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pBT280 evoFERNY-BE4max
  • Sequence Map
    Editing is disabled because sequence edits have been locked.
    tagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgaatcaataattatcattagttaatgccccagtaatcaagtatcgggtatatacctcaaggcgcaatgtattgaatgccatttaccgggcggaccgactggcgggttgctgggggcgggtaactgcagttattactgcatacaagggtatcattgcggttatccctgaaaggtaactCMV promoter, full20406080100120140160
    cgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcagcagttacccacctcataaatgccatttgacgggtgaaccgtcatgtagttcacatagtatacggttcatgcgggggataactgcagttactgccatttaccgggcggaccgtaatacgggtcatgtactggaataccctgaaaggatgaaccgtcatgtagatgcataatcagtNdeISnaBICMV promoter, full180200220240260280300320340
    tcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatagcgataatggtaccactacgccaaaaccgtcatgtagttacccgcacctatcgccaaactgagtgcccctaaaggttcagaggtggggtaactgcagttaccctcaaacaaaaccgtggttttagttgccctgaaaggttttacagcattgttgaggcggggtaactgcgtttaNcoICspCICMV promoter, fullCMV promoter360380400420440460480500520
    gggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagagatccgcggccgctaatacgactcactatagggagagccgccaccatgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcagcccgccatccgcacatgccaccctccagatatattcgtctcgaccaaatcacttggcagtctaggcgatctctaggcgccggcgattatgctgagtgatatccctctcggcggtggtactttgcctgtcggctgccttcgctcaagctcagtggtttcttcttcgcctttcagtcEagINotISacIIS1M1K2R3T4A5D6G7S8E9F10E11S12P13K14K15K16R17K18V19642-698CMV promoter, fullCMV promoterevoFERNY-BE4maxBipartite SV40 NLST7 promotercore promoter540560580600620640660680700
    ttttgagaggaactacgacccccgggagctgagaaaggagacatacctgctgtatgagatcaagtggggcaagtccggcaagctgtggaggcactggtgccagaacaatcgcacacagcacgccgaggtgtacttcctggagaacatctttaatgcccggagattcaatccatctaaaactctccttgatgctgggggccctcgactctttcctctgtatggacgacatactctagttcaccccgttcaggccgttcgacacctccgtgaccacggtcttgttagcgtgtgtcgtgcggctccacatgaaggacctcttgtagaaattacgggcctctaagttaggtagaSmaIXmaITspMIFE2RN4YD6PR8EL10RK12ET14YL16LY18EI20KW22GK24SG26KL28WR30HW32CQ34NN36RT38QH40AE42VY44FL46EN48IF50NA52RR54FN56PS58702-1184evoFERNY-BE4maxevoFERNY720740760780800820840860
    acccactgtagcatcacatggtatctgagctggtccccctgcgccgagtgttctcagaagatcgtggatttcctgaaggagcaccctaacgtgaatctggagatctatgtggcccggctgtactatccagagaacgagaggaataggcAgggccTgcgggatctggtgaattccgtgggtgacatcgtagtgtaccatagactcgaccagggggacgcggctcacaagagtcttctagcacctaaaggacttcctcgtgggattgcacttagacctctagatacaccgggccgacatgataggtctcttgctctccttatccgTcccggAcgccctagaccacttaaggcBglIIEcoRITH60CS62IT64WY66LS68WS70PC72AE74CS76QK78IV80DF82LK84EH86PN88VN90LE92IY94VA96RL98YY100PE102NE104RN106RQ108GL110RD112LV114NS116702-1184evoFERNY-BE4maxevoFERNY8809009209409609801,0001,0201,040
    gcgtgaccatcagaatcAtggaccTgccagattacaactattgctggaagaccttcgtgagcgatcagggaggcgacgaggattactggccaggacacttcgccccttggatcaagcagtatagcctgaagctgtctggcggatctAgcggaggaTcctctggcagcgagacacccgcactggtagtcttagTacctggAcggtctaatgttgataacgaccttctggaagcactcgctagtccctccgctgctcctaatgaccggtcctgtgaagcggggaacctagttcgtcatatcggacttcgacagaccgcctagaTcgcctcctAggagaccgtcgctctgtggBbsIBamHIGV118TI120RI122MD124LP126DY128NY130CW132KT134FV136SD138QG140GD142ED144YW146PG148HF150AP152WI154KQ156YS158LK160L702-1184S1G2G3S4S5G6G7S8S9G10S11E12T13P141185-1280evoFERNY-BE4maxevoFERNYBE4 linker Genscript codonsUS...31,0601,0801,1001,1201,1401,1601,1801,2001,220
    aggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcggcggcagcgacaagaagtacagcatcggcctggCcatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcactccttgttcgctcagtcgttgtggtctctcgtcaccgccgtcgtcgccgccgtcgctgttcttcatgtcgtagccggaccGgtagccgtggttgagacacccgacccggcactagtggctgctcatgttccacgggtcgttctttaagttccacgacccgttgtggctggccgtgBstXIG15T16S17E18S19A20T21P22E23S24S25G26G27S28S29G30G31S321185-1280DK2KY4SI6GL8AI10GT12NS14VG16WA18VI20TD22EY24KV26PS28KK30FK32VL34GN36TD38RH401281-5381evoFERNY-BE4maxnCas9, Genscript codonsBE4 linker Genscript codons1,2401,2601,2801,3001,3201,3401,3601,3801,400
    agcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacatcgtagttcttcttggactagcctcgggacgacaagctgtcgccgctttgtcggctccggtgggccgacttctcttggcggtcttcttctatgtggtctgccttcttggcctagacgatagacgttctctagaagtcgttgctctaccggttccacctgctgtcgaagaaggtgtBglIISI42KK44NL46IG48AL50LF52DS54GE56TA58EA60TR62LK64RT66AR68RR70YT72RR74KN76RI78CY80LQ82EI84FS86NE88MA90KV92DD94SF96FH981281-5381evoFERNY-BE4maxnCas9, Genscript codons1,4201,4401,4601,4801,5001,5201,5401,560
    gactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctctgaccttctcaggaaggaccaccttctcctattcttcgtgctcgccgtggggtagaagccgttgtagcacctgctccaccggatggtgctcttcatggggtggtagatggtggactctttctttgaccacctgtcgtggctgttccggctggacgccgactagatagaccgggaPflMIRL100EE102SF104LV106EE108DK110KH112ER114HP116IF118GN120IV122DE124VA126YH128EK130YP132TI134YH136LR138KK140LV142DS144TD146KA148DL150RL152IY154LA156L1281-5381evoFERNY-BE4maxnCas9, Genscript codons1,5801,6001,6201,6401,6601,6801,7001,7201,740
    ggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgccgggtgtactagttcaaggccccggtgaaggactagctcccgctggacttggggctgttgtcgctgcacctgttcgacaagtaggtcgaccacgtctggatgttggtcgacaagctccttttggggtagttgcggtcgccgcacctgcggttccggtaggacagacggtctgacBmgBIBstXIEcoNIA158HM160IK162FR164GH166FL168IE170GD172LN174PD176NS178DV180DK182LF184IQ186LV188QT190YN192QL194FE196EN198PI200NA202SG204VD206AK208AI210LS212AR214L1281-5381evoFERNY-BE4maxnCas9, Genscript codons1,7601,7801,8001,8201,8401,8601,8801,9001,920
    agcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctcgttctcgtctgccgaccttttagactagcgggtcgacgggccgctcttcttcttaccggacaagcctttggactaacgggactcggacccggactgggggttgaagttctcgttgaagctggaccggctcctacggtttgacgtcgactcgttcctgtggatgctgctgctggEcoNIS216KS218RR220LE222NL224IA226QL228PG230EK232KN234GL236FG238NL240IA242LS244LG246LT248PN250FK252SN254FD256LA258ED260AK262LQ264LS266KD268TY270DD272D1281-5381evoFERNY-BE4maxnCas9, Genscript codons1,9401,9601,9802,0002,0202,0402,0602,0802,100
    tggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgacacctgttggacgaccgggtctagccgctggtcatgcggctggacaaagaccggcggttcttggacaggctgcggtaggacgactcgctgtaggactctcacttgtggctctagtggttccggggggactcgcggagatactagttctctatgctgctcgtggtggtcctggactgL274DN276LL278AQ280IG282DQ284YA286DL288FL290AA292KN294LS296DA298IL300LS302DI304LR306VN308TE310IT312KA314PL316SA318SM320IK322RY324DE326HH328QD330LT3321281-5381evoFERNY-BE4maxnCas9, Genscript codons2,1202,1402,1602,1802,2002,2202,2402,260
    cctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgggacgactttcgagagcacgccgtcgtcgacggactcttcatgtttctctaaaagaagctggtctcgttcttgccgatgcggccgatgtaactgccgcctcggtcggtccttctcaagatgttcaagtagttcgggtaggaccttttctacctgccgtggctccttgacgagcacLL334KA336LV338RQ340QL342PE344KY346KE348IF350FD352QS354KN356GY358AG360YI362DG364GA366SQ368EE370FY372KF374IK376PI378LE380KM382DG384TE386EL388LV3901281-5381evoFERNY-BE4maxnCas9, Genscript codons2,2802,3002,3202,3402,3602,3802,4002,4202,440
    aagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctttcgacttgtctctcctggacgacgccttcgtcgcctggaagctgttgccgtcgtagggggtggtctaggtggaccctctcgacgtgcggtaagacgccgccgtccttctaaaaatgggtaaggacttcctgttggcccttttctagctcttctaggactggaaggcgtaggggaKL392NR394ED396LL398RK400QR402TF404DN406GS408IP410HQ412IH414LG416EL418HA420IL422RR424QE426DF428YP430FL432KD434NR436EK438IE440KI442LT444FR446IP4481281-5381evoFERNY-BE4maxnCas9, Genscript codons2,4602,4802,5002,5202,5402,5602,5802,6002,620
    actacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgcttgatgcacccgggagaccggtcccctttgtcgtctaagcggacctactggtctttctcgctcctttggtagtgggggaccttgaagctccttcaccacctgttcccgcgaaggcgggtctcgaagtagctcgcctactggttgaagctattcttggacgggttgctcttccacgaApaIPspOMIYY450VG452PL454AR456GN458SR460FA462WM464TR466KS468EE470TI472TP474WN476FE478EV480VD482KG484AS486AQ488SF490IE492RM494TN496FD498KN500LP502NE504KV506L1281-5381evoFERNY-BE4maxnCas9, Genscript codons2,6402,6602,6802,7002,7202,7402,7602,7802,800
    gcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagagcgggttcgtgtcggacgacatgctcatgaagtggcacatattgctcgactggtttcactttatgcactggctcccttactctttcgggcggaaggactcgccgctcgtctttttccggtagcacctggacgacaagttctggttggcctttcactggcacttcgtcgactttctcBarIP508KH510SL512LY514EY516FT518VY520NE522LT524KV526KY528VT530EG532MR534KP536AF538LS540GE542QK544KA546IV548DL550LF552KT554NR556KV558TV560KQ562LK564E1281-5381evoFERNY-BE4maxnCas9, Genscript codons2,8202,8402,8602,8802,9002,9202,9402,960
    gactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacacctgatgaagttcttttagctcacgaagctgaggcacctttagaggccgcaccttctagccaagttgcggagggacccgtgtatggtgctagacgacttttaatagttcctgttcctgaaggacctgttactccttttgctcctgtaagaccttctatagcacgactgggactgtgPasIEcoRVD566YF568KK570IE572CF574DS576VE578IS580GV582ED584RF586NA588SL590GT592YH594DL596LK598II600KD602KD604FL606DN608EE610NE612DI614LE616DI618VL620TL622T1281-5381evoFERNY-BE4maxnCas9, Genscript codons2,9803,0003,0203,0403,0603,0803,1003,1203,140
    tgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctacaaactcctgtctctctactagctccttgccgacttttggatacgggtggacaagctgctgtttcactacttcgtcgacttcgccgcctctatgtggccgaccccgtccgactcggccttcgactagttgccgtaggccctgttcgtcaggccgttctgttaggacctaaaggaAhdIL624FE626DR628EM630IE632ER634LK636TY638AH640LF642DD644KV646MK648QL650KR652RR654YT656GW658GR660LS662RK664LI666NG668IR670DK672QS674GK676TI678LD680FL6821281-5381evoFERNY-BE4maxnCas9, Genscript codons3,1603,1803,2003,2203,2403,2603,2803,3003,320
    gaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgcttcaggctgccgaagcggttgtctttgaagtacgtcgactaggtgctgctgtcggactggaaatttctcctgtaggtctttcgggtccacaggccggtcccgctatcggacgtgctcgtgtaacggttagaccggccgtcggggcggtaattcttcccgtaggacgtctgtcacAjuIKS684DG686FA688NR690NF692MQ694LI696HD698DS700LT702FK704ED706IQ708KA710QV712SG714QG716DS718LH720EH722IA724NL726AG728SP730AI732KK734GI736LQ738TV7401281-5381evoFERNY-BE4maxnCas9, Genscript codons3,3403,3603,3803,4003,4203,4403,4603,4803,500
    aaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccttccaccacctgctcgagcactttcactacccggccgtgttcgggctcttgtagcactagctttaccggtctctcttggtctggtgggtcttccctgtcttcttgtcggcgctctcttacttcgcctagcttctcccgtagtttctcgacccgtcggtctaggactttcttgtggSacIEco53kIKV742VD744EL746VK748VM750GR752HK754PE756NI758VI760EM762AR764EN766QT768TQ770KG772QK774NS776RE778RM780KR782IE784EG786IK788EL790GS792QI794LK796EH7981281-5381evoFERNY-BE4maxnCas9, Genscript codons3,5203,5403,5603,5803,6003,6203,6403,660
    ccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgacggcaccttttgtgggtcgacgtcttgctcttcgacatggacatgatggacgtcttacccgccctatacatgcacctggtccttgacctgtagttggccgacaggctgatgctacacctggtatagcacggagtctcgaaagacttcctgctgaggtagctgttgttccacgactgPflMIDraIIIPV800EN802TQ804LQ806NE808KL810YL812YY814LQ816NG818RD820MY822VD824QE826LD828IN830RL832SD834YD836VD838HI840VP842QS844FL846KD848DS850ID852NK854VL856T1281-5381evoFERNY-BE4maxnCas9, Genscript codons3,6803,7003,7203,7403,7603,7803,8003,8203,840
    cagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcgtcttcgctgttcttggccccgttctcgctgttgcacgggaggcttctccagcacttcttctacttcttgatgaccgccgtcgacgacttgcggttcgactaatgggtctctttcaagctgttagactggttccggctctctccgccggactcgcttgacctattccggccgaagR858SD860KN862RG864KS866DN868VP870SE872EV874VK876KM878KN880YW882RQ884LL886NA888KL890IT892QR894KF896DN898LT900KA902ER904GG906LS908EL910DK912AG914F1281-5381evoFERNY-BE4maxnCas9, Genscript codons3,8603,8803,9003,9203,9403,9603,9804,0004,020
    atcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtagttctctgtcgaccacctttgggccgtctagtgtttcgtgcaccgtgtctaggacctgagggcctacttgtgattcatgctgctcttactgttcgactaggcccttcactttcactagtgggacttcaggttcgaccacaggctaaaggccttcctaaaggtcaaaatgtttcPmlICspCII916KR918QL920VE922TR924QI926TK928HV930AQ932IL934DS936RM938NT940KY942DE944ND946KL948IR950EV952KV954IT956LK958SK960LV962SD964FR966KD968FQ970FY972K1281-5381evoFERNY-BE4maxnCas9, Genscript codons4,0404,0604,0804,1004,1204,1404,1604,1804,200
    tgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctacacgcgctctagttgttgatggtggtgcgggtgctgcggatggacttgcggcagcacccttggcgggactagtttttcatgggattcgacctttcgctcaagcacatgccgctgatgttccacatgctgcacgccttctactagcggttctcgctcgtcctttagccgttccgatgBmgBIV974RE976IN978NY980HH982AH984DA986YL988NA990VV992GT994AL996IK998KY1000PK1002LE1004SE1006FV1008YG1010DY1012KV1014YD1016VR1018KM1020IA1022KS1024EQ1026EI1028GK1030AT10321281-5381evoFERNY-BE4maxnCas9, Genscript codons4,2204,2404,2604,2804,3004,3204,3404,360
    cgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagcggttcatgaagaagatgtcgttgtagtacttgaaaaagttctggctctaatgggaccggttgccgctctaggccttcgccggagactagctctgtttgccgctttggcccctctagcacaccctattcccggccctaaaacggtggcacgcctttcacgactcgtacggggttAjuISphIAK1034YF1036FY1038SN1040IM1042NF1044FK1046TE1048IT1050LA1052NG1054EI1056RK1058RP1060LI1062ET1064NG1066ET1068GE1070IV1072WD1074KG1076RD1078FA1080TV1082RK1084VL1086SM1088PQ10901281-5381evoFERNY-BE4maxnCas9, Genscript codons4,3804,4004,4204,4404,4604,4804,5004,5204,540
    gtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggcacttatagcactttttctggctccacgtctgtccgccgaagtcgtttctcagataggacgggttctccttgtcgctattcgactagcggtctttcttcctgaccctgggattcttcatgccgccgaagctgtcggggtggcaccggataagacacgaccaccaccggtttcaccBarIKflIVN1092IV1094KK1096TE1098VQ1100TG1102GF1104SK1106ES1108IL1110PK1112RN1114SD1116KL1118IA1120RK1122KD1124WD1126PK1128KY1130GG1132FD1134SP1136TV1138AY1140SV1142LV1144VA1146KV11481281-5381evoFERNY-BE4maxnCas9, Genscript codons4,5604,5804,6004,6204,6404,6604,6804,7004,720
    aaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaattttcccgttcaggttctttgacttctcacactttctcgacgacccctagtggtagtacctttcttcgtcgaagctcttcttagggtagctgaaagaccttcggttcccgatgtttcttcactttttcctggactagtagttcgacggattcatgagggacaagctcgaccttttBsaBIEK1150GK1152SK1154KL1156KS1158VK1160EL1162LG1164IT1166IM1168ER1170SS1172FE1174KN1176PI1178DF1180LE1182AK1184GY1186KE1188VK1190KD1192LI1194IK1196LP1198KY1200SL1202FE1204LE1206N1281-5381evoFERNY-BE4maxnCas9, Genscript codons4,7404,7604,7804,8004,8204,8404,8604,8804,900
    cggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggccggccttctcttacgaccggagacggccgcttgacgtcttccctttgcttgaccgggacgggaggtttatacacttgaaggacatggaccggtcggtgatactcttcgacttcccgagggggctcctattactcgtctttgtcgacaaacaccttgtcgtgttcgtgatggacBsmIEagIG1208RK1210RM1212LA1214SA1216GE1218LQ1220KG1222NE1224LA1226LP1228SK1230YV1232NF1234LY1236LA1238SH1240YE1242KL1244KG1246SP1248ED1250NE1252QK1254QL1256FV1258EQ1260HK1262HY1264L1281-5381evoFERNY-BE4maxnCas9, Genscript codons4,9204,9404,9604,9805,0005,0205,0405,060
    gacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcactgctctagtagctcgtctagtcgctcaagaggttctctcactaggaccggctgcgattagacctgtttcacgacaggcggatgttgttcgtggccctattcgggtagtctctcgtccggctcttatagtaggtggacaaatgggactggttagaccctcggggacggcggaagtD1266EI1268IE1270QI1272SE1274FS1276KR1278VI1280LA1282DA1284NL1286DK1288VL1290SA1292YN1294KH1296RD1298KP1300IR1302EQ1304AE1306NI1308IH1310LF1312TL1314TN1316LG1318AP1320AA1322F1281-5381evoFERNY-BE4maxnCas9, Genscript codons5,0805,1005,1205,1405,1605,1805,2005,2205,240
    agtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgacagcggcgggagcggcgggagcggggggagcactaatctgagcgatcatgaaactgtggtggtagctggccttctccatgtggtcgtggtttctccacgacctgcggtgggactaggtggtctcgtagtggccggacatgctctgtgcctagctggacagagtcgaccctccactgtcgccgccctcgccgccctcgcccccctcgtgattagactcgctK1324YF1326DT1328TI1330DR1332KR1334YT1336ST1338KE1340VL1342DA1344TL1346IH1348QS1350IT1352GL1354YE1356TR1358ID1360LS1362QL1364GG1366D1281-5381SG2GS4GG6SG8GS10TN12LS14D5382-5951evoFERNY-BE4maxnCas9, Genscript codons2xUGI Genscript codons5,2605,2805,3005,3205,3405,3605,3805,4005,420
    catcattgagaaggagactgggaaacagctggtcattcaggagtccatcctgatgctgcctgaggaggtggaggaagtgatcggcaacaagccagagtctgacatcctggtgcacaccgcctacgacgagtccacagatgagaatgtgatgctgctgacctctgacgcccccgaggtagtaactcttcctctgaccctttgtcgaccagtaagtcctcaggtaggactacgacggactcctccacctccttcactagccgttgttcggtctcagactgtaggaccacgtgtggcggatgctgctcaggtgtctactcttacactacgacgactggagactgcgggggctcBseRIBsu36II16IE18KE20TG22KQ24LV26IQ28ES30IL32ML34PE36EV38EE40VI42GN44KP46ES48DI50LV52HT54AY56DE58ST60DE62NV64ML66LT68SD70AP72E5382-5951evoFERNY-BE4max2xUGI Genscript codons5,4405,4605,4805,5005,5205,5405,5605,5805,600
    tataagccttgggccctggtcatccaggattctaacggcgagaataagatcaagatgctgagcggaggatccggaggatctggaggcagcaccaacctgtctgacatcatcgagaaggagacaggcaagcagctggtcatccaggagagcatcctgatgctgcccgaagaagtcgatattcggaacccgggaccagtaggtcctaagattgccgctcttattctagttctacgactcgcctcctaggcctcctagacctccgtcgtggttggacagactgtagtagctcttcctctgtccgttcgtcgaccagtaggtcctctcgtaggactacgacgggcttcttcagcApaIBamHIPspOMIY74KP76WA78LV80IQ82DS84NG86EN88KI90KM92LS94GG96SG98GS100GG102ST104NL106SD108II110EK112ET114GK116QL118VI120QE122SI124LM126LP128EE130V5382-5951evoFERNY-BE4max2xUGI Genscript codons5,6205,6405,6605,6805,7005,7205,7405,760
    aagaagtgatcggaaacaagcctgagagcgatatcctggtccataccgcctacgacgagagtaccgacgaaaatgtgatgctgctgacatccgacgccccagagtataagccctgggctctggtcatccaggattccaacggagagaacaaaatcaaaatgctgtctggcggctcttcttcactagcctttgttcggactctcgctataggaccaggtatggcggatgctgctctcatggctgcttttacactacgacgactgtaggctgcggggtctcatattcgggacccgagaccagtaggtcctaaggttgcctctcttgttttagttttacgacagaccgccgagBaeIPasIEcoRVE132EV134IG136NK138PE140SD142IL144VH146TA148YD150ES152TD154EN156VM158LL160TS162DA164PE166YK168PW170AL172VI174QD176SN178GE180NK182IK184ML186SG188GS1905382-5951evoFERNY-BE4max2xUGI Genscript codons5,7805,8005,8205,8405,8605,8805,9005,9205,940
    aaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaaccggtcatcatcaccatcaccattgagtttAaacccgcTgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgtttttcttggcggctgccgtcgcttaagctcgggttcttcttctcctttcagattggccagtagtagtggtagtggtaactcaaaTttgggcgActagtcggagctgacacggaagatcaacggtcggtagacaacaaacggggagggggcacggaaggaactgggaccttccacAgeIPmeIEcoRI*1K1R2T3A4D5G6S7E8F9E10P11K12K13K14R15K16V175952-6002bGH PA terminatorevoFERNY-BE4maxSV40 NLSSV40 NLS5,9605,9806,0006,0206,0406,0606,0806,1006,120
    ccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaacggtgagggtgacaggaaaggattattttactcctttaacgtagcgtaacagactcatccacagtaagataagaccccccaccccaccccgtcctgtcgttccccctcctaacccttctgttatcgtccgtacgacccctacgccacccgagataccgaagactccgcctttcttgBbsISphIbGH PA terminator6,1406,1606,1806,2006,2206,2406,2606,2806,300
    cagctggggctcgataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctagggtgcctaatgagtgagctaactcacattaattgcgttggtcgaccccgagctatggcagctggagatcgatctcgaaccgcattagtaccagtatcgacaaaggacacactttaacaataggcgagtgttaaggtgtgttgtatgctcggccttcgtatttcacatttcggatcccacggattactcactcgattgagtgtaattaacgcaacAvrIIlac promoter6,3206,3406,3606,3806,4006,4206,4406,460
    cgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgcActtccgcTtcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgggcgagtgacgggcgaaaggtcagccctttggacagcacggtcgacgtaattacttagccggttgcgcgcccctctccgccaaacgcataacccgcgTgaaggcgAaggagcgagtgactgagcgacgcgagccagcaagccgacgccgctcgccatagtcgagtgagtttccgcc6,4806,5006,5206,5406,5606,5806,6006,6206,640
    taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacattatgccaataggtgtcttagtcccctattgcgtcctttcttgtacactcgttttccggtcgttttccggtccttggcatttttccggcgcaacgaccgcaaaaaggtatccgaggcggggggactgctcgtagtgtttttagctgcgagttcagtctccaccgctttgggctgDrdIPciIAflIIIpUC origin high copy6,6606,6806,7006,7206,7406,7606,7806,8006,820
    aggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgtcctgatatttctatggtccgcaaagggggaccttcgagggagcacgcgagaggacaaggctgggacggcgaatggcctatggacaggcggaaagagggaagcccttcgcaccgcgaaagagtatcgagtgcgacatccatagagtcaagccacatccagcaagcgaggttcgacBciVIpUC origin high copy6,8406,8606,8806,9006,9206,9406,9606,9807,000
    ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaccgacacacgtgcttggggggcaagtcgggctggcgacgcggaataggccattgatagcagaactcaggttgggccattctgtgctgaatagcggtgaccgtcgtcggtgaccattgtcctaatcgtctcgctccatacatccgccacgatgtctcaagaacttcaccaccggatpUC origin high copy7,0207,0407,0607,0807,1007,1207,1407,160
    actacggctacactagaagAacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctgatgccgatgtgatcttcTtgtcataaaccatagacgcgagacgacttcggtcaatggaagcctttttctcaaccatcgagaactaggccgtttgtttggtggcgaccatcgccaccaaaaaaacaaacgttcgtcgtctaatgcgcgtctttttttcctagagttcttctaggpUC origin high copy7,1807,2007,2207,2407,2607,2807,3007,3207,340
    tttgatcttttctacggggtctgacActcagtggaacgaaaactcacgttaagggAttttggtcaTgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaaaactagaaaagatgccccagactgTgagtcaccttgcttttgagtgcaattcccTaaaaccagtActctaatagtttttcctagaagtggatctaggaaaatttaatttttacttcaaaatttagttagatttcatatatactcatttgaaccagactgtcaatggttacgaatDraIII*W286HK2847513-8373pUC origin high copybla...e)US...57,3607,3807,4007,4207,4407,4607,4807,5007,520
    atcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgGgAcccacgcTcaccggctccagatttatcagcaataaaccagccagtagtcactccgtggatagagtcgctagacagataaagcaagtaggtatcaacggactgaggggcagcacatctattgatgctatgccctcccgaatggtagaccggggtcacgacgttactatggcgcCcTgggtgcgAgtggccgaggtctaaatagtcgttatttggtcggtcAhdIKflISacIIIL282SA280GI278EA276IQ274RN272RE270DM268TA266QS264GT262TY260IV258VI256RS254PK252GD250PG248LA246AI244IG242RS240GR238EG236AG234SK232DA230IF228WG226A7513-8373bla beta lactamase (ampicillin/carbenicillin resistance)7,5407,5607,5807,6007,6207,6407,6607,6807,700
    ccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagggccttcccggctcgcgtcttcaccaggacgttgaaataggcggaggtaggtcagataattaacaacggcccttcgatctcattcatcaagcggtcaattatcaaacgcgttgcaacaacggtaacgatgtccgtagcaccacagtgcgagcagcaaaccataccgaagtaagtcAclIFspIP224LA222SR220LL218PG216AV214KD212AE210MW208DI206LQ204QR202SA200LT198LL196EG194TL192LK190RL188TT186AM184AV182PM180TT178DR176ED174NP172IA170EN168L7513-8373bla beta lactamase (ampicillin/carbenicillin resistance)7,7207,7407,7607,7807,8007,8207,8407,860
    ctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgctttgaggccaagggttgctagttccgctcaatgtactagggggtacaacacgttttttcgccaatcgaggaagccaggaggctagcaacagtcttcattcaaccggcgtcacaatagtgagtaccaataccgtcgtgacgtattaagagaatgacagtacggtaggcattctacgaaaPvuIE166PE164WR162DL160RT158VH156DG154MN152HL150FA148TL146EK144PG142GI140TT138LL136LN134AA132TN130DS128MT126IA124AS122CL120ER118VT116MG114DT112LH110K7513-8373bla beta lactamase (ampicillin/carbenicillin resistance)7,8807,9007,9207,9407,9607,9808,0008,0208,040
    tctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgtagacactgaccactcatgagttggttcagtaagactcttatcacatacgccgctggctcaacgagaacgggccgcagttatgccctattatggcgcggtgtatcgtcttgaaattttcacgagtagtaaccttttgcaagaagccccgcttttgagagttcctagaatggcgacaAclIE108TV106PS104YE102VL100DN98QS96YH94IR92RG90LQ88EQ86GA84DI82RS80LV78AG76CL74LV72KF70TS68MM66PF64RE62EP60RF58SE56LI54KG52SN507513-8373bla beta lactamase (ampicillin/carbenicillin resistance)8,0608,0808,1008,1208,1408,1608,1808,2008,220
    tgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagactctaggtcaagctacattgggtgagcacgtgggttgactagaagtcgtagaaaatgaaagtggtcgcaaagacccactcgtttttgtccttccgttttacggcgttttttcccttattcccgctgtgcctttacaacttatgagtatgagaaggaaaaagttataataacttcSspILD48LE46IY44GV42RA40GL38QD36EA34DK32VK30VL28TE26PH24AF22VP20LC18FA16AF14FP12IL10AV8RF6HQ4IS2M7513-8373bla beta lactamase (ampicillin/carbenicillin resistance)8,2408,2608,2808,3008,3208,3408,3608,3808,400
    catttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatcgatctcccgatcccctagggtcgactctcagtacaatctgctctgatgccgtaaatagtcccaataacagagtactcgcctatgtataaacttacataaatctttttatttgtttatccccaaggcgcgtgtaaaggggcttttcacggtggactgcagctgcctagccctctagctagagggctaggggatcccagctgagagtcatgttagacgagactacggClaIAvrIIBciVIBspDISgrDIamp promoter8,4208,4408,4608,4808,5008,5208,5408,560
    gcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgaccgtatcaattcggtcatagacgagggacgaacacacaacctccagcgactcatcacgcgctcgttttaaattcgatgttgttccgttccgaactggctgttaacgtacttcttagacgaatcccaatccgcaaaacgcgacgaagcgctacatgcccggtctatatgcgcaactgMfeIMluINruIAflIII8,5808,6008,6208,6408,6608,6808,7008,7208,740
    attgattattgactaactaataactgSpeI8,760
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    8763 bpS. pyogenes Cas9 D10A nickase, human codon optimizedpBT195 backbone65007000750080008500500100015002000evoFERNY-BE4maxnCas9, Genscript codonsD10AUSER junction 331281-5381CMV promoter, full,NdeI,SnaBI,NcoI,CspCICMV promoter,core promoter,T7 promoter,essential bases,essential base,+12evoFERNY,702-1184H102P,D104N,BglII,EcoRI,BbsIBE4 linker Genscript codons,GGC for SapI Golden Gate cloning,+3BstXIBglIIPflMIEcoNI,BmgBIBstXIEcoNIremoved SapI site,USER jct 26AflIII,PciI,DrdIBciVIpUC origin high copyG>A,USER jct 25,DraIIIAhdIremoved BsaI site,USER jct 31,SacII,KflI,FspIbla beta lactamase (ampicillin/carbenicillin resistance),7513-8373,+2AclIamp promoter,SspI,BciVI,SgrDI,BspDI,ClaI,AvrII,MfeI,NruI,AflIII,MluI,SpeI

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    8763