Addgene PX458_RFXANK_1 Sequencing Result - Sequence Analyzer Skip to main content
Addgene

Sequence Analyzer: PX458_RFXANK_1 Sequencing Result


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8000 6000 4000 2000 End (8476) KpnI (8475) Acc65I (8471) XbaI (8465) NdeI (8215) LKO.1 5' (8208 .. 8227) hU6-F (8037 .. 8057) PciI - AflIII (8031) pBR322ori-F (7876 .. 7895) SacII (7078) FspI (6920) PvuI (6774) Amp-R (6574 .. 6593) AmpR promoter pBRforEco (6165 .. 6183) pGEX 3' (6105 .. 6127) pRS-marker (5986 .. 6005) PsiI (5872) F1ori-F (5811 .. 5832) F1ori-R (5601 .. 5620) PluTI (5444) SfoI (5442) NarI (5441) KasI (5440) SbfI (5437) NotI (5292) BGH-rev (5077 .. 5094) BsrGI (5042) EGFP-C (4986 .. 5007) EXFP-R (4639 .. 4658) FseI (4247) nucleoplasmin NLS BsmI (3755) BsaBI * (3613) KflI (3492) PmlI (2904) EcoRV (1968) PasI (1884) ApaI (1474) PspOMI (1470) BglII (365) SV40 NLS Start (0) 3xFLAG Cas9 EGFP bGH poly(A) signal AAV2 ITR f1 ori AmpR ori U6 promoter gRNA scaffold Cas9 T2A EGFP AAV2 ITR PX458_RFXANK_1 8476 bp

Sequence View

Start (0) 5ʹ 3ʹ 60 1 5 10 15 20 DYKDHDGDYKDHDIDYKDDD 3xFLAG DYKDHDGDYKDHDIDYKDDD VILVVVSVVLIMINIVFVIV 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 g a t g 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 c t a c t g

120 D Cas9 DK MA 130 PKKKRKV (in frame with SV40 NLS) GIHGVPAA D 3xFLAG SV40 NLS Cas9 DKMAPKKKRKVGIHGVPAAD ILHGWLLLPLDTDVSDWCGV 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 g c c g a c 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 c g g c t g

180 5 10 15 20 KKYSIGLDIGTNSVGWAVIT Cas9 5 10 15 20 KKYSIGLDIGTNSVGWAVIT Cas9 KKYSIGLDIGTNSVGWAVIT LLVADAQVDAGVRHAPGHDG 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 a t c a c c 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 t a g t g g

240 25 30 35 40 DEYKVPSKKFKVLGNTDRHS Cas9 25 30 35 40 DEYKVPSKKFKVLGNTDRHS Cas9 DEYKVPSKKFKVLGNTDRHS VLVLHGALFELHQAVGVPVA 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 c a c a g c 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 g t g t c g

300 45 50 55 60 IKKNLIGALLFDSGETAEAT Cas9 45 50 55 60 IKKNLIGALLFDSGETAEAT Cas9 IKKNLIGALLFDSGETAEAT DLLVQDSGQQEVAAFCGLGG 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 c c a c c 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 c g g t g g

360 65 70 75 80 RLKRTARRRYTRRKNRICYL Cas9 65 70 75 80 RLKRTARRRYTRRKNRICYL Cas9 RLKRTARRRYTRRKNRICYL PQLSGGSSSVGSPLVPDAIQ 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 t a t c t 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 a t a g a c

BglII 420 85 90 95 100 QEIFSNEMAKVDDSFFHRLE Cas9 85 90 95 100 QEIFSNEMAKVDDSFFHRLE Cas9 QEIFSNEMAKVDDSFFHRLE LLDEAVLHGLHVVAEEVSQF 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 c t g g a a 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 g a c c t t

480 105 110 115 120 ESFLVEEDKKHERHPIFGNI Cas9 105 110 115 120 ESFLVEEDKKHERHPIFGNI Cas9 ESFLVEEDKKHERHPIFGNI LGEQHFLILLVLPVGDEAVD 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 a a c a t c 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 t t g t a g

540 125 130 135 140 VDEVAYHEKYPTIYHLRKKL Cas9 125 130 135 140 VDEVAYHEKYPTIYHLRKKL Cas9 VDEVAYHEKYPTIYHLRKKL HVLHGVVLLVGGDVVQSLFQ 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 a a a c t 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 t t t g a c

600 145 150 155 160 VDSTDKADLRLIYLALAHMI Cas9 145 150 155 160 VDSTDKADLRLIYLALAHMI Cas9 VDSTDKADLRLIYLALAHMI HVAGVLGVQPQDIQGQGVHD 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 a t g a t 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 t a c t a g

660 165 170 175 180 KFRGHFLIEGDLNPDNSDVD Cas9 165 170 175 180 KFRGHFLIEGDLNPDNSDVD Cas9 KFRGHFLIEGDLNPDNSDVD LEPAVEQDLAVQVGVVAVHV 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 g t g g a c 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 c a c c t g

720 185 190 195 200 KLFIQLVQTYNQLFEENPIN Cas9 185 190 195 200 KLFIQLVQTYNQLFEENPIN Cas9 KLFIQLVQTYNQLFEENPIN LQEDLQHLGVVLQELFVGDV 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 a t c a a c 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 t a g t t g

780 205 210 215 220 ASGVDAKAILSARLSKSRRL Cas9 205 210 215 220 ASGVDAKAILSARLSKSRRL Cas9 ASGVDAKAILSARLSKSRRL GAAHVGLGDQRGSQALASPQ 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 c g g c 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 g c c g a c

840 225 230 235 240 ENLIAQLPGEKKNGLFGNLI Cas9 225 230 235 240 ENLIAQLPGEKKNGLFGNLI Cas9 ENLIAQLPGEKKNGLFGNLI FIQDGLQGALLLIAQESVQN 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 c t g a t t 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 g a c t a a

900 245 250 255 260 ALSLGLTPNFKSNFDLAEDA Cas9 245 250 255 260 ALSLGLTPNFKSNFDLAEDA Cas9 ALSLGLTPNFKSNFDLAEDA GQAQAQGGVELAVEVQGLIG 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 t g c c 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 c t a c g g

960 265 270 275 280 KLQLSKDTYDDDLDNLLAQI Cas9 265 270 275 280 KLQLSKDTYDDDLDNLLAQI Cas9 KLQLSKDTYDDDLDNLLAQI FQLQALVGVVVVQVVQQGLD 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 a g a t c 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 g t c t a g

1020 285 290 295 300 GDQYADLFLAAKNLSDAILL Cas9 285 290 295 300 GDQYADLFLAAKNLSDAILL Cas9 GDQYADLFLAAKNLSDAILL AVLVGVQKQGGLVQGVGDQQ 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 c t g c t 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 g a c g a c

1080 305 310 315 320 SDILRVNTEITKAPLSASMI Cas9 305 310 315 320 SDILRVNTEITKAPLSASMI Cas9 SDILRVNTEITKAPLSASMI AVDQSHVGLDGLGGQAGRHD 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 a t g a t 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 t a c t a g

1140 325 330 335 340 KRYDEHHQDLTLLKALVRQQ Cas9 325 330 335 340 KRYDEHHQDLTLLKALVRQQ Cas9 KRYDEHHQDLTLLKALVRQQ LSVVLVVLVQGQQFSEHPLL 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 c a g c 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 g t c g t c

1200 345 350 355 360 LPEKYKEIFFDQSKNGYAGY Cas9 345 350 355 360 LPEKYKEIFFDQSKNGYAGY Cas9 LPEKYKEIFFDQSKNGYAGY QRLLVFLNEEVLALVAVGAV 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 g c t a 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 c c g a t g

1260 365 370 375 380 IDGGASQEEFYKFIKPILEK Cas9 365 370 375 380 IDGGASQEEFYKFIKPILEK Cas9 IDGGASQEEFYKFIKPILEK NVASGALFLEVLEDLGDQFL 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 g a a a a 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 c t t t t c

1320 385 390 395 400 MDGTEELLVKLNREDLLRKQ Cas9 385 390 395 400 MDGTEELLVKLNREDLLRKQ Cas9 MDGTEELLVKLNREDLLRKQ HVAGLFQEHLQVSLVQQPLL 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 a a g c a g 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 t t c g t c

1380 405 410 415 420 RTFDNGSIPHQIHLGELHAI Cas9 405 410 415 420 RTFDNGSIPHQIHLGELHAI Cas9 RTFDNGSIPHQIHLGELHAI PGEVVAADGVLDVQSLQVGN 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 g c c a t t 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 c g g t a a

1440 425 430 435 440 LRRQEDFYPFLKDNREKIEK Cas9 425 430 435 440 LRRQEDFYPFLKDNREKIEK Cas9 LRRQEDFYPFLKDNREKIEK QPPLFIKVWEQLVVPFLDLL 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 g a g a a g 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 c t c t t c

PspOMIApaI 1500 445 450 455 460 ILTFRIPYYVGPLARGNSRF Cas9 445 450 455 460 ILTFRIPYYVGPLARGNSRF Cas9 ILTFRIPYYVGPLARGNSRF DQGEADGVVHARQGPSVASE 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 a g a 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 t c t a a g

1560 465 470 475 480 AWMTRKSEETITPWNFEEVV Cas9 465 470 475 480 AWMTRKSEETITPWNFEEVV Cas9 AWMTRKSEETITPWNFEEVV GPHGSLALFGDGGPVELFHH 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 g t g g t 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 c a c c a c

1620 485 490 495 500 DKGASAQSFIERMTNFDKNL Cas9 485 490 495 500 DKGASAQSFIERMTNFDKNL Cas9 DKGASAQSFIERMTNFDKNL VLASGGLAEDLPHGVEILVQ 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 a a c c t g 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 t t g g a c

1680 505 510 515 520 PNEKVLPKHSLLYEYFTVYN Cas9 505 510 515 520 PNEKVLPKHSLLYEYFTVYN Cas9 PNEKVLPKHSLLYEYFTVYN GVLLHQGLVAQQVLVEGHIV 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 a t a 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 a t a t t g

1740 525 530 535 540 ELTKVKYVTEGMRKPAFLSG Cas9 525 530 535 540 ELTKVKYVTEGMRKPAFLSG Cas9 ELTKVKYVTEGMRKPAFLSG LQGFHFVHGLSHSLGGEQAA 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 g c g g c 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 t c g c c g

1800 545 550 555 560 EQKKAIVDLLFKTNRKVTVK Cas9 545 550 555 560 EQKKAIVDLLFKTNRKVTVK Cas9 EQKKAIVDLLFKTNRKVTVK LLFLGDHVQQELGVPFHGHL 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 g t g a a 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 c a c t t c

1860 565 570 575 580 QLKEDYFKKIECFDSVEISG Cas9 565 570 575 580 QLKEDYFKKIECFDSVEISG Cas9 QLKEDYFKKIECFDSVEISG LQFLVVELFDLAEVGHFDGA 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 t c c g g 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 a g g c c g

PasI 1920 585 590 595 600 VEDRFNASLGTYHDLLKIIK Cas9 585 590 595 600 VEDRFNASLGTYHDLLKIIK Cas9 VEDRFNASLGTYHDLLKIIK HFIPEVGGQACVVIQQFNDL 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 t c a a 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 t a g t t c

EcoRV 1980 605 610 615 620 DKDFLDNEENEDILEDIVLT Cas9 605 610 615 620 DKDFLDNEENEDILEDIVLT Cas9 DKDFLDNEENEDILEDIVLT VLVEQVILFVLVNQFIDHQG 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 c t g a c 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 g a c t g g

2040 625 630 635 640 LTLFEDREMIEERLKTYAHL Cas9 625 630 635 640 LTLFEDREMIEERLKTYAHL Cas9 LTLFEDREMIEERLKTYAHL *AWR QCQKLVSLHDLFPQFGIGVQ 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 c a c c t 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 g t g g a c

2100 645 650 655 660 FDDKVMKQLKRRRYTGWGRL Cas9 645 650 655 660 FDDKVMKQLKRRRYTGWGRL Cas9 FDDKVMKQLKRRRYTGWGRL NSSLTIFCSFRRLYVPQPLS EVVFHHLLQLPPSVGAPAPQ 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 a g g c t g 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 t c c g a c

2160 665 670 675 680 SRKLINGIRDKQSGKTILDF Cas9 665 670 675 680 SRKLINGIRDKQSGKTILDF Cas9 SRKLINGIRDKQSGKTILDF LRFSILPMRSLCDPLVIRSK APLQDVADPVLLGALCDQIE 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 g a t t t 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 c t a a a g

2220 685 690 695 700 LKSDGFANRNFMQLIHDDSL Cas9 685 690 695 700 LKSDGFANRNFMQLIHDDSL Cas9 LKSDGFANRNFMQLIHDDSL RFDSPKALLFKMCSIWSSLR QLGVAEGVSVEHLQDVVVAQ 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 a g c c t 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 t c g g a c

2280 705 710 715 720 TFKEDIQKAQVSGQGDSLHE Cas9 705 710 715 720 TFKEDIQKAQVSGQGDSLHE Cas9 TFKEDIQKAQVSGQGDSLHE VKLSSMWFAWTDPWPSLRCS GKFLVDLFGLHGALAIAQVL 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 c a c g a g 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 g t g c t c

2340 725 730 735 740 HIANLAGSPAIKKGILQTVK Cas9 725 730 735 740 HIANLAGSPAIKKGILQTVK Cas9 HIANLAGSPAIKKGILQTVK CMALRAPLGAMLFPMRCVTF VNGIQGAAGGNLLADQLCHL 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 a a g 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 c a c t t c

2400 745 750 755 760 VVDELVKVMGRHKPENIVIE Cas9 745 750 755 760 VVDELVKVMGRHKPENIVIE Cas9 VVDELVKVMGRHKPENIVIE TTSSSTFTIPRCLGSFMTIS HHVLEHFHHAPVLGLVDHDF 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 a t c g a a 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 t a g c t t

2460 765 770 775 780 MARENQTTQKGQKNSRERMK Cas9 765 770 775 780 MARENQTTQKGQKNSRERMK Cas9 MARENQTTQKGQKNSRERMK IALSFWVVWFPCFFLRSLIF HGSLVLGGLLSLLVAALSHL 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 a t g a a g 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 t a c t t c

2520 785 790 795 800 RIEEGIKELGSQILKEHPVE Cas9 785 790 795 800 RIEEGIKELGSQILKEHPVE Cas9 RIEEGIKELGSQILKEHPVE RISSPM PDFLADFLQAALDQFFVGHF 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 g t g g a a 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 c a c c t t

2580 805 810 815 820 NTQLQNEKLYLYYLQNGRDM Cas9 805 810 815 820 NTQLQNEKLYLYYLQNGRDM Cas9 NTQLQNEKLYLYYLQNGRDM VGLQLVLLQVQVVQLIPPIH 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 g a t a t g 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 c t a t a c

2640 825 830 835 840 YVDQELDINRLSDYDVDHIV Cas9 825 830 835 840 YVDQELDINRLSDYDVDHIV Cas9 YVDQELDINRLSDYDVDHIV VHVLFQVDVPQGVVIHVM 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 a t c g t g 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 t a g c a c

2700 845 850 855 860 PQSFLKDDSIDNKVLTRSDK Cas9 845 850 855 860 PQSFLKDDSIDNKVLTRSDK Cas9 PQSFLKDDSIDNKVLTRSDK 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 g a c a a g 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 c t g t t c

2760 865 870 875 880 NRGKSDNVPSEEVVKKMKNY Cas9 865 870 875 880 NRGKSDNVPSEEVVKKMKNY Cas9 NRGKSDNVPSEEVVKKMKNY 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 a a c t a 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 t t g a t g

2820 885 890 895 900 WRQLLNAKLITQRKFDNLTK Cas9 885 890 895 900 WRQLLNAKLITQRKFDNLTK Cas9 WRQLLNAKLITQRKFDNLTK 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 a c c a a 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 t g g t t c

2880 905 910 915 920 AERGGLSELDKAGFIKRQLV Cas9 905 910 915 920 AERGGLSELDKAGFIKRQLV Cas9 AERGGLSELDKAGFIKRQLV 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 c t g g t g 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 g a c c a c

PmlI 2940 925 930 935 940 ETRQITKHVAQILDSRMNTK Cas9 925 930 935 940 ETRQITKHVAQILDSRMNTK Cas9 ETRQITKHVAQILDSRMNTK 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 a c t a a g 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 t g a t t c

3000 945 950 955 960 YDENDKLIREVKVITLKSKL Cas9 945 950 955 960 YDENDKLIREVKVITLKSKL Cas9 YDENDKLIREVKVITLKSKL 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 a a g c t g 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 t t c g a c

3060 965 970 975 980 VSDFRKDFQFYKVREINNYH Cas9 965 970 975 980 VSDFRKDFQFYKVREINNYH Cas9 VSDFRKDFQFYKVREINNYH 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 a c c 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 a t g g t g

3120 985 990 995 1000 HAHDAYLNAVVGTALIKKYP Cas9 985 990 995 1000 HAHDAYLNAVVGTALIKKYP Cas9 HAHDAYLNAVVGTALIKKYP 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 t a c c c t 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 a t g g g a

3180 1005 1010 1015 1020 KLESEFVYGDYKVYDVRKMI Cas9 1005 1010 1015 1020 KLESEFVYGDYKVYDVRKMI Cas9 KLESEFVYGDYKVYDVRKMI 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 a t c 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 t a c t a g

3240 1025 1030 1035 1040 AKSEQEIGKATAKYFFYSNI Cas9 1025 1030 1035 1040 AKSEQEIGKATAKYFFYSNI Cas9 AKSEQEIGKATAKYFFYSNI 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 a a c a t c 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 t t g t a g

3300 1045 1050 1055 1060 MNFFKTEITLANGEIRKRPL Cas9 1045 1050 1055 1060 MNFFKTEITLANGEIRKRPL Cas9 MNFFKTEITLANGEIRKRPL 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 c c t c t 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 g g a g a c

3360 1065 1070 1075 1080 IETNGETGEIVWDKGRDFAT Cas9 1065 1070 1075 1080 IETNGETGEIVWDKGRDFAT Cas9 IETNGETGEIVWDKGRDFAT 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 c c a c 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 c g g t g g

3420 1085 1090 1095 1100 VRKVLSMPQVNIVKKTEVQT Cas9 1085 1090 1095 1100 VRKVLSMPQVNIVKKTEVQT Cas9 VRKVLSMPQVNIVKKTEVQT 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 a g a c a 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 g t c t g t

3480 1105 1110 1115 1120 GGFSKESILPKRNSDKLIAR Cas9 1105 1110 1115 1120 GGFSKESILPKRNSDKLIAR Cas9 GGFSKESILPKRNSDKLIAR 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 c c a g a 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 c g g t c t

KflI 3540 1125 1130 1135 1140 KKDWDPKKYGGFDSPTVAYS Cas9 1125 1130 1135 1140 KKDWDPKKYGGFDSPTVAYS Cas9 KKDWDPKKYGGFDSPTVAYS 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 t a t 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 a t a a g a

3600 1145 1150 1155 1160 VLVVAKVEKGKSKKLKSVKE Cas9 1145 1150 1155 1160 VLVVAKVEKGKSKKLKSVKE Cas9 VLVVAKVEKGKSKKLKSVKE 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 a a g a g 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 t t t c t c

BsaBI* 3660 1165 1170 1175 1180 LLGITIMERSSFEKNPIDFL Cas9 1165 1170 1175 1180 LLGITIMERSSFEKNPIDFL Cas9 LLGITIMERSSFEKNPIDFL 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 g 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 a a a g a c

3720 1185 1190 1195 1200 EAKGYKEVKKDLIIKLPKYS Cas9 1185 1190 1195 1200 EAKGYKEVKKDLIIKLPKYS Cas9 EAKGYKEVKKDLIIKLPKYS 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 t a c t c 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 a t g a g g

BsmI 3780 1205 1210 1215 1220 LFELENGRKRMLASAGELQK Cas9 1205 1210 1215 1220 LFELENGRKRMLASAGELQK Cas9 LFELENGRKRMLASAGELQK 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 c a g a a 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 g t c t t c

3840 1225 1230 1235 1240 GNELALPSKYVNFLYLASHY Cas9 1225 1230 1235 1240 GNELALPSKYVNFLYLASHY Cas9 GNELALPSKYVNFLYLASHY 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 c a c t a t 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 g t g a t a

3900 1245 1250 1255 1260 EKLKGSPEDNEQKQLFVEQH Cas9 1245 1250 1255 1260 EKLKGSPEDNEQKQLFVEQH Cas9 EKLKGSPEDNEQKQLFVEQH 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 c a g c a c 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 g t c g t g

3960 1265 1270 1275 1280 KHYLDEIIEQISEFSKRVIL Cas9 1265 1270 1275 1280 KHYLDEIIEQISEFSKRVIL Cas9 KHYLDEIIEQISEFSKRVIL 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 a t c c 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 t a g g a c

4020 1285 1290 1295 1300 ADANLDKVLSAYNKHRDKPI Cas9 1285 1290 1295 1300 ADANLDKVLSAYNKHRDKPI Cas9 ADANLDKVLSAYNKHRDKPI 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 c c c a t 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 g g g t a g

4080 1305 1310 1315 1320 REQAENIIHLFTLTNLGAPA Cas9 1305 1310 1315 1320 REQAENIIHLFTLTNLGAPA Cas9 REQAENIIHLFTLTNLGAPA 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 c c t g c c 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 g g a c g g

4140 1325 1330 1335 1340 AFKYFDTTIDRKRYTSTKEV Cas9 1325 1330 1335 1340 AFKYFDTTIDRKRYTSTKEV Cas9 AFKYFDTTIDRKRYTSTKEV 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 a g g t 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 c t c c a c

4200 1345 1350 1355 1360 LDATLIHQSITGLYETRIDL Cas9 1345 1350 1355 1360 LDATLIHQSITGLYETRIDL Cas9 LDATLIHQSITGLYETRIDL 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 g a c c t g 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 c t g g a c

FseI 4260 1365 SQLGGD (in frame with Cas9) KRPAATKKAGQAKK Cas9 1365 SQLGGD 1 5 10 KRPAATKKAGQAKK Cas9 nucleoplasmin NLS SQLGGDKRPAATKKAGQAKK 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 a a a a a g 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 t t t t t c

4320 (in frame with Cas9) KKEFGSGEGRGSLLTCGDVE 15 KK EFGSG 140 145 150 EGRGSLLTCGDVE nucleoplasmin NLS T2A KKEFGSGEGRGSLLTCGDVE 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 g t c g a g 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 c a g c t c

4380 (in frame with Cas9) ENPGP 1a V 5 10 15 SKGEELFTGVVPIL EGFP 155 ENPGP 1a V 5 10 15 SKGEELFTGVVPIL T2A EGFP ENPGPVSKGEELFTGVVPIL g a g a a t c c t g g c c c a g t g a g c a a g g g c g a g g a g c t g t t c a c c g g g g t g g t g c c c a t c c t g c t c t t a g g a c c g g g t c a c t c g t t c c c g c t c c t c g a c a a g t g g c c c c a c c a c g g g t a g g a c

4440 20 25 30 35 VELDGDVNGHKFSVSGEGEG EGFP 20 25 30 35 VELDGDVNGHKFSVSGEGEG EGFP VELDGDVNGHKFSVSGEGEG g t c g a g c t g g a c g g c g a c g t a a a c g g c c a c a a g t t c a g c g t g t c c g g c g a g g g c g a g g g c c a g c t c g a c c t g c c g c t g c a t t t g c c g g t g t t c a a g t c g c a c a g g c c g c t c c c g c t c c c g

4500 40 45 50 55 DATYGKLTLKFICTTGKLPV EGFP 40 45 50 55 DATYGKLTLKFICTTGKLPV EGFP DATYGKLTLKFICTTGKLPV g a t g c c a c c t a c g g c a a g c t g a c c c t g a a g t t c a t c t g c a c c a c c g g c a a g c t g c c c g t g c t a c g g t g g a t g c c g t t c g a c t g g g a c t t c a a g t a g a c g t g g t g g c c g t t c g a c g g g c a c

4560 60 65 70 75 PWPTLVTTLTYGVQCFSRYP EGFP 60 65 70 75 PWPTLVTTLTYGVQCFSRYP EGFP PWPTLVTTLTYGVQCFSRYP c c c t g g c c c a c c c t c g t g a c c a c c c t g a c c t a c g g c g t g c a g t g c t t c a g c c g c t a c c c c g g g a c c g g g t g g g a g c a c t g g t g g g a c t g g a t g c c g c a c g t c a c g a a g t c g g c g a t g g g g

4620 80 85 90 95 DHMKQHDFFKSAMPEGYVQE EGFP 80 85 90 95 DHMKQHDFFKSAMPEGYVQE EGFP DHMKQHDFFKSAMPEGYVQE g a c c a c a t g a a g c a g c a c g a c t t c t t c a a g t c c g c c a t g c c c g a a g g c t a c g t c c a g g a g c t g g t g t a c t t c g t c g t g c t g a a g a a g t t c a g g c g g t a c g g g c t t c c g a t g c a g g t c c t c

4680 100 105 110 115 RTIFFKDDGNYKTRAEVKFE EGFP 100 105 110 115 RTIFFKDDGNYKTRAEVKFE EGFP C T G C T G C C G T T G A T G T T C T G EXFP-R RTIFFKDDGNYKTRAEVKFE c g c a c c a t c t t c t t c a a g g a c g a c g g c a a c t a c a a g a c c c g c g c c g a g g t g a a g t t c g a g g c g t g g t a g a a g a a g t t c c t g c t g c c g t t g a t g t t c t g g g c g c g g c t c c a c t t c a a g c t c

4740 120 125 130 135 GDTLVNRIELKGIDFKEDGN EGFP 120 125 130 135 GDTLVNRIELKGIDFKEDGN EGFP GDTLVNRIELKGIDFKEDGN g g c g a c a c c c t g g t g a a c c g c a t c g a g c t g a a g g g c a t c g a c t t c a a g g a g g a c g g c a a c c c g c t g t g g g a c c a c t t g g c g t a g c t c g a c t t c c c g t a g c t g a a g t t c c t c c t g c c g t t g

4800 140 145 150 155 ILGHKLEYNYNSHNVYIMAD EGFP 140 145 150 155 ILGHKLEYNYNSHNVYIMAD EGFP ILGHKLEYNYNSHNVYIMAD a t c c t g g g g c a c a a g c t g g a g t a c a a c t a c a a c a g c c a c a a c g t c t a t a t c a t g g c c g a c t a g g a c c c c g t g t t c g a c c t c a t g t t g a t g t t g t c g g t g t t g c a g a t a t a g t a c c g g c t g

4860 160 165 170 175 KQKNGIKVNFKIRHNIEDGS EGFP 160 165 170 175 KQKNGIKVNFKIRHNIEDGS EGFP KQKNGIKVNFKIRHNIEDGS a a g c a g a a g a a c g g c a t c a a g g t g a a c t t c a a g a t c c g c c a c a a c a t c g a g g a c g g c a g c t t c g t c t t c t t g c c g t a g t t c c a c t t g a a g t t c t a g g c g g t g t t g t a g c t c c t g c c g t c g

4920 180 185 190 195 VQLADHYQQNTPIGDGPVLL EGFP 180 185 190 195 VQLADHYQQNTPIGDGPVLL EGFP VQLADHYQQNTPIGDGPVLL g t g c a g c t c g c c g a c c a c t a c c a g c a g a a c a c c c c c a t c g g c g a c g g c c c c g t g c t g c t g c a c g t c g a g c g g c t g g t g a t g g t c g t c t t g t g g g g g t a g c c g c t g c c g g g g c a c g a c g a c

4980 200 205 210 215 PDNHYLSTQSALSKDPNEKR EGFP 200 205 210 215 PDNHYLSTQSALSKDPNEKR EGFP PDNHYLSTQSALSKDPNEKR c c c g a c a a c c a c t a c c t g a g c a c c c a g t c c g c c c t g a g c a a a g a c c c c a a c g a g a a g c g c g g g c t g t t g g t g a t g g a c t c g t g g g t c a g g c g g g a c t c g t t t c t g g g g t t g c t c t t c g c g

5040 220 225 230 235 DHMVLLEFVTAAGITLGMDE EGFP 220 225 230 235 DHMVLLEFVTAAGITLGMDE EGFP C A T G G T C C T G C T G G A G T T C G T G EGFP-C DHMVLLEFVTAAGITLGMDE g a t c a c a t g g t c c t g c t g g a g t t c g t g a c c g c c g c c g g g a t c a c t c t c g g c a t g g a c g a g c t a g t g t a c c a g g a c g a c c t c a a g c a c t g g c g g c g g c c c t a g t g a g a g c c g t a c c t g c t c

BsrGI 5100 LYK EF* EGFP bGH poly(A) signal LYK EF* EGFP G G A G C T G A C A C G G A A G A T BGH-rev LYKEF* 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 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 g a c a t g t t c c t t a a g a t t 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

5160 bGH poly(A) signal a g c c a t c t g t 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 t c g g t a g a c a 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

5220 bGH poly(A) signal c t g t c c t t t c 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 g a c a g g a a a 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

5280 bGH poly(A) signal t t c t g g g g g g 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 a g a c c c c c c 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

NotI 5340 bGH poly(A) signal AAV2 ITR AAV2 ITR a t g c t g g g g a 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 t a c g a c c c 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

5400 AAV2 ITR AAV2 ITR 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 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 g a g c g a g c g a 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

SbfIKasINarISfoIPluTI 5460 AAV2 ITR AAV2 ITR c g g c c t c a g t 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 g c c g g a g t 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

5520 f1 ori 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 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 a g g a a t g c g t 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

5580 f1 ori 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 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 g a c a t c g c c g 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

5640 f1 ori G A G G A A A G C G A A A G A A G G G A F1ori-R t g c c a g c g c c t 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 a c g g t c g c g g a 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

5700 f1 ori c g g c t t t c c c 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 g c c g a a a g g g 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

5760 f1 ori a c g g c a c c t c 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 t g c c g t g g a 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

5820 f1 ori G T G G A C T C T T F1ori-F c t g a t a g a c g 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 a c t a t c t g c 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

PsiI 5880 f1 ori G T T C C A A A C T G G F1ori-F 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 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 c a a g g t t t g a c c t t g t t g t g a 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

5940 f1 ori t t t g c c g a t t t c g g t 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 a a a c g g c t a a a g c c a 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

6000 f1 ori A T G T T A G A C G A G A C T pRS-marker t t t t a a c a a a 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 a a a a t t g t t t 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

6060 A C G G C pRS-marker t g c c g c a t a g 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 a c g g c g t a 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

6120 C C G G G A G C T G C A T G T G pGEX 3' t t g t c t g c t c 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 a a c a g a c g a g 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

6180 T C A G A G G pGEX 3' C G G A G C A C T A T G C G G A pBRforEco 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 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 g t c t c c a a a 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

6240 T A A pBRforEco a t t t t t a t a g 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 t a a a a a t a t c 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

6300 AmpR promoter 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 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 c c c t t t a c a c 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

6360 1 signal... MS AmpR promoter AmpR MS 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 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 c g a g t a c t c 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

6420 5 10 15 20 signal sequence IQHFRVALIPFFAAFCLPVF AmpR IQHFRVALIPFFAAFCLPVF 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 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 a t a a g t t g t 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

6480 signa... A 25 30 35 40 HPETLVKVKDAEDQLGARV AmpR AHPETLVKVKDAEDQLGARV 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 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 a c g a g t g g g t 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

6540 45 50 55 60 GYIELDLNSGKILESFRPEE AmpR GYIELDLNSGKILESFRPEE g g g t t a c a t c 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 c c c a a t g t a g 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

6600 65 70 75 80 RFPMMSTFKVLLCGAVLSRI AmpR C G A T A C A C C G C G C C A T A A T A Amp-R RFPMMSTFKVLLCGAVLSRI a c g t t t t c c a 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 c a a a a g g t 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

6660 85 90 95 100 DAGQEQLGRRIHYSQNDLVE AmpR DAGQEQLGRRIHYSQNDLVE t g a c g c c g g g 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 a c t g c g g c c c 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

6720 105 110 115 120 YSPVTEKHLTDGMTVRELCS AmpR YSPVTEKHLTDGMTVRELCS g t a c t c a c c a 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 c a t g a g t g g t 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

PvuI 6780 125 130 135 140 AAITMSDNTAANLLLTTIGG AmpR AAITMSDNTAANLLLTTIGG t g c t g c c a t a 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 g a c g g t a t 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

6840 145 150 155 160 PKELTAFLHNMGDHVTRLDR AmpR PKELTAFLHNMGDHVTRLDR 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 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 g g c t t c c t c 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

6900 165 170 175 180 WEPELNEAIPNDERDTTMPV AmpR WEPELNEAIPNDERDTTMPV 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 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 a c c c t t g g c 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

FspI 6960 185 190 195 200 AMATTLRKLLTGELLTLASR AmpR AMATTLRKLLTGELLTLASR a g c a a t g g c a 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 t c g t t a c c g t 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

7020 205 210 215 220 QQLIDWMEADKVAGPLLRSA AmpR QQLIDWMEADKVAGPLLRSA g c a a c a a t t a 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 g t t g t t a a 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

SacII 7080 225 230 235 240 LPAGWFIADKSGAGERGSRG AmpR LPAGWFIADKSGAGERGSRG c c t t c c g g c t 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 g g a a g g c c g 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

7140 245 250 255 260 IIAALGPDGKPSRIVVIYTT AmpR IIAALGPDGKPSRIVVIYTT t a t c a t t g c a 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 a t a g t a a c g t 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

7200 265 270 275 280 GSQATMDERNRQIAEIGASL AmpR GSQATMDERNRQIAEIGASL 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 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 c c c c t c a g t c 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

7260 285 IKHW* AmpR IKHW* g a t t a a g c a t 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 c t a a t t c g t a 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

7320 a c t t c a t t t t 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 t g a a g t a a 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

7380 a a t c c c t t a a 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 t t a g g g a a t t 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

7440 ori 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 a a c a a a a a a a c c a 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 g t t t g t t t t t t t g g t g g

7500 ori 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 c t t t t t c c g a a g g t a a c 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 g a a a a a g g c t t c c a t t g

7560 ori 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 t a g c c g t a g t t a g g c c a 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 a t c g g c a t c a a t c c g g t

7620 ori 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 c t a a t c c t g t t a c c a 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 g a t t a g g a c a a t g g t c a

7680 ori 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 t c a a g a c g a t a g t t a c c 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 a g t t c t g c t a t c a a t g g

7740 ori 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 c a g c c c a g c t t g g a g c 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 g t c g g g t c g a a c c t c g c

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

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

7920 ori G G G A A A C G C C T G G T A T C T T T pBR322ori-F 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 g t c g g g t t t c g c c a c c t 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 c a g c c c a a a g c g g t g g a

7980 ori 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 a g c c t a t g g a a a a a c g c 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 t c g g a t a c c t t t t t g c g

AflIIIPciI 8040 U6 promoter G A G G hU6-F 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 t t g c t c a c a t g t g a g 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 a a a c g a g t g t a c a c t c c

8100 U6 promoter G C C T A T T T C C C A T G A T T hU6-F 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 a t a a 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 t a t t

8160 U6 promoter 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 a a g 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 t t c a

NdeI 8220 U6 promoter G A C T A T C A T A T G C LKO.1 5' 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 a t g c 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 t a c g

8280 U6 promoter T T A C C G T LKO.1 5' 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 c g a a 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 g c t t

8340 U6 promoter gRNA scaffold a c a c c g a g t g t c t g a g t c c c c g g c 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 g t g g c t c a c a g a c t c a g g g g c c g 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

8400 gRNA scaffold 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 a 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 t c t

8460 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 a a a 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 t t t

XbaIAcc65IKpnIEnd (8476) 3ʹ 5ʹ 8476 t g g c t c t a g a g g t a c c a c c g a g a t c t c c a t 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.