Addgene pLentiCRISPR sgAMDHD1 Sequencing Result - Sequence Analyzer Skip to main content
Addgene

Sequence Analyzer: pLentiCRISPR sgAMDHD1 Sequencing Result


Map View

L4440 (11,549 .. 11,566) PciI (11,451) pBR322ori-F (11,296 .. 11,315) Amp-R (9994 .. 10,013) AmpR promoter F1ori-F (9487 .. 9508) F1ori-R (9277 .. 9296) M13/pUC Forward (9041 .. 9063) M13 Forward (9032 .. 9049) M13 fwd T7 (9003 .. 9022) T7 promoter AvrII (8982) SfiI (8935) SV40 ori EBV-rev (8778 .. 8797) SV40 poly(A) signal SV40pA-R (8724 .. 8743) SnaBI (8613) 3' LTR (ΔU3) WPRE-R (7769 .. 7789) MluI (7701) CsiI - SexAI * (7668) Puro-F (7597 .. 7617) BstEII (7235) RsrII (7217) BsiWI (7157) PflFI - Tth111I (7143) Puro-R (7101 .. 7120) P2A BmtI (7033) NheI (7029) nucleoplasmin NLS PshAI (6982) BsaBI * (6375) CAP binding site lac promoter M13/pUC Reverse (172 .. 194) lac operator M13 Reverse (191 .. 207) M13 rev T3 (226 .. 246) T3 promoter 5' LTR (truncated) HIV-1 Ψ NruI * (880) NotI (1191) BbvCI (1467) AleI (1620) gp41 peptide hU6-F (1986 .. 2006) LKO.1 5' (2157 .. 2176) NdeI (2164) gRNA scaffold EcoRI (2338) XbaI * (2342) cPPT/CTS PaeR7I - PspXI - XhoI (2539) AgeI (2624) EF-1α core promoter BamHI (2790) FLAG SV40 NLS PspOMI (4232) ApaI (4236) EcoRV (4730) PmlI (5666) pLentiCRISPR sgAMDHD1 11,590 bp

Sequence View

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

120 CAP binding site g a c t g g a a a g c g g g c a g t g a g c g c a a c g c a a t t a a t g t g a g t t a g c t c a c t c a t t a g g c a c t g a c c t t t c g c c c g t c a c t c g c g t t g c g t t a a t t a c a c t c a a t c g a g t g a g t a a t c c g t

180 -35 -10 lac promoter lac operator A G C G G A T A A M13/pUC Reverse c c c c a g g c t t t a c a c t t t a t g c t t c c g g c t c g t a t g t t g t g t g g a a t t g t g a g c g g a t a a g g g g t c c g a a a t g t g a a a t a c g a a g g c c g a g c a t a c a a c a c a c c t t a a c a c t c g c c t a t t

240 lac operator M13 rev T3 promoter C A A T T T C A C A C A G G M13/pUC Reverse C A G G A A A C A G C T A T G A C M13 Reverse G C A A T T A A C C C T C A C T3 c a a t t t c a c a c a g g a a a c a g c t a t g a c c a t g a t t a c g c c a a g c g c g c a a t t a a c c c t c a c g t t a a a g t g t g t c c t t t g t c g a t a c t g g t a c t a a t g c g g t t c g c g c g t t a a t t g g g a g t g

300 T3 promoter RSV promoter T A A A G G T3 t a a a g g g a a c a a a a g c t g g a g c t g c a a g c t t a a t g t a g t c t t a t g c a a t a c t c t t g t a g t a t t t c c c t t g t t t t c g a c c t c g a c g t t c g a a t t a c a t c a g a a t a c g t t a t g a g a a c a t c a

360 RSV promoter c t t g c a a c a t g g t a a c g a t g a g t t a g c a a c a t g c c t t a c a a g g a g a g a a a a a g c a c c g t g g a a c g t t g t a c c a t t g c t a c t c a a t c g t t g t a c g g a a t g t t c c t c t c t t t t t c g t g g c a c

420 RSV promoter c a t g c c g a t t g g t g g a a g t a a g g t g g t a c g a t c g t g c c t t a t t a g g a a g g c a a c a g a c g g g t a c g g c t a a c c a c c t t c a t t c c a c c a t g c t a g c a c g g a a t a a t c c t t c c g t t g t c t g c c

480 RSV promoter g t c t g a c a t g g a t t g g a c g a a c c a c t g a a t t g c c g c a t t g c a g a g a t a t t g t a t t t a a g t c a g a c t g t a c c t a a c c t g c t t g g t g a c t t a a c g g c g t a a c g t c t c t a t a a c a t a a a t t c a

540 RSV promoter 5' LTR (truncated) g c c t a g c t c g a t a c a t a a a c g g g t c t c t c t g g t t a g a c c a g a t c t g a g c c t g g g a g c t c t c g g a t c g a g c t a t g t a t t t g c c c a g a g a g a c c a a t c t g g t c t a g a c t c g g a c c c t c g a g a

600 5' LTR (truncated) c t g g c t a a c t a g g g a a c c c a c t g c t t a a g c c t c a a t a a a g c t t g c c t t g a g t g c t t c a a g g a c c g a t t g a t c c c t t g g g t g a c g a a t t c g g a g t t a t t t c g a a c g g a a c t c a c g a a g t t c

660 5' LTR (truncated) t a g t g t g t g c c c g t c t g t t g t g t g a c t c t g g t a a c t a g a g a t c c c t c a g a c c c t t t t a g t a t c a c a c a c g g g c a g a c a a c a c a c t g a g a c c a t t g a t c t c t a g g g a g t c t g g g a a a a t c a

720 5' LTR (truncated) c a g t g t g g a a a a t c t c t a g c a g t g g c g c c c g a a c a g g g a c t t g a a a g c g a a a g g g a a a c c g t c a c a c c t t t t a g a g a t c g t c a c c g c g g g c t t g t c c c t g a a c t t t c g c t t t c c c t t t g g

780 HIV-1 Ψ a g a g g a g c t c t c t c g a c g c a g g a c t c g g c t t g c t g a a g c g c g c a c g g c a a g a g g c g a g g g t c t c c t c g a g a g a g c t g c g t c c t g a g c c g a a c g a c t t c g c g c g t g c c g t t c t c c g c t c c c

840 HIV-1 Ψ g c g g c g a c t g g t g a g t a c g c c a a a a a t t t t g a c t a g c g g a g g c t a g a a g g a g a g a g a t g g c g c c g c t g a c c a c t c a t g c g g t t t t t a a a a c t g a t c g c c t c c g a t c t t c c t c t c t c t a c c

NruI* 900 HIV-1 Ψ g t g c g a g a g c g t c a g t a t t a a g c g g g g g a g a a t t a g a t c g c g a t g g g a a a a a a t t c g g t t c a c g c t c t c g c a g t c a t a a t t c g c c c c c t c t t a a t c t a g c g c t a c c c t t t t t t a a g c c a a

960 a a g g c c a g g g g g a a a g a a a a a a t a t a a a t t a a a a c a t a t a g t a t g g g c a a g c a g g g a g c t t t c c g g t c c c c c t t t c t t t t t t a t a t t t a a t t t t g t a t a t c a t a c c c g t t c g t c c c t c g a

1020 a g a a c g a t t c g c a g t t a a t c c t g g c c t g t t a g a a a c a t c a g a a g g c t g t a g a c a a a t a c t t c t t g c t a a g c g t c a a t t a g g a c c g g a c a a t c t t t g t a g t c t t c c g a c a t c t g t t t a t g a

1080 g g g a c a g c t a c a a c c a t c c c t t c a g a c a g g a t c a g a a g a a c t t a g a t c a t t a t a t a a t a c c c c t g t c g a t g t t g g t a g g g a a g t c t g t c c t a g t c t t c t t g a a t c t a g t a a t a t a t t a t g

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

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

1260 MRDNWRSELYKY t c t t c a g a c c t g g a g g a g g a g a t a t g a g g g a c a a t t g g a g a a g t g a a t t a t a t a a a t a t a a g a a g t c t g g a c c t c c t c c t c t a t a c t c c c t g t t a a c c t c t t c a c t t a a t a t a t t t a t a t

1320 KVVKIEPLGVAPTKAKRRVV a a g t a g t a a a a a t t g a a c c a t t a g g a g t a g c a c c c a c c a a g g c a a a g a g a a g a g t g g t g c t t c a t c a t t t t t a a c t t g g t a a t c c t c a t c g t g g g t g g t t c c g t t t c t c t t c t c a c c a c g

1380 RRE QREKRAVGIGALFLGFLGAA a g a g a g a a a a a a g a g c a g t g g g a a t a g g a g c t t t g t t c c t t g g g t t c t t g g g a g c a g c a g t c t c t c t t t t t t c t c g t c a c c c t t a t c c t c g a a a c a a g g a a c c c a a g a a c c c t c g t c g t c

1440 RRE GSTMGAASMTLTVQARQLLS g a a g c a c t a t g g g c g c a g c g t c a a t g a c g c t g a c g g t a c a g g c c a g a c a a t t a t t g t c t g c t t c g t g a t a c c c g c g t c g c a g t t a c t g c g a c t g c c a t g t c c g g t c t g t t a a t a a c a g a c

BbvCI 1500 RRE GIVQQQNNLLRAIEAQQHLL g t a t a g t g c a g c a g c a g a a c a a t t t g c t g a g g g c t a t t g a g g c g c a a c a g c a t c t g t t g c c a t a t c a c g t c g t c g t c t t g t t a a a c g a c t c c c g a t a a c t c c g c g t t g t c g t a g a c a a c g

1560 RRE QLTVWGIKQLQARILAVERY a a c t c a c a g t c t g g g g c a t c a a g c a g c t c c a g g c a a g a a t c c t g g c t g t g g a a a g a t a c c t t g a g t g t c a g a c c c c g t a g t t c g t c g a g g t c c g t t c t t a g g a c c g a c a c c t t t c t a t g g

AleI 1620 RRE LKDQQLLGIWGCSGKLICTT t a a a g g a t c a a c a g c t c c t g g g g a t t t g g g g t t g c t c t g g a a a a c t c a t t t g c a c c a c t g a t t t c c t a g t t g t c g a g g a c c c c t a a a c c c c a a c g a g a c c t t t t g a g t a a a c g t g g t g a c

1680 AVPWNASWSNKSLEQIWNHT c t g t g c c t t g g a a t g c t a g t t g g a g t a a t a a a t c t c t g g a a c a g a t t t g g a a t c a c a c g a g a c a c g g a a c c t t a c g a t c a a c c t c a t t a t t t a g a g a c c t t g t c t a a a c c t t a g t g t g c t

1740 TWMEWDREINNYTSLIHSLI c c t g g a t g g a g t g g g a c a g a g a a a t t a a c a a t t a c a c a a g c t t a a t a c a c t c c t t a a t t g g g a c c t a c c t c a c c c t g t c t c t t t a a t t g t t a a t g t g t t c g a a t t a t g t g a g g a a t t a a c

1800 1 5 10 KNEQELLELDKW gp41 peptide EESQNQQEKNEQELLELDKW a a g a a t c g c a a a a c c a g c a a g a a a a g a a t g a a c a a g a a t t a t t g g a a t t a g a t a a a t g g g t t c t t a g c g t t t t g g t c g t t c t t t t c t t a c t t g t t c t t a a t a a c c t t a a t c t a t t t a c c c

1860 15 ASL (in frame with gp41 peptide) WNWFNITNWLWYIKLFI gp41 peptide ASLWNWFNITNWLWYIKLFI c a a g t t t g t g g a a t t g g t t t a a c a t a a c a a a t t g g c t g t g g t a t a t a a a a t t a t t c a t a a g t t c a a a c a c c t t a a c c a a a t t g t a t t g t t t a a c c g a c a c c a t a t a t t t t a a t a a g t a t t

1920 (in frame with gp41 peptide) MIVGGLVGLRIVFAVLSIVN MIVGGLVGLRIVFAVLSIVN t g a t a g t a g g a g g c t t g g t a g g t t t a a g a a t a g t t t t t g c t g t a c t t t c t a t a g t g a a t a a c t a t c a t c c t c c g a a c c a t c c a a a t t c t t a t c a a a a a c g a c a t g a a a g a t a t c a c t t a t

1980 (in frame with gp41 peptide) RVRQGYSPLSFQTHLPTPRG RVRQGYSPLSFQTHLPTPRG g a g t t a g g c a g g g a t a t t c a c c a t t a t c g t t t c a g a c c c a c c t c c c a a c c c c g a g g g g a c c t c a a t c c g t c c c t a t a a g t g g t a a t a g c a a a g t c t g g g t g g a g g g t t g g g g c t c c c c t g

2040 (in frame with gp41 peptide) PREGLFPMIPSYLHIRYKAV 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 PREGLFPMIPSYLHIRYKAV c c a g a 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 g t c t 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

2100 (in frame with gp41 peptide) REIIRINLTVNTKILVQNT* U6 promoter REIIRINLTVNTKILVQNT* g a g a g a t a a t t a 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 c t c t c t a t t a a t 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

2160 U6 promoter G A C T LKO.1 5' g t a g a a a g t a a t a a t t t c t t g g g t a g t t t g c a g t t t t a a a a t t a t g t t t t a a a a t g g a c t c a t c t t t c a t t a t t a a a g a a c c c a t c a a a c g t c a a a a t t t t a a t a c a a a a t t t t a c c t g a

NdeI 2220 U6 promoter A T C A T A T G C T T A C C G T LKO.1 5' a t c a t a t g c t t a c c g t a a c t t g a a a g t a t t t c g a t t t c t t g g c t t t a t a t a t c t t g t g g a t a g t a t a c g a a t g g c a t t g a a c t t t c a t a a a g c t a a a g a a c c g a a a t a t a t a g a a c a c c t

2280 U6 promoter gRNA scaffold a a g g a c g a a a c a c c g a t g g a a a t t c a c c a g g c c g g 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 t t c c t g c t t t g t g g c t a c c t t t a a g t g g t c c g g c c 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

EcoRI 2340 gRNA scaffold a a a a t a a g g c t a g t c c g t t a t c a a c t t g a a a a a g t g g c a c c g a g t c g g t g c t t t t t t g a a t t t t a t t c c g a t c a g g c a a t a g t t g a a c t t t t t c a c c g t g g c t c a g c c a c g a a a a a a c t t

XbaI* 2400 cPPT/CTS t t c t a g a t c t t g a g a c a a a t g g c a g t a t t c a t c c a c a a t t t t a a a a g a a a a g g g g g g a t t a a g a t c t a g a a c t c t g t t t a c c g t c a t a a g t a g g t g t t a a a a t t t t c t t t t c c c c c c t a a

2460 cPPT/CTS g g g g g g t a c a g t g c a g g g g a a a g a a t a g t a g a c a t a a t a g c a a c a g a c a t a c a a a c t a a a c c c c c c a t g t c a c g t c c c c t t t c t t a t c a t c t g t a t t a t c g t t g t c t g t a t g t t t g a t t t

2520 cPPT/CTS *LSLL g a a t t a c a a a a a c a a a t t a c a a a a a t t c a a a a t t t t c g g g t t t a t t a c a g g g a c a g c a g a c t t a a t g t t t t t g t t t a a t g t t t t t a a g t t t t a a a a g c c c a a a t a a t g t c c c t g t c g t c t

PaeR7IPspXIXhoI 2580 EF-1 α core promoter SGSQRRSSHSRHGDTPLACM g a t c c a c t t t g g c g c c g g c t c g a g t g g c t c c g g t g c c c g t c a g t g g g c a g a g c g c a c a t c c t a g g t g a a a c c g c g g c c g a g c t c a c c g a g g c c a c g g g c a g t c a c c c g t c t c g c g t g t a g

AgeI 2640 EF-1 α core promoter AWLGRSTPPLPRCNFRHRSF g c c c a c a g t c c c c g a g a a g t t g g g g g g a g g g g t c g g c a a t t g a a c c g g t g c c t a g a g a a g c g g g t g t c a g g g g c t c t t c a a c c c c c c t c c c c a g c c g t t a a c t t g g c c a c g g a t c t c t t c

2700 EF-1 α core promoter TARPLSPFHHRTSAGGKERP g t g g c g c g g g g t a a a c t g g g a a a g t g a t g t c g t g t a c t g g c t c c g c c t t t t t c c c g a g g g c a c c g c g c c c c a t t t g a c c c t t t c a c t a c a g c a c a t g a c c g a g g c g g a a a a a g g g c t c c c

2760 EF-1 α core promoter HPLVTYLHLLRRSREKECRT t g g g g g a g a a c c g t a t a t a a g t g c a g t a g t c g c c g t g a a c g t t c t t t t t c g c a a c g g g t t a c c c c c t c t t g g c a t a t a t t c a c g t c a t c a g c g g c a c t t g c a a g a a a a a g c g t t g c c c a a

BamHI 2820 1 5 DYKDD EF-1 α core promoter FLAG Kozak sequence MDYKDD QRWFVPTTVRSGGGHIVFVI t g c c g c c a g a a c a c a g g t g t c g t g a c g c g g g a t c c g c c a c c a t g g a t t a c a a a g a c g a t g a c g g c g g t c t t g t g t c c a c a g c a c t g c g c c c t a g g c g g t g g t a c c t a a t g t t t c t g c t a c

2880 DDK MA 130 PKKKRKV (in frame with SV40 NLS) GIHGVPAA FLAG SV40 NLS Cas9 Cas9 DDKMAPKKKRKVGIHGVPAA VILHGWLLLPLDTDVSDWCG a c g a t a a g a t g g c c c c a a a g a a g a a g c g g a a g g t c g g t a t c c a c g g a g t c c c a g c a g c c g t g c t a t t c t a c c g g g g t t t c t t c t t c g c c t t c c a g c c a t a g g t g c c t c a g g g t c g t c g g c

2940 5 10 15 20 DKKYSIGLDIGTNSVGWAVI Cas9 5 10 15 20 DKKYSIGLDIGTNSVGWAVI Cas9 DKKYSIGLDIGTNSVGWAVI VLLVADAQVDAGVRHAPGHD a c a a g a a g t a c a g c a t c g g c c t g g a c a t c g g c a c c a a c t c t g t g g g c t g g g c c g t g a t c a t g t t c t t c a t g t c g t a g c c g g a c c t g t a g c c g t g g t t g a g a c a c c c g a c c c g g c a c t a g t

3000 25 30 35 40 TDEYKVPSKKFKVLGNTDRH Cas9 25 30 35 40 TDEYKVPSKKFKVLGNTDRH Cas9 TDEYKVPSKKFKVLGNTDRH GVLVLHGALFELHQAVGVPV c c g a c g a g t a c a a g g t g c c c a g c a a g a a a t t c a a g g t g c t g g g c a a c a c c g a c c g g c a c a g g c t g c t c a t g t t c c a c g g g t c g t t c t t t a a g t t c c a c g a c c c g t t g t g g c t g g c c g t g t

3060 45 50 55 60 SIKKNLIGALLFDSGETAEA Cas9 45 50 55 60 SIKKNLIGALLFDSGETAEA Cas9 SIKKNLIGALLFDSGETAEA ADLLVQDSGQQEVAAFCGLG g c a t c a a g a a g a a c c t g a t c g g a g c c c t g c t g t t c g a c a g c g g c g a a a c a g c c g a g g c c a c g t a g t t c t t c t t g g a c t a g c c t c g g g a c g a c a a g c t g t c g c c g c t t t g t c g g c t c c g g t

3120 65 70 75 80 TRLKRTARRRYTRRKNRICY Cas9 65 70 75 80 TRLKRTARRRYTRRKNRICY Cas9 TRLKRTARRRYTRRKNRICY GPQLSGGSSSVGSPLVPDAI c c c g g c t g a a g a g a a c c g c c a g a a g a a g a t a c a c c a g a c g g a a g a a c c g g a t c t g c t a t c g g g c c g a c t t c t c t t g g c g g t c t t c t t c t a t g t g g t c t g c c t t c t t g g c c t a g a c g a t a g

3180 85 90 95 100 LQEIFSNEMAKVDDSFFHRL Cas9 85 90 95 100 LQEIFSNEMAKVDDSFFHRL Cas9 LQEIFSNEMAKVDDSFFHRL QLLDEAVLHGLHVVAEEVSQ t g c a a g a g a t c t t c a g c a a c g a g a t g g c c a a g g t g g a c g a c a g c t t c t t c c a c a g a c t g g a c g t t c t c t a g a a g t c g t t g c t c t a c c g g t t c c a c c t g c t g t c g a a g a a g g t g t c t g a c c

3240 105 110 115 120 EESFLVEEDKKHERHPIFGN Cas9 105 110 115 120 EESFLVEEDKKHERHPIFGN Cas9 EESFLVEEDKKHERHPIFGN FLGEQHFLILLVLPVGDEAV a a g a g t c c t t c c t g g t g g a a g a g g a t a a g a a g c a c g a g c g g c a c c c c a t c t t c g g c a a c a t t c t c a g g a a g g a c c a c c t t c t c c t a t t c t t c g t g c t c g c c g t g g g g t a g a a g c c g t t g t

3300 125 130 135 140 IVDEVAYHEKYPTIYHLRKK Cas9 125 130 135 140 IVDEVAYHEKYPTIYHLRKK Cas9 IVDEVAYHEKYPTIYHLRKK DHVLHGVVLLVGGDVVQSLF t c g t g g a c g a g g t g g c c t a c c a c g a g a a g t a c c c c a c c a t c t a c c a c c t g a g a a a g a a a c a g c a c c t g c t c c a c c g g a t g g t g c t c t t c a t g g g g t g g t a g a t g g t g g a c t c t t t c t t t g

3360 145 150 155 160 LVDSTDKADLRLIYLALAHM Cas9 145 150 155 160 LVDSTDKADLRLIYLALAHM Cas9 LVDSTDKADLRLIYLALAHM QHVAGVLGVQPQDIQGQGVH t g g t g g a c a g c a c c g a c a a g g c c g a c c t g c g g c t g a t c t a t c t g g c c c t g g c c c a c a t g a a c c a c c t g t c g t g g c t g t t c c g g c t g g a c g c c g a c t a g a t a g a c c g g g a c c g g g t g t a c t

3420 165 170 175 180 IKFRGHFLIEGDLNPDNSDV Cas9 165 170 175 180 IKFRGHFLIEGDLNPDNSDV Cas9 IKFRGHFLIEGDLNPDNSDV DLEPAVEQDLAVQVGVVAVH t c a a g t t c c g g g g c c a c t t c c t g a t c g a g g g c g a c c t g a a c c c c g a c a a c a g c g a c g t g g a g t t c a a g g c c c c g g t g a a g g a c t a g c t c c c g c t g g a c t t g g g g c t g t t g t c g c t g c a c c

3480 185 190 195 200 DKLFIQLVQTYNQLFEENPI Cas9 185 190 195 200 DKLFIQLVQTYNQLFEENPI Cas9 DKLFIQLVQTYNQLFEENPI VLQEDLQHLGVVLQELFVGD a c a a g c t g t t c a t c c a g c t g g t g c a g a c c t a c a a c c a g c t g t t c g a g g a a a a c c c c a t c a t g t t c g a c a a g t a g g t c g a c c a c g t c t g g a t g t t g g t c g a c a a g c t c c t t t t g g g g t a g t

3540 205 210 215 220 NASGVDAKAILSARLSKSRR Cas9 205 210 215 220 NASGVDAKAILSARLSKSRR Cas9 NASGVDAKAILSARLSKSRR VGAAHVGLGDQRGSQALASP a c g c c a g c g g c g t g g a c g c c a a g g c c a t c c t g t c t g c c a g a c t g a g c a a g a g c a g a 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

3600 225 230 235 240 LENLIAQLPGEKKNGLFGNL Cas9 225 230 235 240 LENLIAQLPGEKKNGLFGNL Cas9 LENLIAQLPGEKKNGLFGNL QFIQDGLQGALLLIAQESVQ t g g a a a a t c t g a t c g c c c a g c t g c c c g g c g a g a a g a a g a a t g g c c t g t t c g g a a a c c t g a a c c t t t t a g a c t a g c g g g t c g a c g g g c c g c t c t t c t t c t t a c c g g a c a a g c c t t t g g a c t

3660 245 250 255 260 IALSLGLTPNFKSNFDLAED Cas9 245 250 255 260 IALSLGLTPNFKSNFDLAED Cas9 IALSLGLTPNFKSNFDLAED NGQAQAQGGVELAVEVQGLI t t g c c c t g a g c c t g g g c c t g a c c c c c a a c t t c a a g a g c a a c t t c g a c c t g g c c g a g g a t g a a c g g g a c t c g g a c c c g g a c t g g g g g t t g a a g t t c t c g t t g a a g c t g g a c c g g c t c c t a c

3720 265 270 275 280 AKLQLSKDTYDDDLDNLLAQ Cas9 265 270 275 280 AKLQLSKDTYDDDLDNLLAQ Cas9 AKLQLSKDTYDDDLDNLLAQ GFQLQALVGVVVVQVVQQGL c c a a a c t g c a g c t g a g c a a g g a c a c c t a c g a c g a c g a c c t g g a c a a c c t g c t g g c c c a g a g g t t t g a c g t c g a c t c g t t c c t g t g g a t g c t g c t g c t g g a c c t g t t g g a c g a c c g g g t c t

3780 285 290 295 300 IGDQYADLFLAAKNLSDAIL Cas9 285 290 295 300 IGDQYADLFLAAKNLSDAIL Cas9 IGDQYADLFLAAKNLSDAIL DAVLVGVQKQGGLVQGVGDQ t c g g c g a c c a g t a c g c c g a c c t g t t t c t g g c c g c c a a g a a c c t g t c c g a c g c c a t c c t g c a g c c g c t g g t c a t g c g g c t g g a c a a a g a c c g g c g g t t c t t g g a c a g g c t g c g g t a g g a c g

3840 305 310 315 320 LSDILRVNTEITKAPLSASM Cas9 305 310 315 320 LSDILRVNTEITKAPLSASM Cas9 LSDILRVNTEITKAPLSASM QAVDQSHVGLDGLGGQAGRH t g a g c g a c a t c c t g a g a g t g a a c a c c g a g a t c a c c a a g g c c c c c c t g a g c g c c t c t a t g a a c t c g c t g t a g g a c t c t c a c t t g t g g c t c t a g t g g t t c c g g g g g g a c t c g c g g a g a t a c t

3900 325 330 335 340 IKRYDEHHQDLTLLKALVRQ Cas9 325 330 335 340 IKRYDEHHQDLTLLKALVRQ Cas9 IKRYDEHHQDLTLLKALVRQ DLSVVLVVLVQGQQFSEHPL t c a a g a g a t a c g a c g a g c a c c a c c a g g a c c t g a c c c t g c t g a a a g c t c t c g t g c g g 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

3960 345 350 355 360 QLPEKYKEIFFDQSKNGYAG Cas9 345 350 355 360 QLPEKYKEIFFDQSKNGYAG Cas9 QLPEKYKEIFFDQSKNGYAG LQRLLVFLNEEVLALVAVGA a g c t g c c t g a g a a g t a c a a a g a g a t t t t c t t c g a c c a g a g c a a g a a c g g c t a c g c c g g c t t c g a c g g a c t c t t c a t g t t t c t c t a a a a g a a g c t g g t c t c g t t c t t g c c g a t g c g g c c g a

4020 365 370 375 380 YIDGGASQEEFYKFIKPILE Cas9 365 370 375 380 YIDGGASQEEFYKFIKPILE Cas9 YIDGGASQEEFYKFIKPILE VNVASGALFLEVLEDLGDQF a c a t t g a c g g c g g a g c c a g c c a g g a a g a g t t c t a c a a g t t c a t c a a g c c c a t c c t g g a a a t g t a a c t g c c g c c t c g g t c g g t c c t t c t c a a g a t g t t c a a g t a g t t c g g g t a g g a c c t t t

4080 385 390 395 400 KMDGTEELLVKLNREDLLRK Cas9 385 390 395 400 KMDGTEELLVKLNREDLLRK Cas9 KMDGTEELLVKLNREDLLRK LHVAGLFQEHLQVSLVQQPL a g a t g g a c g g c a c c g a g g a a c t g c t c g t g a a g c t g a a c a g a g a g g a c c t g c t g c g g a a g c t c t a c c t g c c g t g g c t c c t t g a c g a g c a c t t c g a c t t g t c t c t c c t g g a c g a c g c c t t c g

4140 405 410 415 420 QRTFDNGSIPHQIHLGELHA Cas9 405 410 415 420 QRTFDNGSIPHQIHLGELHA Cas9 QRTFDNGSIPHQIHLGELHA LPGEVVAADGVLDVQSLQVG a g c g g a c c t t c g a c a a c g g c a g c a t c c c c c a c c a g a t c c a c c t g g g a g a g c t g c a c g c c a t c g c c t g g a a g c t g t t g c c g t c g t a g g g g g t g g t c t a g g t g g a c c c t c t c g a c g t g c g g t

4200 425 430 435 440 ILRRQEDFYPFLKDNREKIE Cas9 425 430 435 440 ILRRQEDFYPFLKDNREKIE Cas9 ILRRQEDFYPFLKDNREKIE NQPPLFIKVWEQLVVPFLDL t t c t g c g g c g g c a g g a a g a t t t t t a c c c a t t c c t g a a g g a c a a c c g g g a a a a g a t c g a g a a a g a c g c c g c c g t c c t t c t a a a a a t g g g t a a g g a c t t c c t g t t g g c c c t t t t c t a g c t c t

PspOMIApaI 4260 445 450 455 460 KILTFRIPYYVGPLARGNSR Cas9 445 450 455 460 KILTFRIPYYVGPLARGNSR Cas9 KILTFRIPYYVGPLARGNSR LDQGEADGVVHARQGPSVAS a g a t c c t g a c c t t c c g c a t c c c c t a c t a c g t g g g c c c t c t g g c c a g g g g a a a c a g c 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

4320 465 470 475 480 FAWMTRKSEETITPWNFEEV Cas9 465 470 475 480 FAWMTRKSEETITPWNFEEV Cas9 FAWMTRKSEETITPWNFEEV EGPHGSLALFGDGGPVELFH t c g c c t g g a t g a c c a g a a a g a g c g a g g a a a c c a t c a c c c c c t g g a a c t t c g a g g a a g t g g a g c g g a c c t a c t g g t c t t t c t c g c t c c t t t g g t a g t g g g g g a c c t t g a a g c t c c t t c a c c

4380 485 490 495 500 VDKGASAQSFIERMTNFDKN Cas9 485 490 495 500 VDKGASAQSFIERMTNFDKN Cas9 VDKGASAQSFIERMTNFDKN HVLASGGLAEDLPHGVEILV t g g a c a a g g g c g c t t c c g c c c a g a g c t t c a t c g a g c g g a t g a c c a a c t t c g a t a a g a a c c a c c t g t t c c c g c g a a g g c g g g t c t c g a a g t a g c t c g c c t a c t g g t t g a a g c t a t t c t t g g

4440 505 510 515 520 LPNEKVLPKHSLLYEYFTVY Cas9 505 510 515 520 LPNEKVLPKHSLLYEYFTVY Cas9 LPNEKVLPKHSLLYEYFTVY QGVLLHQGLVAQQVLVEGHI t g c c c a a c g a g a a g g t g c t g c c c a a g c a c a g c c t g c t g t a c g a g t a c t t c a c c g t g t 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

4500 525 530 535 540 NELTKVKYVTEGMRKPAFLS Cas9 525 530 535 540 NELTKVKYVTEGMRKPAFLS Cas9 NELTKVKYVTEGMRKPAFLS VLQGFHFVHGLSHSLGGEQA a c g a g c t g a c c a a a g t g a a a t a c g t g a c c g a g g g a a t g a g a a a g c c c g c c t t c c t g a g c g t g c t c g a c t g g t t t c a c t t t a t g c a c t g g c t c c c t t a c t c t t t c g g g c g g a a g g a c t c g c

4560 545 550 555 560 GEQKKAIVDLLFKTNRKVTV Cas9 545 550 555 560 GEQKKAIVDLLFKTNRKVTV Cas9 GEQKKAIVDLLFKTNRKVTV ALLFLGDHVQQELGVPFHGH g c g a g c a g a a a a a g g c c a t c g t g g a c c t g c t g t t c a a g a c c a a c c g g a a a g t g a c c g t g a c g c t c g t c t t t t t c c g g t a g c a c c t g g a c g a c a a g t t c t g g t t g g c c t t t c a c t g g c a c t

4620 565 570 575 580 KQLKEDYFKKIECFDSVEIS Cas9 565 570 575 580 KQLKEDYFKKIECFDSVEIS Cas9 KQLKEDYFKKIECFDSVEIS LLQFLVVELFDLAEVGHFDG a g c a g c t g a a a g a g g a c t a c t t c a a g a a a a t c g a g t g c t t c g a c t c c g t g g a a a t c t c c g t c g t c g a c t t t c t c c t g a t g a a g t t c t t t t a g c t c a c g a a g c t g a g g c a c c t t t a g a g g c

4680 585 590 595 600 GVEDRFNASLGTYHDLLKII Cas9 585 590 595 600 GVEDRFNASLGTYHDLLKII Cas9 GVEDRFNASLGTYHDLLKII AHFIPEVGGQACVVIQQFND g c g t g g a a g a t c g g t t c a a c g c c t c c c t g g g c a c a t a c c a c g a t c t g c t g a a a a t t a t c a c g c a c c t t c t a g c c a a g t t g c g g a g g g a c c c g t g t a t g g t g c t a g a c g a c t t t t a a t a g t

EcoRV 4740 605 610 615 620 KDKDFLDNEENEDILEDIVL Cas9 605 610 615 620 KDKDFLDNEENEDILEDIVL Cas9 KDKDFLDNEENEDILEDIVL LVLVEQVILFVLVNQFIDHQ a g g a c a a g g a c t t c c t g g a c a a t g a g g a a a a c g a g g a c a t t c t g g a a g a t a t c g t g c t g a t c c t g t t c c t g a a g g a c c t g t t a c t c c t t t t g c t c c t g t a a g a c c t t c t a t a g c a c g a c t

4800 625 630 635 640 TLTLFEDREMIEERLKTYAH Cas9 625 630 635 640 TLTLFEDREMIEERLKTYAH Cas9 TLTLFEDREMIEERLKTYAH GQCQKLVSLHDLFPQFGIGV *AWR c c c t g a c a c t g t t t g a g g a c a g a g a g a t g a t c g a g g a a c g g c t g a a a a c c t a t g c c c a c c g g g a c t g t g a c a a a c t c c t g t c t c t c t a c t a g c t c c t t g c c g a c t t t t g g a t a c g g g t g g

4860 645 650 655 660 LFDDKVMKQLKRRRYTGWGR Cas9 645 650 655 660 LFDDKVMKQLKRRRYTGWGR Cas9 LFDDKVMKQLKRRRYTGWGR QEVVFHHLLQLPPSVGAPAP NSSLTIFCSFRRLYVPQPLS t g t t c g a c g a c a a a g t g a t g a a g c a g c t g a a g c g g c g g a g a t a c a c c g g c t g g g g c a g g c a c a a g c t g c t g t t t c a c t a c t t c g t c g a c t t c g c c g c c t c t a t g t g g c c g a c c c c g t c c g

4920 665 670 675 680 LSRKLINGIRDKQSGKTILD Cas9 665 670 675 680 LSRKLINGIRDKQSGKTILD Cas9 LSRKLINGIRDKQSGKTILD QAPLQDVADPVLLGALCDQI LRFSILPMRSLCDPLVIRSK t g a g c c g g a a g c t g a t c a a c g g c a t c c g g g a c a a g c a g t c c g g c a a g a c a a t c c t g g a t t a c t c g g c c t t c g a c t a g t t g c c g t a g g c c c t g t t c g t c a g g c c g t t c t g t t a g g a c c t a a

4980 685 690 695 700 FLKSDGFANRNFMQLIHDDS Cas9 685 690 695 700 FLKSDGFANRNFMQLIHDDS Cas9 FLKSDGFANRNFMQLIHDDS EQLGVAEGVSVEHLQDVVVA RFDSPKALLFKMCSIWSSLR t c c t g a a g t c c g a c g g c t t c g c c a a c a g a a a c t t c a t g c a g c t g a t c c a c g a c g a c a g c c a g g a c t t c a g g c t g c c g a a g c g g t t g t c t t t g a a g t a c g t c g a c t a g g t g c t g c t g t c g g

5040 705 710 715 720 LTFKEDIQKAQVSGQGDSLH Cas9 705 710 715 720 LTFKEDIQKAQVSGQGDSLH Cas9 LTFKEDIQKAQVSGQGDSLH QGKFLVDLFGLHGALAIAQV VKLSSMWFAWTDPWPSLRCS t g a c c t t t a a a g a g g a c a t c c a g a a a g c c c a g g t g t c c g g c c a g g g c g a t a g c c t g c a c g a c t g g a a a t t t c t c c t g t a g g t c t t t c g g g t c c a c a g g c c g g t c c c g c t a t c g g a c g t g c

5100 725 730 735 740 EHIANLAGSPAIKKGILQTV Cas9 725 730 735 740 EHIANLAGSPAIKKGILQTV Cas9 EHIANLAGSPAIKKGILQTV LVNGIQGAAGGNLLADQLCH CMALRAPLGAMLFPMRCVTF a g c a c a t t g c c a a t c t g g c c g g c a g c c c c g c c a t t a a g a a g g g c a t c c t g c a g a c a g t g a t c g t g t a a c g g t t a g a c c g g c c g t c g g g g c g g t a a t t c t t c c c g t a g g a c g t c t g t c a c t

5160 745 750 755 760 KVVDELVKVMGRHKPENIVI Cas9 745 750 755 760 KVVDELVKVMGRHKPENIVI Cas9 KVVDELVKVMGRHKPENIVI LHHVLEHFHHAPVLGLVDHD TTSSSTFTIPRCLGSFMTIS a g g t g g t g g a c g a g c t c g t g a a a g t g a t g g g c c g g c a c a a g c c c g a g a a c a t c g t g a t c g t c c a c c a c c t g c t c g a g c a c t t t c a c t a c c c g g c c g t g t t c g g g c t c t t g t a g c a c t a g c

5220 765 770 775 780 EMARENQTTQKGQKNSRERM Cas9 765 770 775 780 EMARENQTTQKGQKNSRERM Cas9 EMARENQTTQKGQKNSRERM FHGSLVLGGLLSLLVAALSH IALSFWVVWFPCFFLRSLIF a a a t g g c c a g a g a g a a c c a g a c c a c c c a g a a g g g a c a g a a g a a c a g c c g c g a g a g a a t g a t t t a c c g g t c t c t c t t g g t c t g g t g g g t c t t c c c t g t c t t c t t g t c g g c g c t c t c t t a c t

5280 785 790 795 800 KRIEEGIKELGSQILKEHPV Cas9 785 790 795 800 KRIEEGIKELGSQILKEHPV Cas9 KRIEEGIKELGSQILKEHPV LPDFLADFLQAALDQFFVGH RISSPM a g c g g a t c g a a g a g g g c a t c a a a g a g c t g g g c a g c c a g a t c c t g a a a g a a c a c c c c g t g g t c g c c t a g c t t c t c c c g t a g t t t c t c g a c c c g t c g g t c t a g g a c t t t c t t g t g g g g c a c c

5340 805 810 815 820 ENTQLQNEKLYLYYLQNGRD Cas9 805 810 815 820 ENTQLQNEKLYLYYLQNGRD Cas9 ENTQLQNEKLYLYYLQNGRD FVGLQLVLLQVQVVQLIPPI a a a a c a c c c a g c t g c a g a a c g a g a a g c t g t a c c t g t a c t a c c t g c a g a a t g g g c g g g a t a t t t t g t g g g t c g a c g t c t t g c t c t t c g a c a t g g a c a t g a t g g a c g t c t t a c c c g c c c t a t

5400 825 830 835 840 MYVDQELDINRLSDYDVDHI Cas9 825 830 835 840 MYVDQELDINRLSDYDVDHI Cas9 MYVDQELDINRLSDYDVDHI HVHVLFQVDVPQGVVIHVM t g t a c g t g g a c c a g g a a c t g g a c a t c a a c c g g c t g t c c g a c t a c g a t g t g g a c c a t a t c g a c a t g c a c c t g g t c c t t g a c c t g t a g t t g g c c g a c a g g c t g a t g c t a c a c c t g g t a t a g c

5460 845 850 855 860 VPQSFLKDDSIDNKVLTRSD Cas9 845 850 855 860 VPQSFLKDDSIDNKVLTRSD Cas9 VPQSFLKDDSIDNKVLTRSD t g c c t c a g a g c t t t c t g a a g g a c g a c t c c a t c g a c a a c a a g g t g c t g a c c a g a a g c g a c a a c g g a g t c t c g a a a g a c t t c c t g c t g a g g t a g c t g t t g t t c c a c g a c t g g t c t t c g c t g t

5520 865 870 875 880 KNRGKSDNVPSEEVVKKMKN Cas9 865 870 875 880 KNRGKSDNVPSEEVVKKMKN Cas9 KNRGKSDNVPSEEVVKKMKN a g a a c c g g g g c a a g a g c g a c a a c g t g c c c t c c g a a g a g g t c g t g a a g a a g a t g a a g a a c t t c t t g g c c c c g t t c t c g c t g t t g c a c g g g a g g c t t c t c c a g c a c t t c t t c t a c t t c t t g a

5580 885 890 895 900 YWRQLLNAKLITQRKFDNLT Cas9 885 890 895 900 YWRQLLNAKLITQRKFDNLT Cas9 YWRQLLNAKLITQRKFDNLT a c t g g c g g c a g c t g c t g a a c g c c a a g c t g a t t a c c c a g a g a a a g t t c g a c a a t c t g a c c a t g a c c g c c g t c g a c g a c t t g c g g t t c g a c t a a t g g g t c t c t t t c a a g c t g t t a g a c t g g t

5640 905 910 915 920 KAERGGLSELDKAGFIKRQL Cas9 905 910 915 920 KAERGGLSELDKAGFIKRQL Cas9 KAERGGLSELDKAGFIKRQL a g g c c g a g a g a g g c g g c c t g a g c g a a c t g g a t a a g g c c g g c t t c a t c a a g a g a c a g c t g g t c c g g c t c t c t c c g c c g g a c t c g c t t g a c c t a t t c c g g c c g a a g t a g t t c t c t g t c g a c c

PmlI 5700 925 930 935 940 VETRQITKHVAQILDSRMNT Cas9 925 930 935 940 VETRQITKHVAQILDSRMNT Cas9 VETRQITKHVAQILDSRMNT t g g a a a c c c g g c a g a t c a c a a a g c a c g t g g c a c a g a t c c t g g a c t c c c g g a t g a a c a c t a a c c t t t g g g c c g t c t a g t g t t t c g t g c a c c g t g t c t a g g a c c t g a g g g c c t a c t t g t g a t

5760 945 950 955 960 KYDENDKLIREVKVITLKSK Cas9 945 950 955 960 KYDENDKLIREVKVITLKSK Cas9 KYDENDKLIREVKVITLKSK a g t a c g a c g a g a a t g a c a a g c t g a t c c g g g a a g t g a a a g t g a t c a c c c t g a a g t c c a a g c t c a t g c t g c t c t t a c t g t t c g a c t a g g c c c t t c a c t t t c a c t a g t g g g a c t t c a g g t t c g

5820 965 970 975 980 LVSDFRKDFQFYKVREINNY Cas9 965 970 975 980 LVSDFRKDFQFYKVREINNY Cas9 LVSDFRKDFQFYKVREINNY t g g t g t c c g a t t t c c g g a a g g a t t t c c a g t t t t a c a a a g t g c g c g a g a t c a a c a a c t 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

5880 985 990 995 1000 HHAHDAYLNAVVGTALIKKY Cas9 985 990 995 1000 HHAHDAYLNAVVGTALIKKY Cas9 HHAHDAYLNAVVGTALIKKY a c c a c g c c c a c g a c g c c t a c c t g a a c g c c g t c g t g g g a a c c g c c c t g a t c a a a a a g t a c c t g g t g c g g g t g c t g c g g a t g g a c t t g c g g c a g c a c c c t t g g c g g g a c t a g t t t t t c a t g g

5940 1005 1010 1015 1020 PKLESEFVYGDYKVYDVRKM Cas9 1005 1010 1015 1020 PKLESEFVYGDYKVYDVRKM Cas9 PKLESEFVYGDYKVYDVRKM c t a a g c t g g a a a g c g a g t t c g t g t a c g g c g a c t a c a a g g t g t a c g a c g t g c g g a a g a t g a g a t t c g a c c t t t c g c t c a a g c a c a t g c c g c t g a t g t t c c a c a t g c t g c a c g c c t t c t a c t

6000 1025 1030 1035 1040 IAKSEQEIGKATAKYFFYSN Cas9 1025 1030 1035 1040 IAKSEQEIGKATAKYFFYSN Cas9 IAKSEQEIGKATAKYFFYSN t c g c c a a g a g c g a g c a g g a a a t c g g c a a g g c t a c c g c c a a g t a c t t c t t c t a c a g c a a c a a g c g g t t c t c g c t c g t c c t t t a g c c g t t c c g a t g g c g g t t c a t g a a g a a g a t g t c g t t g t

6060 1045 1050 1055 1060 IMNFFKTEITLANGEIRKRP Cas9 1045 1050 1055 1060 IMNFFKTEITLANGEIRKRP Cas9 IMNFFKTEITLANGEIRKRP t c a t g a a c t t t t t c a a g a c c g a g a t t a c c c t g g c c a a c g g c g a g a t c c g g a a g c g g c c t c a g t a c t t g a a a a a g t t c t g g c t c t a a t g g g a c c g g t t g c c g c t c t a g g c c t t c g c c g g a g

6120 1065 1070 1075 1080 LIETNGETGEIVWDKGRDFA Cas9 1065 1070 1075 1080 LIETNGETGEIVWDKGRDFA Cas9 LIETNGETGEIVWDKGRDFA t g a t c g a g a c a a a c g g c g a a a c c g g g g a g a t c g t g t g g g a t a a g g g c c g g g a t t t t g c c a a c t a g c t c t g t t t g c c g c t t t g g c c c c t c t a g c a c a c c c t a t t c c c g g c c c t a a a a c g g t

6180 1085 1090 1095 1100 TVRKVLSMPQVNIVKKTEVQ Cas9 1085 1090 1095 1100 TVRKVLSMPQVNIVKKTEVQ Cas9 TVRKVLSMPQVNIVKKTEVQ c c g t g c g g a a a g t g c t g a g c a t g c c c c a a g t g a a t a t c g t g a a a a a g a c c g a g g t g c a g a g g c a c g c c t t t c a c g a c t c g t a c g g g g t t c a c t t a t a g c a c t t t t t c t g g c t c c a c g t c t

6240 1105 1110 1115 1120 TGGFSKESILPKRNSDKLIA Cas9 1105 1110 1115 1120 TGGFSKESILPKRNSDKLIA Cas9 TGGFSKESILPKRNSDKLIA c a g g c g g c t t c a g c a a a g a g t c t a t c c t g c c c a a g a g g a a c a g c g a t a a g c t g a t c g c c a g t c c g c c g a a g t c g t t t c t c a g a t a g g a c g g g t t c t c c t t g t c g c t a t t c g a c t a g c g g t

6300 1125 1130 1135 1140 RKKDWDPKKYGGFDSPTVAY Cas9 1125 1130 1135 1140 RKKDWDPKKYGGFDSPTVAY Cas9 RKKDWDPKKYGGFDSPTVAY g a a a g a a g g a c t g g g a c c c t a a g a a g t a c g g c g g c t t c g a c a g c c c c a c c g t g g c c 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

6360 1145 1150 1155 1160 SVLVVAKVEKGKSKKLKSVK Cas9 1145 1150 1155 1160 SVLVVAKVEKGKSKKLKSVK Cas9 SVLVVAKVEKGKSKKLKSVK c t g t g c t g g t g g t g g c c a a a g t g g a a a a g g g c a a g t c c a a g a a a c t g a a g a g t g t g a a a g g a c a c g a c c a c c a c c g g t t t c a c c t t t t c c c g t t c a g g t t c t t t g a c t t c t c a c a c t t t c

BsaBI* 6420 1165 1170 1175 1180 ELLGITIMERSSFEKNPIDF Cas9 1165 1170 1175 1180 ELLGITIMERSSFEKNPIDF Cas9 ELLGITIMERSSFEKNPIDF a g c t g c t g g g g a t c a c c a t c a t g g a a a g a a g c a g c t t c g a g a a g a a t c c c a t c g a c t t t c t c g a c g a c c c c t a g t g g t a g t a c c t t t c t t c g t c g a a g c t c t t c t t a g g g t a g c t g a a a g

6480 1185 1190 1195 1200 LEAKGYKEVKKDLIIKLPKY Cas9 1185 1190 1195 1200 LEAKGYKEVKKDLIIKLPKY Cas9 LEAKGYKEVKKDLIIKLPKY t g g a a g c c a a g g g c t a c a a a g a a g t g a a a a a g g a c c t g a t c a t c a a g c t g c c t a a g t a c t a c c t t c g g t t c c c g a t g t t t c t t c a c t t t t t c c t g g a c t a g t a g t t c g a c g g a t t c a t g a

6540 1205 1210 1215 1220 SLFELENGRKRMLASAGELQ Cas9 1205 1210 1215 1220 SLFELENGRKRMLASAGELQ Cas9 SLFELENGRKRMLASAGELQ c c c t g t t c g a g c t g g a a a a c g g c c g g a a g a g a a t g c t g g c c t c t g c c g g c g a a c t g c a g a g g g a c a a g c t c g a c c t t t t g c c g g c c t t c t c t t a c g a c c g g a g a c g g c c g c t t g a c g t c t

6600 1225 1230 1235 1240 KGNELALPSKYVNFLYLASH Cas9 1225 1230 1235 1240 KGNELALPSKYVNFLYLASH Cas9 KGNELALPSKYVNFLYLASH a g g g a a a c g a a c t g g c c c t g c c c t c c a a a t a t g t g a a c t t c c t g t a c c t g g c c a g c c a c t t c c c t t t g c t t g a c c g g g a c g g g a g g t t t a t a c a c t t g a a g g a c a t g g a c c g g t c g g t g a

6660 1245 1250 1255 1260 YEKLKGSPEDNEQKQLFVEQ Cas9 1245 1250 1255 1260 YEKLKGSPEDNEQKQLFVEQ Cas9 YEKLKGSPEDNEQKQLFVEQ a t g a g a a g c t g a a g g g c t c c c c c g a g g a t a a t g a g c a g a a a c a g c t g t t t g t g g a a c a g c t a c t c t t c g a c t t c c c g a g g g g g c t c c t a t t a c t c g t c t t t g t c g a c a a a c a c c t t g t c g

6720 1265 1270 1275 1280 HKHYLDEIIEQISEFSKRVI Cas9 1265 1270 1275 1280 HKHYLDEIIEQISEFSKRVI Cas9 HKHYLDEIIEQISEFSKRVI a c a a g c a c t a c c t g g a c g a g a t c a t c g a g c a g a t c a g c g a g t t c t c c a a g a g a g t g 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

6780 1285 1290 1295 1300 LADANLDKVLSAYNKHRDKP Cas9 1285 1290 1295 1300 LADANLDKVLSAYNKHRDKP Cas9 LADANLDKVLSAYNKHRDKP t g g c c g a c g c t a a t c t g g a c a a a g t g c t g t c c g c c t a c a a c a a g c a c c g g g a t a a g c c c a a c c g g c t g c g a t t a g a c c t g t t t c a c g a c a g g c g g a t g t t g t t c g t g g c c c t a t t c g g g t

6840 1305 1310 1315 1320 IREQAENIIHLFTLTNLGAP Cas9 1305 1310 1315 1320 IREQAENIIHLFTLTNLGAP Cas9 IREQAENIIHLFTLTNLGAP t c a g a g a g c a g g c c g a g a a t a t c a t c c a c c t g t t t a c c c t g a c c a a t c t g g g a g c c c c t g a g t c t c t c g t c c g g c t c t t a t a g t a g g t g g a c a a a t g g g a c t g g t t a g a c c c t c g g g g a c

6900 1325 1330 1335 1340 AAFKYFDTTIDRKRYTSTKE Cas9 1325 1330 1335 1340 AAFKYFDTTIDRKRYTSTKE Cas9 AAFKYFDTTIDRKRYTSTKE c c g c c t t c a a g t a c t t t g a c a c c a c c a t c g a c c g g a a g a g g t a c a c c a g c a c c a a a g a g g g g c g g a a g t t c a t g a a a c t g t g g t g g t a g c t g g c c t t c t c c a t g t g g t c g t g g t t t c t c c

6960 1345 1350 1355 1360 VLDATLIHQSITGLYETRID Cas9 1345 1350 1355 1360 VLDATLIHQSITGLYETRID Cas9 VLDATLIHQSITGLYETRID t g c t g g a c g c c a c c c t g a t c c a c c a g a g c a t c a c c g g c c t g t a c g a g a c a c g g a t c g a c c a c g a c c t g c g g t g g g a c t a g g t g g t c t c g t a g t g g c c g g a c a t g c t c t g t g c c t a g c t g g

PshAI 7020 1365 LSQLGGD 1 5 10 KRPAATKKAGQAK Cas9 nucleoplasmin NLS 1365 LSQLGGD (in frame with Cas9) KRPAATKKAGQAK Cas9 LSQLGGDKRPAATKKAGQAK t g t c t c a g c t g g g a g g c g a c a a g c g t c c t g c t g c t a c t a a g a a a g c t g g t c a a g c t a a g a a c a g a g t c g a c c c t c c g c t g t t c g c a g g a c g a c g a t g a t t c t t t c g a c c a g t t c g a t t c t

BmtINheI 7080 (in frame with Cas9) KKKASGSG 1 5 10 ATNFSLLKQAGD P2A 15 KKK (in frame with nucleoplasmin NLS) ASGSGATNFSLLKQAGD nucleoplasmin NLS KKKASGSGATNFSLLKQAGD a a a a g a a a g c t a g c g g c a g c g g c g c c a c c a a c t t c a g c c t g c t g a a g c a g g c c g g c g a c g t t t t c t t t c g a t c g c c g t c g c c g c g g t g g t t g a a g t c g g a c g a c t t c g t c c g g c c g c t g c

7140 (in frame with nucleoplasmin NLS) VEENPGPMTEYKPTVRLATR 15 VEENPGP 1 5 10 MTEYKPTVRLATR P2A PuroR T A C T G G C T C A T G T T C G G G T G Puro-R VEENPGPMTEYKPTVRLATR t g g a g g a g a a c c c c g g c c c c a t g a c c g a g t a c a a g c c c a c g g t g c g c c t c g c c a c c c g c g a c c t c c t c t t g g g g c c g g g g t a c t g g c t c a t g t t c g g g t g c c a c g c g g a g c g g t g g g c g c

BsiWITth111IPflFI 7200 (in frame with nucleoplasmin NLS) DDVPRAVRTLAAAFADYPAT 15 20 25 30 DDVPRAVRTLAAAFADYPAT PuroR DDVPRAVRTLAAAFADYPAT a c g a c g t c c c c a g g g c c g t a c g c a c c c t c g c c g c c g c g t t c g c c g a c t a c c c c g c c a c g c t g c t g c a g g g g t c c c g g c a t g c g t g g g a g c g g c g g c g c a a g c g g c t g a t g g g g c g g t g c g

BstEIIRsrII 7260 (in frame with nucleoplasmin NLS) RHTVDPDRHIERVTELQELF 35 40 45 50 RHTVDPDRHIERVTELQELF PuroR RHTVDPDRHIERVTELQELF g c c a c a c c g t c g a t c c g g a c c g c c a c a t c g a g c g g g t c a c c g a g c t g c a a g a a c t c t t c c c g g t g t g g c a g c t a g g c c t g g c g g t g t a g c t c g c c c a g t g g c t c g a c g t t c t t g a g a a g g

7320 (in frame with nucleoplasmin NLS) LTRVGLDIGKVWVADDGAAV 55 60 65 70 LTRVGLDIGKVWVADDGAAV PuroR LTRVGLDIGKVWVADDGAAV t c a c g c g c g t c g g g c t c g a c a t c g g c a a g g t g t g g g t c g c g g a c g a c g g c g c c g c g g t g g a g t g c g c g c a g c c c g a g c t g t a g c c g t t c c a c a c c c a g c g c c t g c t g c c g c g g c g c c a c c

7380 (in frame with nucleoplasmin NLS) AVWTTPESVEAGAVFAEIGP 75 80 85 90 AVWTTPESVEAGAVFAEIGP PuroR AVWTTPESVEAGAVFAEIGP c g g t c t g g a c c a c g c c g g a g a g c g t c g a a g c g g g g g c g g t g t t c g c c g a g a t c g g c c c g c g c c a g a c c t g g t g c g g c c t c t c g c a g c t t c g c c c c c g c c a c a a g c g g c t c t a g c c g g g c g

7440 (in frame with nucleoplasmin NLS) RMAELSGSRLAAQQQMEGLL 95 100 105 110 RMAELSGSRLAAQQQMEGLL PuroR RMAELSGSRLAAQQQMEGLL g c a t g g c c g a g t t g a g c g g t t c c c g g c t g g c c g c g c a g c a a c a g a t g g a a g g c c t c c t g g c g t a c c g g c t c a a c t c g c c a a g g g c c g a c c g g c g c g t c g t t g t c t a c c t t c c g g a g g a c c

7500 (in frame with nucleoplasmin NLS) APHRPKEPAWFLATVGVSPD 115 120 125 130 APHRPKEPAWFLATVGVSPD PuroR APHRPKEPAWFLATVGVSPD c g c c g c a c c g g c c c a a g g a g c c c g c g t g g t t c c t g g c c a c c g t c g g a g t c t c g c c c g a c c g c g g c g t g g c c g g g t t c c t c g g g c g c a c c a a g g a c c g g t g g c a g c c t c a g a g c g g g c t g g

7560 (in frame with nucleoplasmin NLS) HQGKGLGSAVVLPGVEAAER 135 140 145 150 HQGKGLGSAVVLPGVEAAER PuroR HQGKGLGSAVVLPGVEAAER a c c a g g g c a a g g g t c t g g g c a g c g c c g t c g t g c t c c c c g g a g t g g a g g c g g c c g a g c g c g t g g t c c c g t t c c c a g a c c c g t c g c g g c a g c a c g a g g g g c c t c a c c t c c g c c g g c t c g c g c

7620 (in frame with nucleoplasmin NLS) AGVPAFLETSAPRNLPFYER 155 160 165 170 AGVPAFLETSAPRNLPFYER PuroR G C A A C C T C C C C T T C T A C G A G C Puro-F AGVPAFLETSAPRNLPFYER c c g g g g t g c c c g c c t t c c t g g a g a c c t c c g c g c c c c g c a a c c t c c c c t t c t a c g a g c g g c g g c c c c a c g g g c g g a a g g a c c t c t g g a g g c g c g g g g c g t t g g a g g g g a a g a t g c t c g c c g

SexAI*CsiI 7680 (in frame with nucleoplasmin NLS) LGFTVTADVEVPEGPRTWCM 175 180 185 190 LGFTVTADVEVPEGPRTWCM PuroR LGFTVTADVEVPEGPRTWCM t c g g c t t c a c c g t c a c c g c c g a c g t c g a g g t g c c c g a a g g a c c g c g c a c c t g g t g c a t g a a g c c g a a g t g g c a g t g g c g g c t g c a g c t c c a c g g g c t t c c t g g c g c g t g g a c c a c g t a c t

MluI 7740 (in frame with nucleoplasmin NLS) TRKPGA* WPRE 195 200 TRKPGA* PuroR TRKPGA* c c c g c a a g c c c g g t g c c t g a a c g c g t t a a g t c g a c a a t c a a c c t c t g g a t t a c a a a a t t t g g g c g t t c g g g c c a c g g a c t t g c g c a a t t c a g c t g t t a g t t g g a g a c c t a a t g t t t t a a a

7800 WPRE A C A A C G A G G A A A A T G C G A T A C WPRE-R g t g a a a g a t t g a c t g g t a t t c t t a a c t a t g t t g c t c c t t t t a c g c t a t g t g g a t a c g c t g c a c t t t c t a a c t g a c c a t a a g a a t t g a t a c a a c g a g g a a a a t g c g a t a c a c c t a t g c g a c

7860 WPRE MLLLPVWLSFSPPC MPLYHAIASRMAFIFSSL c t t t a a t g c c t t t g t a t c a t g c t a t t g c t t c c c g t a t g g c t t t c a t t t t c t c c t c c t t g t g a a a t t a c g g a a a c a t a g t a c g a t a a c g a a g g g c a t a c c g a a a g t a a a a g a g g a g g a a c a

7920 WPRE INPGCCLFMRSCGPLSGNVA YKSWLLSLYEELWPVVRQRG a t a a a t c c t g g t t g c t g t c t c t t t a t g a g g a g t t g t g g c c c g t t g t c a g g c a a c g t g g c g t a t t t a g g a c c a a c g a c a g a g a a a t a c t c c t c a a c a c c g g g c a a c a g t c c g t t g c a c c g c

7980 WPRE WCALCLLTQPPLVGALPPPV VVCTVFADATPTGWGIATTC t g g t g t g c a c t g t g t t t g c t g a c g c a a c c c c c a c t g g t t g g g g c a t t g c c a c c a c c t g t c a c c a c a c g t g a c a c a a a c g a c t g c g t t g g g g g t g a c c a a c c c c g t a a c g g t g g t g g a c a g

8040 WPRE SSFPGLSLSPSLLPRRNSSP QLLSGTFAFPLPIATAELIA a g c t c c t t t c c g g g a c t t t c g c t t t c c c c c t c c c t a t t g c c a c g g c g g a a c t c a t c g c c g t c g a g g a a a g g c c c t g a a a g c g a a a g g g g g a g g g a t a a c g g t g c c g c c t t g a g t a g c g g c

8100 WPRE PALPAAGQGLGCWALTIPWC ACLARCWTGARLLGTDNSVV c c t g c c t t g c c c g c t g c t g g a c a g g g g c t c g g c t g t t g g g c a c t g a c a a t t c c g t g g t g t g g a c g g a a c g g g c g a c g a c c t g t c c c c g a g c c g a c a a c c c g t g a c t g t t a a g g c a c c a c a

8160 WPRE (in frame with Factor Xa site) *RGKRPQEGTNGGPNQ CRGNHRPFLGCSPVLPPGFC LSGKSSSFPWLLACVATWIL t g t c g g g g a a a t c a t c g t c c t t t c c t t g g c t g c t c g c c t g t g t t g c c a c c t g g a t t c t g c a c a g c c c c t t t a g t a g c a g g a a a g g a a c c g a c g a g c g g a c a c a a c g g t g g a c c t a a g a c g

8220 WPRE (in frame with Factor Xa site) APRGEAVDR 1 RGEI Factor Xa site AGRPSATSLRPSIQRTFLPA RGTSFCYVPSALNPADLPSR g c g g g a c g t c c t t c t g c t a c g t c c c t t c g g c c c t c a a t c c a g c g g a c c t t c c t t c c c g c g c g c c c t g c a g g a a g a c g a t g c a g g g a a g c c g g g a g t t a g g t c g c c t g g a a g g a a g g g c g c

8280 WPRE ACCRLCGLFRVFAFALRRVG GLLPALRPLPRLRLRPQTSR g c c t g c t g c c g g c t c t g c g g c c t c t t c c g c g t c t t c g c c t t c g c c c t c a g a c g a g t c g g a c g g a c g a c g g c c g a g a c g c c g g a g a a g g c g c a g a a g c g g a a g c g g g a g t c t g c t c a g c c t

8340 WPRE SPFGPPPRVDFKTNDLQGSC ISLWAASPRRL* t c t c c c t t t g g g c c g c c t c c c c g c g t c g a c t t t a a g a c c a a t g a c t t a c a a g g c a g c t g t a g a g g g a a a c c c g g c g g a g g g g c g c a g c t g a a a t t c t g g t t a c t g a a t g t t c c g t c g a c a

8400 3' LTR ( Δ U3) RS* a g a t c t t a g c c a c t t t t t a a a a g a a a a g g g g g g a c t g g a a g g g c t a a t t c a c t c c c a a c g t c t a g a a t c g g t g a a a a a t t t t c t t t t c c c c c c t g a c c t t c c c g a t t a a g t g a g g g t t g c

8460 3' LTR ( Δ U3) a a g a c a a g a t c t g c t t t t t g c t t g t a c t g g g t c t c t c t g g t t a g a c c a g a t c t g a g c c t g t t c t g t t c t a g a c g a a a a a c g a a c a t g a c c c a g a g a g a c c a a t c t g g t c t a g a c t c g g a c

8520 3' LTR ( Δ U3) g g a g c t c t c t g g c t a a c t a g g g a a c c c a c t g c t t a a g c c t c a a t a a a g c t t g c c t t g a g t c c t c g a g a g a c c g a t t g a t c c c t t g g g t g a c g a a t t c g g a g t t a t t t c g a a c g g a a c t c a

8580 3' LTR ( Δ U3) g c t t c a a g t a g t g t g t g c c c g t c t g t t g t g t g a c t c t g g t a a c t a g a g a t c c c t c a g a c c c g a a g t t c a t c a c a c a c g g g c a g a c a a c a c a c t g a g a c c a t t g a t c t c t a g g g a g t c t g g

SnaBI 8640 3' LTR ( Δ U3) c t t t t a g t c a g t g t g g a a a a t c t c t a g c a g t a c g t a t a g t a g t t c a t g t c a t c t t a t t a t g a a a a t c a g t c a c a c c t t t t a g a g a t c g t c a t g c a t a t c a t c a a g t a c a g t a g a a t a a t a

8700 SV40 poly(A) signal t c a g t a t t t a t a a c t t g c a a a g a a a t g a a t a t c a g a g a g t g a g a g g a a c t t g t t t a t t g c a g t c a t a a a t a t t g a a c g t t t c t t t a c t t a t a g t c t c t c a c t c t c c t t g a a c a a a t a a c g

8760 SV40 poly(A) signal C G T T A T C G T A G T G T T T A A A G SV40pA-R a g c t t a t a a t g g t t a c a a a t a a a g c a a t a g c a t c a c a a a t t t c a c a a a t a a a g c a t t t t t t c g a a t a t t a c c a a t g t t t a t t t c g t t a t c g t a g t g t t t a a a g t g t t t a t t t c g t a a a a a

8820 SV40 poly(A) signal G T G G T T T G T C C A A A C T C A T C EBV-rev t t c a c t g c a t t c t a g t t g t g g t t t g t c c a a a c t c a t c a a t g t a t c t t a t c a t g t c t g g c t a a g t g a c g t a a g a t c a a c a c c a a a c a g g t t t g a g t a g t t a c a t a g a a t a g t a c a g a c c g a

8880 SV40 ori c t a g c t a t c c c g c c c c t a a c t c c g c c c a t c c c g c c c c t a a c t c c g c c c a g t t c c g c c c a t g a t c g a t a g g g c g g g g a t t g a g g c g g g t a g g g c g g g g a t t g a g g c g g g t c a a g g c g g g t a

SfiI 8940 SV40 ori t c t c c g c c c c a t g g c t g a c t a a t t t t t t t t a t t t a t g c a g a g g c c g a g g c c g c c t c g g c c a g a g g c g g g g t a c c g a c t g a t t a a a a a a a a t a a a t a c g t c t c c g g c t c c g g c g g a g c c g g

AvrII 9000 SV40 ori t c t g a g c t a t t c c a g a a g t a g t g a g g a g g c t t t t t t g g a g g c c t a g g g a c g t a c c c a a t t a g a c t c g a t a a g g t c t t c a t c a c t c c t c c g a a a a a a c c t c c g g a t c c c t g c a t g g g t t a a

9060 T7 promoter M13 fwd G G G A T A T C A C T C A G C A T A A T T7 T G A C C G G C A G C A A A A T G T M13 Forward G C A A A A T G T T G C A G C A C T G A M13/pUC Forward c g c c c t a t a g t g a g t c g t a t t a c g c g c g c t c a c t g g c c g t c g t t t t a c a a c g t c g t g a c t g c g g g a t a t c a c t c a g c a t a a t g c g c g c g a g t g a c c g g c a g c a a a a t g t t g c a g c a c t g a

9120 C C C M13/pUC Forward g g g a a a a c c c t g g c g t t a c c c a a c t t a a t c g c c t t g c a g c a c a t c c c c c t t t c g c c a g c t c c c t t t t g g g a c c g c a a t g g g t t g a a t t a g c g g a a c g t c g t g t a g g g g g a a a g c g g t c g a

9180 g g c g t a a t a g c g a a g a g g c c c g c a c c g a t c g c c c t t c c c a a c a g t t g c g c a g c c t g a a t g c c g c a t t a t c g c t t c t c c g g g c g t g g c t a g c g g g a a g g g t t g t c a a c g c g t c g g a c t t a c

9240 f1 ori g c g a a t g g g a c g c g c c c t g t a g c g g c g c a t t a a g c g c g g c g g g t g t g g t g g t t a c g c g c a c g c t t a c c c t g c g c g g g a c a t c g c c g c g t a a t t c g c g c c g c c c a c a c c a c c a a t g c g c g t

9300 f1 ori G A G G A A A G C G A A A G A A G G G A F1ori-R 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 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 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 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

9360 f1 ori t t c t c g c c a c g t t c g c c g g c t t t c c c c g t c a a g c t c t a a a t c g g g g g c t c c c t t t a g g g t a a g a g c g g t g c a a g c g g c c g a a a g g g g c a g t t c g a g a t t t a g c c c c c g a g g g a a a t c c c a

9420 f1 ori t c c g a t t t a g t g c t t t a c g g c a c c t c g a c c c c a a a a a a c t t g a t t a g g g t g a t g g t t c a c a g g c t a a a t c a c g a a a t g c c g t g g a g c t g g g g t t t t t t g a a c t a a t c c c a c t a c c a a g t g

9480 f1 ori g t a g t g g g c c a t c g c c c t g a t a g a c g g t t t t t c g c c c t t t g a c g t t g g a g t c c a c g t t c t c a t c a c c c g g t a g c g g g a c t a t c t g c c a a a a a g c g g g a a a c t g c a a c c t c a g g t g c a a g a

9540 f1 ori G T G G A C T C T T G T T C C A A A C T G G F1ori-F t t a a t a g t g g a c t c t t g t t c c a a a c t g g a a c a a c a c t c a a c c c t a t c t c g g t c t a t t c t t a a t t a t c a c c t g a g a a c a a g g t t t g a c c t t g t t g t g a g t t g g g a t a g a g c c a g a t a a g a a

9600 f1 ori t t g a t t t a t a a g g g a t t t t g c c g a t t t c g g 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 c t a a a t a t t c c c t a a a a c g g c t a a a g c c 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

9660 f1 ori a a a a a t t t a a c g c g a a t t t t a a c a a a a t a t t a a c g c t t a c a a t t t a g g t g g c a c t t t t c g t t t t t a a a t t g c g c t t a a a a t t g t t t t a t a a t t g c g a a t g t t a a a t c c a c c g t g a a a a g c

9720 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

9780 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

9840 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

9900 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

9960 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

10,020 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

10,080 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

10,140 105 110 115 120 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

10,200 125 130 135 140 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

10,260 145 150 155 160 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

10,320 165 170 175 180 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

10,380 185 190 195 200 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

10,440 205 210 215 220 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

10,500 225 230 235 240 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 g t c t 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 c a g a g c g c c

10,560 245 250 255 260 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

10,620 265 270 275 280 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

10,680 285 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

10,740 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

10,800 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

10,860 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

10,920 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

10,980 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

11,040 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

11,100 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

11,160 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

11,220 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

11,280 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

11,340 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

11,400 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

PciI 11,460 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 t c t t 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 a g a a

11,520 t c c t g c g t t a t c c c c t g a t t c t g t g g a t a a c c g t a t t a c c g c c t t t g a g t g a g c t g a t a c a g g a c g c a a t a g g g g a c t a a g a c a c c t a t t g g c a t a a t g g c g g a a a c t c a c t c g a c t a t g

11,580 A G C G A G T C A G T G A G C G A G L4440 c g c t c g c c g c a g c c g a a c g a c c g a g c g c a g c g a g t c a g t g a g c g a g g a a g c g g a a g a g c g g c g a g c g g c g t c g g c t t g c t g g c t c g c g t c g c t c a g t c a c t c g c t c c t t c g c c t t c t c g c

3ʹ 5ʹ 11,590 c c c a a t a c g c g g g t t a t g c g

Restriction Enzymes

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

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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.