKDDSIDNKVLTRSDKNR 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 g a c a a g a a c c g g g 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 c t g t t c t t g g c c c

4020 865 870 875 880 GKSDNVPSEEVVKKMKNYWR Cas9 865 870 875 880 GKSDNVPSEEVVKKMKNYWR Cas9(H840A) GKSDNVPSEEVVKKMKNYWR 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 a a c t a c t g g c g g 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 t t g a t g a c c g c c g

4080 885 890 895 900 QLLNAKLITQRKFDNLTKAE Cas9 885 890 895 900 QLLNAKLITQRKFDNLTKAE Cas9(H840A) QLLNAKLITQRKFDNLTKAE 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 a c c a a g g c c g a g a 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 t g g t t c c g g c t c t

4140 905 910 915 920 RGGLSELDKAGFIKRQLVET Cas9 905 910 915 920 RGGLSELDKAGFIKRQLVET Cas9(H840A) RGGLSELDKAGFIKRQLVET 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 c t g g t g g a a a c c c 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 g a c c a c c t t t g g g

PmlI 4200 925 930 935 940 RQITKHVAQILDSRMNTKYD Cas9 925 930 935 940 RQITKHVAQILDSRMNTKYD Cas9(H840A) RQITKHVAQILDSRMNTKYD 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 a c t a a g t a c g a c 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 t g a t t c a t g c t g c

4260 945 950 955 960 ENDKLIREVKVITLKSKLVS Cas9 945 950 955 960 ENDKLIREVKVITLKSKLVS Cas9(H840A) ENDKLIREVKVITLKSKLVS 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 a a g c t g g t g t c c g 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 t t c g a c c a c a g g c

4320 965 970 975 980 DFRKDFQFYKVREINNYHHA Cas9 965 970 975 980 DFRKDFQFYKVREINNYHHA Cas9(H840A) DFRKDFQFYKVREINNYHHA 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 a c c a c c a c g c c 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 a t g g t g g t g c g g g

4380 985 990 995 1000 HDAYLNAVVGTALIKKYPKL Cas9 985 990 995 1000 HDAYLNAVVGTALIKKYPKL Cas9(H840A) HDAYLNAVVGTALIKKYPKL 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 t a c c c t a a g c t 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 a t g g g a t t c g a c c

4440 1005 1010 1015 1020 ESEFVYGDYKVYDVRKMIAK Cas9 1005 1010 1015 1020 ESEFVYGDYKVYDVRKMIAK Cas9(H840A) ESEFVYGDYKVYDVRKMIAK 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 a t c g c c a a g a 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 t a c t a g c g g t t c t

4500 1025 1030 1035 1040 SEQEIGKATAKYFFYSNIMN Cas9 1025 1030 1035 1040 SEQEIGKATAKYFFYSNIMN Cas9(H840A) SEQEIGKATAKYFFYSNIMN 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 a a c a t c a t g a a 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 t t g t a g t a c t t g a

4560 1045 1050 1055 1060 FFKTEITLANGEIRKRPLIE Cas9 1045 1050 1055 1060 FFKTEITLANGEIRKRPLIE Cas9(H840A) FFKTEITLANGEIRKRPLIE 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 c c t c t g a t c g a 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 g g a g a c t a g c t c t

4620 1065 1070 1075 1080 TNGETGEIVWDKGRDFATVR Cas9 1065 1070 1075 1080 TNGETGEIVWDKGRDFATVR Cas9(H840A) TNGETGEIVWDKGRDFATVR 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 c c a c c g t g c g g a 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 c g g t g g c a c g c c t

4680 1085 1090 1095 1100 KVLSMPQVNIVKKTEVQTGG Cas9 1085 1090 1095 1100 KVLSMPQVNIVKKTEVQTGG Cas9(H840A) KVLSMPQVNIVKKTEVQTGG 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 a g a c a g g c g g 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 g t c t g t c c g c c g a

4740 1105 1110 1115 1120 FSKESILPKRNSDKLIARKK Cas9 1105 1110 1115 1120 FSKESILPKRNSDKLIARKK Cas9(H840A) FSKESILPKRNSDKLIARKK 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 c c a g a a a g a a g g 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 c g g t c t t t c t t c c

KflI 4800 1125 1130 1135 1140 DWDPKKYGGFDSPTVAYSVL Cas9 1125 1130 1135 1140 DWDPKKYGGFDSPTVAYSVL Cas9(H840A) DWDPKKYGGFDSPTVAYSVL 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 t a t t c t g t g c t g g 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 a t a a g a c a c g a c c

4860 1145 1150 1155 1160 VVAKVEKGKSKKLKSVKELL Cas9 1145 1150 1155 1160 VVAKVEKGKSKKLKSVKELL Cas9(H840A) VVAKVEKGKSKKLKSVKELL 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 a a g a g c t g c t g g 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 t t t c t c g a c g a c c

BsaBI* 4920 1165 1170 1175 1180 GITIMERSSFEKNPIDFLEA Cas9 1165 1170 1175 1180 GITIMERSSFEKNPIDFLEA Cas9(H840A) GITIMERSSFEKNPIDFLEA 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 g g a a g c c a 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 a a a g a c c t t c g g t

4980 1185 1190 1195 1200 KGYKEVKKDLIIKLPKYSLF Cas9 1185 1190 1195 1200 KGYKEVKKDLIIKLPKYSLF Cas9(H840A) KGYKEVKKDLIIKLPKYSLF 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 t a c t c c c t g t t c g 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 a t g a g g g a c a a g c

BsmI 5040 1205 1210 1215 1220 ELENGRKRMLASAGELQKGN Cas9 1205 1210 1215 1220 ELENGRKRMLASAGELQKGN Cas9(H840A) ELENGRKRMLASAGELQKGN 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 c a g a a g g g a a a c g 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 g t c t t c c c t t t g c

5100 1225 1230 1235 1240 ELALPSKYVNFLYLASHYEK Cas9 1225 1230 1235 1240 ELALPSKYVNFLYLASHYEK Cas9(H840A) ELALPSKYVNFLYLASHYEK 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 c a c t a t g a g a a 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 g t g a t a c t c t t c g

5160 1245 1250 1255 1260 LKGSPEDNEQKQLFVEQHKH Cas9 1245 1250 1255 1260 LKGSPEDNEQKQLFVEQHKH Cas9(H840A) LKGSPEDNEQKQLFVEQHKH 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 c a g c a c a a g c a c t 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 g t c g t g t t c g t g a

5220 1265 1270 1275 1280 YLDEIIEQISEFSKRVILAD Cas9 1265 1270 1275 1280 YLDEIIEQISEFSKRVILAD Cas9(H840A) YLDEIIEQISEFSKRVILAD 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 a t c c t g g c c g a c g 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 t a g g a c c g g c t g c

5280 1285 1290 1295 1300 ANLDKVLSAYNKHRDKPIRE Cas9 1285 1290 1295 1300 ANLDKVLSAYNKHRDKPIRE Cas9(H840A) ANLDKVLSAYNKHRDKPIRE 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 c c c a t c a g a g a 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 g g g t a g t c t c t c g

5340 1305 1310 1315 1320 QAENIIHLFTLTNLGAPAAF Cas9 1305 1310 1315 1320 QAENIIHLFTLTNLGAPAAF Cas9(H840A) QAENIIHLFTLTNLGAPAAF 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 c c t g c c g c c t t c a 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 g g a c g g c g g a a g t

5400 1325 1330 1335 1340 KYFDTTIDRKRYTSTKEVLD Cas9 1325 1330 1335 1340 KYFDTTIDRKRYTSTKEVLD Cas9(H840A) KYFDTTIDRKRYTSTKEVLD 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 a g g t g c t g g a c g 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 c t c c a c g a c c t g c

5460 1345 1350 1355 1360 ATLIHQSITGLYETRIDLSQ Cas9 1345 1350 1355 1360 ATLIHQSITGLYETRIDLSQ Cas9(H840A) ATLIHQSITGLYETRIDLSQ 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 g a c c t g t c t c a 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 c t g g a c a g a g t c g

FseI 5520 1365 LGGD (in frame with Cas9) KRPAATKKAGQAKKKK Cas9 1365 LGGD 1 5 10 15 KRPAATKKAGQAKKKK Cas9(H840A) nucleoplasmin NLS LGGDKRPAATKKAGQAKKKK 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 a a a a a g a a a a a g t 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 t t t t t c t t t t t c a

EcoRI 5580 * bGH poly(A) signal * G G A G C T G A C A C G G A A G A T BGH-rev * a a g a a t t c c t a g a g 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 t c t t a a g g a t c t 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

5640 bGH poly(A) signal t g t t t g c c c c t c c c 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 a c a a a c g g g g a g 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

5700 bGH poly(A) signal c t a a t a a a a t g a g g 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 g a t t a t t t t a c t c c 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

5760 bGH poly(A) signal t g g g g t g g g g c a g g 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 a c c c c a c c c c g t c 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

NotI 5820 AAV2 ITR AAV2 ITR g c g g c c g c a g g a a c 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 g c c g g c g t c c t t g 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

5880 AAV2 ITR AAV2 ITR c a c t g a g g c c g g g c 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 t g a c t c c g g c c c g 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

SbfIKasINarISfoIPluTI 5940 AAV2 ITR AAV2 ITR g a g c g a g c g a g c g c 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 c t c g c t c g c t c g c g 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

6000 f1 ori t c t g t g c g g t a t t t 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 a g a c a c g c c a t a a a 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

6060 f1 ori g c a t t a a g c g c g g c 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 c g t a a t t c g c g c c g 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

6120 f1 ori G A G G A A A G C G A A A G A A G G G A F1ori-R c t a g c g c c c g c t c c 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 g a t c g c g g g c g a g g 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

6180 f1 ori c g t c a a g c t c t a a a 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 c a g t t c g a g a t t 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

6240 f1 ori g a c c c c a a a a a a c t 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 c t g g g g t t t t t t g a 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

6300 f1 ori G T G G A C T C T T G T T C C A A A C T F1ori-F g t t t t t c g c c c t t t 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 c a a a a a g c g g g a 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

PsiI 6360 f1 ori G G F1ori-F g g a a c a a c a c t c a a c c 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 c c t t g t t g t g a g t t g g 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

6420 f1 ori t c g g c c t a t t g g t t 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 g c c g g a t a a c c a a 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

6480 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 t a t t a a c g t t t a c 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 a t a a t t g c a a a t 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

6540 t t a a g c c a g c c c c g 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 a a t t c g g t c g g g g c 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

6600 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 c g g c a t c c g c t t a 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 g g c c g t a g g c g a a t 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

6660 C G G A G C A C T A T G C G G A T A A pBRforEco t c a c c g t c a t c a c c 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 a g t g g c a g t a g t g g 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

6720 g t t a a t g t c a t g a t 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 a a t t a c a g t a c t a 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

6780 AmpR promoter c g c g g a a c c c c t a t 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 g c g c c t t g g g 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

6840 1 5 signal sequence MSIQH AmpR promoter AmpR MSIQH c a a t a a c c c t g a t a 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 g t t a t t g g g a c t a 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

6900 10 15 20 signal sequence FRVALIPFFAAFCLPVFA 25 HP AmpR FRVALIPFFAAFCLPVFAHP t t c c g t g t c g c c c t 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 a a g g c a c a g c g g g 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

6960 30 35 40 45 ETLVKVKDAEDQLGARVGYI AmpR ETLVKVKDAEDQLGARVGYI g a a a c g c t g g t g a a 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 c t t t g c g a c c a c t t 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

7020 50 55 60 65 ELDLNSGKILESFRPEERFP AmpR ELDLNSGKILESFRPEERFP g a a c t g g a t c t c a a 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 c t t g a c c t a g a g 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

7080 70 75 80 85 MMSTFKVLLCGAVLSRIDAG AmpR C G A T A C A C C G C G C C A T A A T A Amp-R MMSTFKVLLCGAVLSRIDAG a t g a t g a g c a c t t t 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 t a c t a c t c g t g a a a 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

7140 90 95 100 105 QEQLGRRIHYSQNDLVEYSP AmpR QEQLGRRIHYSQNDLVEYSP c a a g a g c a a c t c g g 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 t c t c g t t g a g c c 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

7200 110 115 120 125 VTEKHLTDGMTVRELCSAAI AmpR VTEKHLTDGMTVRELCSAAI g t c a c a g a a a a g c a 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 c a g t g t c t t t t c g t 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

PvuI 7260 130 135 140 145 TMSDNTAANLLLTTIGGPKE AmpR TMSDNTAANLLLTTIGGPKE a c c a t g a g t g a t a a 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 t g g t a c t c a c t a 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

7320 150 155 160 165 LTAFLHNMGDHVTRLDRWEP AmpR LTAFLHNMGDHVTRLDRWEP c t a a c c g c t t t t t t 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 t t g g c g a a a a a a 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

7380 170 175 180 185 ELNEAIPNDERDTTMPVAMA AmpR ELNEAIPNDERDTTMPVAMA g a g c t g a a t g a a g c 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 c t c g a c t t a c t t c g 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

FspI 7440 190 195 200 205 TTLRKLLTGELLTLASRQQL AmpR TTLRKLLTGELLTLASRQQL a c a a c g t t g c g c a a 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 t g t t g c a a c g c g t t 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

7500 210 215 220 225 IDWMEADKVAGPLLRSALPA AmpR IDWMEADKVAGPLLRSALPA a t a g a c t g g a t g g a 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 t a t c t g a c c t a c c t 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

SacII 7560 230 235 240 245 GWFIADKSGAGERGSRGIIA AmpR GWFIADKSGAGERGSRGIIA g g c t g g t t t a t t g c 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 c c g a c c a a a t a a c g 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

7620 250 255 260 265 ALGPDGKPSRIVVIYTTGSQ AmpR ALGPDGKPSRIVVIYTTGSQ g c a c t g g g g c c a g a 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 c g t g a c c c c g g t c t 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

7680 270 275 280 285 ATMDERNRQIAEIGASLIKH AmpR ATMDERNRQIAEIGASLIKH g c a a c t a t g g a t g a 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 c g t t g a t a c c t a c t 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

7740 W* AmpR W* t g g t a a c t g t c a g a 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 a c c a t t g a c a g t c 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

7800 t a a t t t a a a a g g a t 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 a t t a a a t t t t c c t a 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

7860 ori c g t g a g t t t t c g t t c c a c t g a g c g t c a g a c c c c 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 c a c t c a a a a g c a a g g t g a c t c g c a g t c t g g g 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

7920 ori 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 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 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 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

7980 ori 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 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 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 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

8040 ori a g a g c g c a g a t a c c a a a t a c t g t c c t t c t a g t g 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 t c t c g c g t c t a t g g t t t a t g a c a g g a a g a t c a c 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

8100 ori 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 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 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 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

8160 ori 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 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 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 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

8220 ori 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 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 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 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

8280 ori 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 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 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 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

8340 ori 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 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 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 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

8400 ori G G G A A A C G C C T G G T A T C T T T pBR322ori-F 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 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 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 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

8460 ori 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 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 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 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

AflIIIPciI 8506 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 t t g c t c a c a t g t 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 a a a c g a g t g t a c a

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.