                         SEQUENCE LISTING

<110>  President and Fellows of Harvard College
 
<120>  SPLIT CAS9 PROTEINS

<130>  010498_00690

<140>  PCT/US16/012570
<141>  2016-01-08

<150>  US 62/246321
<151>  2015-10-26

<150>  US 62/101043
<151>  2015-01-08

<160>  66    

<170>  PatentIn version 3.5

<210>  1
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Mstn on target M3 protospacer and PAM

<400>  1
aagtctctcc gggacctctt ggg                                               23


<210>  2
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  chr16:+3906202 protospacer and PAM

<400>  2
aaggctctcc aggacctctt ggg                                               23


<210>  3
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Locus chr4:-55206243 protospacer and PAM

<400>  3
aagtatctct gggacctctt cag                                               23


<210>  4
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Locus chr13:+38093999 protospacer and PAM

<400>  4
gagtccctcc gggagctctt ggg                                               23


<210>  5
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Locus Mstn on target M4 protospacer and PAM

<400>  5
gtgctgccgc taccccctca cgg                                               23


<210>  6
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Locus chr2:-49015581 protospacer and PAM

<400>  6
gtgctgcagc tgccacctca ggg                                               23


<210>  7
<211>  23
<212>  DNA
<213>  Artificial

<220>
<223>  Locus chr5:+132154281 protospacer and PAM

<400>  7
gtgcagccgc taccaccaca aag                                               23


<210>  8
<211>  1368
<212>  PRT
<213>  Streptococcus pyogenes

<400>  8

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 
1               5                   10                  15      


Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 
            20                  25                  30          


Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 
        35                  40                  45              


Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 
    50                  55                  60                  


Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 
65                  70                  75                  80  


Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 
                85                  90                  95      


Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 
            100                 105                 110         


His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 
        115                 120                 125             


His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 
    130                 135                 140                 


Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 
145                 150                 155                 160 


Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 
                165                 170                 175     


Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 
            180                 185                 190         


Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 
        195                 200                 205             


Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 
    210                 215                 220                 


Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 
225                 230                 235                 240 


Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 
                245                 250                 255     


Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 
            260                 265                 270         


Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 
        275                 280                 285             


Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 
    290                 295                 300                 


Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 
305                 310                 315                 320 


Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 
                325                 330                 335     


Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 
            340                 345                 350         


Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 
        355                 360                 365             


Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 
    370                 375                 380                 


Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 
385                 390                 395                 400 


Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 
                405                 410                 415     


Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 
            420                 425                 430         


Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 
        435                 440                 445             


Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 
    450                 455                 460                 


Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 
465                 470                 475                 480 


Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 
                485                 490                 495     


Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 
            500                 505                 510         


Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 
        515                 520                 525             


Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 
    530                 535                 540                 


Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 
545                 550                 555                 560 


Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 
                565                 570                 575     


Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 
            580                 585                 590         


Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 
        595                 600                 605             


Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 
    610                 615                 620                 


Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 
625                 630                 635                 640 


His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 
                645                 650                 655     


Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 
            660                 665                 670         


Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 
        675                 680                 685             


Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 
    690                 695                 700                 


Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 
705                 710                 715                 720 


His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 
                725                 730                 735     


Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 
            740                 745                 750         


Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 
        755                 760                 765             


Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 
    770                 775                 780                 


Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 
785                 790                 795                 800 


Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 
                805                 810                 815     


Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 
            820                 825                 830         


Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 
        835                 840                 845             


Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 
    850                 855                 860                 


Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 
865                 870                 875                 880 


Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 
                885                 890                 895     


Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 
            900                 905                 910         


Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 
        915                 920                 925             


Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 
    930                 935                 940                 


Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 
945                 950                 955                 960 


Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 
                965                 970                 975     


Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 
            980                 985                 990         


Val Gly Thr Ala Leu Ile Lys Lys  Tyr Pro Lys Leu Glu  Ser Glu Phe 
        995                 1000                 1005             


Val Tyr  Gly Asp Tyr Lys Val  Tyr Asp Val Arg Lys  Met Ile Ala 
    1010                 1015                 1020             


Lys Ser  Glu Gln Glu Ile Gly  Lys Ala Thr Ala Lys  Tyr Phe Phe 
    1025                 1030                 1035             


Tyr Ser  Asn Ile Met Asn Phe  Phe Lys Thr Glu Ile  Thr Leu Ala 
    1040                 1045                 1050             


Asn Gly  Glu Ile Arg Lys Arg  Pro Leu Ile Glu Thr  Asn Gly Glu 
    1055                 1060                 1065             


Thr Gly  Glu Ile Val Trp Asp  Lys Gly Arg Asp Phe  Ala Thr Val 
    1070                 1075                 1080             


Arg Lys  Val Leu Ser Met Pro  Gln Val Asn Ile Val  Lys Lys Thr 
    1085                 1090                 1095             


Glu Val  Gln Thr Gly Gly Phe  Ser Lys Glu Ser Ile  Leu Pro Lys 
    1100                 1105                 1110             


Arg Asn  Ser Asp Lys Leu Ile  Ala Arg Lys Lys Asp  Trp Asp Pro 
    1115                 1120                 1125             


Lys Lys  Tyr Gly Gly Phe Asp  Ser Pro Thr Val Ala  Tyr Ser Val 
    1130                 1135                 1140             


Leu Val  Val Ala Lys Val Glu  Lys Gly Lys Ser Lys  Lys Leu Lys 
    1145                 1150                 1155             


Ser Val  Lys Glu Leu Leu Gly  Ile Thr Ile Met Glu  Arg Ser Ser 
    1160                 1165                 1170             


Phe Glu  Lys Asn Pro Ile Asp  Phe Leu Glu Ala Lys  Gly Tyr Lys 
    1175                 1180                 1185             


Glu Val  Lys Lys Asp Leu Ile  Ile Lys Leu Pro Lys  Tyr Ser Leu 
    1190                 1195                 1200             


Phe Glu  Leu Glu Asn Gly Arg  Lys Arg Met Leu Ala  Ser Ala Gly 
    1205                 1210                 1215             


Glu Leu  Gln Lys Gly Asn Glu  Leu Ala Leu Pro Ser  Lys Tyr Val 
    1220                 1225                 1230             


Asn Phe  Leu Tyr Leu Ala Ser  His Tyr Glu Lys Leu  Lys Gly Ser 
    1235                 1240                 1245             


Pro Glu  Asp Asn Glu Gln Lys  Gln Leu Phe Val Glu  Gln His Lys 
    1250                 1255                 1260             


His Tyr  Leu Asp Glu Ile Ile  Glu Gln Ile Ser Glu  Phe Ser Lys 
    1265                 1270                 1275             


Arg Val  Ile Leu Ala Asp Ala  Asn Leu Asp Lys Val  Leu Ser Ala 
    1280                 1285                 1290             


Tyr Asn  Lys His Arg Asp Lys  Pro Ile Arg Glu Gln  Ala Glu Asn 
    1295                 1300                 1305             


Ile Ile  His Leu Phe Thr Leu  Thr Asn Leu Gly Ala  Pro Ala Ala 
    1310                 1315                 1320             


Phe Lys  Tyr Phe Asp Thr Thr  Ile Asp Arg Lys Arg  Tyr Thr Ser 
    1325                 1330                 1335             


Thr Lys  Glu Val Leu Asp Ala  Thr Leu Ile His Gln  Ser Ile Thr 
    1340                 1345                 1350             


Gly Leu  Tyr Glu Thr Arg Ile  Asp Leu Ser Gln Leu  Gly Gly Asp 
    1355                 1360                 1365             


<210>  9
<211>  40
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  9
gggccatgtg gacatccatg aggtgagaca gtgccagcgt                             40


<210>  10
<211>  37
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  10
ggcctgaagc cactacagct gctggagatc aaggctc                                37


<210>  11
<211>  38
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  11
ggcctgaagc cactacagct gctggagatc aaggctcg                               38


<210>  12
<211>  30
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  12
gccattgcag ctgttagaag tgaaagcaag                                        30


<210>  13
<211>  27
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  13
ggccctagca tctaagttct cgcaggc                                           27


<210>  14
<211>  35
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  14
ggtcattcca tctcagctgt gacagcagcg cagaa                                  35


<210>  15
<211>  34
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  15
ggaagtcaag gtgacagaca cacccaagag gtcc                                   34


<210>  16
<211>  31
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  16
ggacacaccc aagaggtccc ggagagactt t                                      31


<210>  17
<211>  30
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  17
gtcaagccca aagtctctcc gggacctctt                                        30


<210>  18
<211>  29
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  18
ggaatcccgg tgctgccgct accccctca                                         29


<210>  19
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  19
gctagagaat aggaacttct t                                                 21


<210>  20
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  20
gaaagaattg atttgatacc g                                                 21


<210>  21
<211>  19
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  21
gatccccatc aagctgatc                                                    19


<210>  22
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  22
ggtatgctat acgaagttat t                                                 21


<210>  23
<211>  402
<212>  DNA
<213>  Artificial

<220>
<223>  chimeric gRNA scaffold

<400>  23
tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc       60

gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct      120

gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg      180

tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg      240

gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg      300

tggaaaggac gaaacaccgg ttttagagct agaaatagca agttaaaata aggctagtcc      360

gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tt                         402


<210>  24
<211>  54
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  24
ctttccctac acgacgctct tccgatctnn nnnnctggag tgttagagtg ggcg             54


<210>  25
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer

<400>  25
ggagttcaga cgtgtgctct tccgatctga ctgccccatg gaaagaca                    48


<210>  26
<211>  58
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  26
ctttccctac acgacgctct tccgatctnn nnnngggcca tgaaaggaaa aatgaagt         58


<210>  27
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer

<400>  27
ggagttcaga cgtgtgctct tccgatctgc ctctggggtt tgcttggt                    48


<210>  28
<211>  67
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  28
ctttccctac acgacgctct tccgatctnn nnnngagata taagctgaat aaggccaatg       60

acatact                                                                 67


<210>  29
<211>  67
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  29
ctttccctac acgacgctct tccgatctnn nnnnggtatg tttattgaaa ttccctagtc       60

tatctac                                                                 67


<210>  30
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer

<400>  30
ggagttcaga cgtgtgctct tccgatctct actgctcttt cctgccga                    48


<210>  31
<211>  54
<212>  DNA
<213>  Artificial

<220>
<223>  Amplification primer

<400>  31
ggagttcaga cgtgtgctct tccgatctaa atacagaagt agatagacta ggga             54


<210>  32
<211>  130
<212>  DNA
<213>  Artificial

<220>
<223>  AAV ITR sequence

<400>  32
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt       60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact      120

aggggttcct                                                             130


<210>  33
<211>  831
<212>  PRT
<213>  Artificial

<220>
<223>  Coding sequence for SphCas9N-RmaIntN

<400>  33

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 
1               5                   10                  15      


Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 
            20                  25                  30          


Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 
        35                  40                  45              


Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 
    50                  55                  60                  


Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 
65                  70                  75                  80  


Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 
                85                  90                  95      


Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 
            100                 105                 110         


Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 
        115                 120                 125             


His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 
    130                 135                 140                 


His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 
145                 150                 155                 160 


Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 
                165                 170                 175     


Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 
            180                 185                 190         


Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 
        195                 200                 205             


Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 
    210                 215                 220                 


Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 
225                 230                 235                 240 


Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 
                245                 250                 255     


Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 
            260                 265                 270         


Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 
        275                 280                 285             


Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 
    290                 295                 300                 


Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 
305                 310                 315                 320 


Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 
                325                 330                 335     


Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 
            340                 345                 350         


Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 
        355                 360                 365             


Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 
    370                 375                 380                 


Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 
385                 390                 395                 400 


Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 
                405                 410                 415     


Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 
            420                 425                 430         


Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 
        435                 440                 445             


Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 
    450                 455                 460                 


Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 
465                 470                 475                 480 


Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 
                485                 490                 495     


Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 
            500                 505                 510         


Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 
        515                 520                 525             


Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 
    530                 535                 540                 


Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 
545                 550                 555                 560 


Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 
                565                 570                 575     


Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 
            580                 585                 590         


Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 
        595                 600                 605             


Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 
    610                 615                 620                 


Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 
625                 630                 635                 640 


Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 
                645                 650                 655     


His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 
            660                 665                 670         


Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 
        675                 680                 685             


Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 
    690                 695                 700                 


Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 
705                 710                 715                 720 


Lys Glu Asp Ile Gln Lys Ala Gln Val Cys Leu Ala Gly Asp Thr Leu 
                725                 730                 735     


Ile Thr Leu Ala Asp Gly Arg Arg Val Pro Ile Arg Glu Leu Val Ser 
            740                 745                 750         


Gln Gln Asn Phe Ser Val Trp Ala Leu Asn Pro Gln Thr Tyr Arg Leu 
        755                 760                 765             


Glu Arg Ala Arg Val Ser Arg Ala Phe Cys Thr Gly Ile Lys Pro Val 
    770                 775                 780                 


Tyr Arg Leu Thr Thr Arg Leu Gly Arg Ser Ile Arg Ala Thr Ala Asn 
785                 790                 795                 800 


His Arg Phe Leu Thr Pro Gln Gly Trp Lys Arg Val Asp Glu Leu Gln 
                805                 810                 815     


Pro Gly Asp Tyr Leu Ala Leu Pro Arg Arg Ile Pro Thr Ala Ser 
            820                 825                 830     


<210>  34
<211>  2496
<212>  DNA
<213>  Artificial

<220>
<223>  Coding sequence for SphCas9N-RmaIntN

<400>  34
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag       60

tactccattg ggctcgatat cggcacaaac agcgtcggct gggccgtcat tacggacgag      120

tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag      180

aagaacctca ttggcgccct cctgttcgac tccggggaaa cggccgaagc cacgcggctc      240

aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag      300

atctttagta atgagatggc taaggtggat gactctttct tccataggct ggaggagtcc      360

tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa tatcgtggac      420

gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa gcttgtagac      480

agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat gatcaaattt      540

cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt cgacaaactc      600

tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat caacgcatcc      660

ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg gctcgaaaac      720

ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct tatcgccctg      780

tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga tgccaagctt      840

caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca gatcggcgac      900

cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct gctgagtgat      960

attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat gatcaagcgc     1020

tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca gcaactgcct     1080

gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg atacattgac     1140

ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga aaaaatggac     1200

ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa acagcgcact     1260

ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc tatcctcagg     1320

cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga gaaaatcctc     1380

acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg     1440

atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt cgtggataag     1500

ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa tctgcctaac     1560

gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta taacgagctc     1620

accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc tggagagcag     1680

aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt gaaacagctc     1740

aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag cggagtggag     1800

gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat taaagacaag     1860

gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct cacccttacg     1920

ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca tctcttcgac     1980

gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg gctgtcaaga     2040

aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga ttttcttaag     2100

tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc tctcaccttt     2160

aaggaggaca tccagaaagc acaagtttgt ctggctggcg atactctcat taccctggcc     2220

gatggacgac gagtgcctat tagagaactg gtgtcacagc agaatttttc cgtgtgggct     2280

ctgaatcctc agacttaccg cctggagagg gctagagtga gtagagcttt ctgtaccggc     2340

atcaaacctg tgtaccgcct caccactaga ctggggagat ccattagggc cactgccaac     2400

caccgatttc tcacacctca gggctggaaa cgagtcgatg aactccagcc tggagattac     2460

ctggctctgc ctaggagaat ccctactgcc tcctga                               2496


<210>  35
<211>  970
<212>  PRT
<213>  Artificial

<220>
<223>  Coding sequence for RmaIntC-SphCas9C-P2A-turboGFP

<400>  35

Met Ala Ala Ala Cys Pro Glu Leu Arg Gln Leu Ala Gln Ser Asp Val 
1               5                   10                  15      


Tyr Trp Asp Pro Ile Val Ser Ile Glu Pro Asp Gly Val Glu Glu Val 
            20                  25                  30          


Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val Ala Asn Asp Ile 
        35                  40                  45              


Ile Ala His Asn Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala 
    50                  55                  60                  


Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val 
65                  70                  75                  80  


Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu 
                85                  90                  95      


Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly 
            100                 105                 110         


Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys 
        115                 120                 125             


Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln 
    130                 135                 140                 


Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp 
145                 150                 155                 160 


Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp 
                165                 170                 175     


Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp 
            180                 185                 190         


Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn 
        195                 200                 205             


Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln 
    210                 215                 220                 


Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr 
225                 230                 235                 240 


Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile 
                245                 250                 255     


Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln 
            260                 265                 270         


Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu 
        275                 280                 285             


Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp 
    290                 295                 300                 


Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr 
305                 310                 315                 320 


His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu 
                325                 330                 335     


Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr 
            340                 345                 350         


Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 
        355                 360                 365             


Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe 
    370                 375                 380                 


Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro 
385                 390                 395                 400 


Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly 
                405                 410                 415     


Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn 
            420                 425                 430         


Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser 
        435                 440                 445             


Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp 
    450                 455                 460                 


Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr 
465                 470                 475                 480 


Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu 
                485                 490                 495     


Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 
            500                 505                 510         


Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu 
        515                 520                 525             


Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu 
    530                 535                 540                 


Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln 
545                 550                 555                 560 


Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr 
                565                 570                 575     


Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu 
            580                 585                 590         


Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 
        595                 600                 605             


Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala 
    610                 615                 620                 


Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro 
625                 630                 635                 640 


Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn 
                645                 650                 655     


Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg 
            660                 665                 670         


Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His 
        675                 680                 685             


Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu 
    690                 695                 700                 


Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Ser Arg 
705                 710                 715                 720 


Ala Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 
                725                 730                 735     


Val Glu Glu Asn Pro Gly Pro Met Pro Ala Met Lys Ile Glu Cys Arg 
            740                 745                 750         


Ile Thr Gly Thr Leu Asn Gly Val Glu Phe Glu Leu Val Gly Gly Gly 
        755                 760                 765             


Glu Gly Thr Pro Glu Gln Gly Arg Met Thr Asn Lys Met Lys Ser Thr 
    770                 775                 780                 


Lys Gly Ala Leu Thr Phe Ser Pro Tyr Leu Leu Ser His Val Met Gly 
785                 790                 795                 800 


Tyr Gly Phe Tyr His Phe Gly Thr Tyr Pro Ser Gly Tyr Glu Asn Pro 
                805                 810                 815     


Phe Leu His Ala Ile Asn Asn Gly Gly Tyr Thr Asn Thr Arg Ile Glu 
            820                 825                 830         


Lys Tyr Glu Asp Gly Gly Val Leu His Val Ser Phe Ser Tyr Arg Tyr 
        835                 840                 845             


Glu Ala Gly Arg Val Ile Gly Asp Phe Lys Val Val Gly Thr Gly Phe 
    850                 855                 860                 


Pro Glu Asp Ser Val Ile Phe Thr Asp Lys Ile Ile Arg Ser Asn Ala 
865                 870                 875                 880 


Thr Val Glu His Leu His Pro Met Gly Asp Asn Val Leu Val Gly Ser 
                885                 890                 895     


Phe Ala Arg Thr Phe Ser Leu Arg Asp Gly Gly Tyr Tyr Ser Phe Val 
            900                 905                 910         


Val Asp Ser His Met His Phe Lys Ser Ala Ile His Pro Ser Ile Leu 
        915                 920                 925             


Gln Asn Gly Gly Pro Met Phe Ala Phe Arg Arg Val Glu Glu Leu His 
    930                 935                 940                 


Ser Asn Thr Glu Leu Gly Ile Val Glu Tyr Gln His Ala Phe Lys Thr 
945                 950                 955                 960 


Pro Ile Ala Phe Ala Arg Ser Arg Ala Arg 
                965                 970 


<210>  36
<211>  2913
<212>  DNA
<213>  Artificial

<220>
<223>  Coding sequence for RmaIntC-SphCas9C-P2A-turboGFP

<400>  36
atggcggcgg cgtgcccgga actgcgtcag ctggcgcaga gcgatgtgta ttgggatccg       60

attgtgagca ttgaaccgga tggcgtggaa gaagtgtttg atctgaccgt gccgggcccg      120

cataactttg tggcgaacga tattattgcg cataactctg gccaggggga cagtcttcac      180

gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt      240

aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc      300

gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg      360

aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt      420

gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac      480

atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc      540

gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat      600

aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat      660

tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact      720

aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt      780

gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc      840

aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag      900

ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac      960

caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat     1020

cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg     1080

atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat     1140

attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca     1200

cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg     1260

acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag     1320

accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca     1380

cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac     1440

agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag     1500

gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt     1560

ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac     1620

tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag     1680

aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac     1740

tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa     1800

cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc     1860

ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc     1920

atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct     1980

gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag     2040

gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac     2100

ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa ggtgtctcga     2160

gctggatccg gagccacgaa cttctctctg ttaaagcaag caggggacgt ggaagaaaac     2220

cccggtccta tgcccgccat gaagatcgag tgccgcatca ccggcaccct gaacggcgtg     2280

gagttcgagc tggtgggcgg cggagagggc acccccgagc agggccgcat gaccaacaag     2340

atgaagagca ccaaaggcgc cctgaccttc agcccctacc tgctgagcca cgtgatgggc     2400

tacggcttct accacttcgg cacctacccc agcggctacg agaacccctt cctgcacgcc     2460

atcaacaacg gcggctacac caacacccgc atcgagaagt acgaggacgg cggcgtgctg     2520

cacgtgagct tcagctaccg ctacgaggcc ggccgcgtga tcggcgactt caaggtggtg     2580

ggcaccggct tccccgagga cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc     2640

accgtggagc acctgcaccc catgggcgat aacgtgctgg tgggcagctt cgcccgcacc     2700

ttcagcctgc gcgacggcgg ctactacagc ttcgtggtgg acagccacat gcacttcaag     2760

agcgccatcc accccagcat cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg     2820

gaggagctgc acagcaacac cgagctgggc atcgtggagt accagcacgc cttcaagacc     2880

cccatcgcct tcgccagatc tcgagctcga tga                                  2913


<210>  37
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  qPCR primers

<400>  37
ggaaccccta gtgatggagt t                                                 21


<210>  38
<211>  16
<212>  DNA
<213>  Artificial

<220>
<223>  qPCR primer

<400>  38
cggcctcagt gagcga                                                       16


<210>  39
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  qPCR probe

<400>  39
cactccctct ctgcgcgctc g                                                 21


<210>  40
<211>  54
<212>  DNA
<213>  Artificial

<220>
<223>  Primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  40
ctttccctac acgacgctct tccgatctnn nnnnctggag tgttagagtg ggcg             54


<210>  41
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  41
ggagttcaga cgtgtgctct tccgatctga ctgccccatg gaaagaca                    48


<210>  42
<211>  58
<212>  DNA
<213>  Artificial

<220>
<223>  Primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  42
ctttccctac acgacgctct tccgatctnn nnnngggcca tgaaaggaaa aatgaagt         58


<210>  43
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  43
ggagttcaga cgtgtgctct tccgatctgc ctctggggtt tgcttggt                    48


<210>  44
<211>  67
<212>  DNA
<213>  Artificial

<220>
<223>  Primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  44
ctttccctac acgacgctct tccgatctnn nnnngagata taagctgaat aaggccaatg       60

acatact                                                                 67


<210>  45
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  45
ggagttcaga cgtgtgctct tccgatctct actgctcttt cctgccga                    48


<210>  46
<211>  40
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  46
gggccatgtg gacatccatg aggtgagaca gtgccagcgt                             40


<210>  47
<211>  38
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  47
ggcctgaagc cactacagct gctggagatc aaggctcg                               38


<210>  48
<211>  27
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  48
ggccctagca tctaagttct cgcaggc                                           27


<210>  49
<211>  35
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  49
ggtcattcca tctcagctgt gacagcagcg cagaa                                  35


<210>  50
<211>  30
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  50
gtcaagccca aagtctctcc gggacctctt                                        30


<210>  51
<211>  29
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  51
ggaatcccgg tgctgccgct accccctca                                         29


<210>  52
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  52
gctagagaat aggaacttct t                                                 21


<210>  53
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  53
gaaagaattg atttgatacc g                                                 21


<210>  54
<211>  19
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  54
gatccccatc aagctgatc                                                    19


<210>  55
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  gRNA spacer sequence

<400>  55
ggtatgctat acgaagttat t                                                 21


<210>  56
<211>  58
<212>  DNA
<213>  Artificial

<220>
<223>  Primer


<220>
<221>  misc_feature
<222>  (29)..(34)
<223>  n is a, c, g, or t

<400>  56
ctttccctac acgacgctct tccgatctnn nnnngggcca tgaaaggaaa aatgaagt         58


<210>  57
<211>  48
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  57
ggagttcaga cgtgtgctct tccgatctgc ctctggggtt tgcttggt                    48


<210>  58
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  58
gcctactcgc tgctgcccat t                                                 21


<210>  59
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  59
cctggagacc cccaaaagct c                                                 21


<210>  60
<211>  19
<212>  DNA
<213>  Artificial

<220>
<223>  Probe

<400>  60
agatcttccc acttcaggt                                                    19


<210>  61
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  61
ggaaccccta gtgatggagt t                                                 21


<210>  62
<211>  16
<212>  DNA
<213>  Artificial

<220>
<223>  Primer

<400>  62
cggcctcagt gagcga                                                       16


<210>  63
<211>  21
<212>  DNA
<213>  Artificial

<220>
<223>  Probe

<400>  63
cactccctct ctgcgcgctc g                                                 21


<210>  64
<211>  3181
<212>  DNA
<213>  Artificial

<220>
<223>  scAAV-Promoter-Cas9N-RmaIntN-synpA

<400>  64
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg       60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaaac      120

gttgacattg attattgact agccgctagc aggactcacg gggatttcca agtctccacc      180

ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc      240

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata      300

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatccg gactctaagg      360

taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttctctct      420

tttagattcc aacctttgga actgaattcg ccgccaccat ggccccaaag aagaagcgga      480

aggtcggtat ccacggagtc ccagcagccg acaagaagta ctccattggg ctcgatatcg      540

gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat      600

tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc      660

tgttcgactc cggggaaacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat      720

atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta      780

aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa      840

agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt      900

acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc      960

ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg     1020

gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt     1080

acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc     1140

tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg     1200

agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact     1260

ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg     1320

atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg     1380

cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga     1440

tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact     1500

tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct     1560

tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat     1620

tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa     1680

agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc     1740

accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct     1800

ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg     1860

taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga     1920

ccatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca     1980

tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact     2040

ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag     2100

aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc     2160

tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga     2220

ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg     2280

gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga     2340

acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga     2400

ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca     2460

agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag     2520

acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga     2580

acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac     2640

aagtttgtct ggctggcgat actctcatta ccctggccga tggacgacga gtgcctatta     2700

gagaactggt gtcacagcag aatttttccg tgtgggctct gaatcctcag acttaccgcc     2760

tggagagggc tagagtgagt agagctttct gtaccggcat caaacctgtg taccgcctca     2820

ccactagact ggggagatcc attagggcca ctgccaacca ccgatttctc acacctcagg     2880

gctggaaacg agtcgatgaa ctccagcctg gagattacct ggctctgcct aggagaatcc     2940

ctactgcctc ctgacaataa aatatcttta ttttcattac atctgtgtgt tggttttttg     3000

tgttagctag cgcgtagata agtagcatgg cgggttaatc attaactaca aggaacccct     3060

agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc     3120

aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcca     3180

g                                                                     3181


<210>  65
<211>  2842
<212>  DNA
<213>  Artificial

<220>
<223>  scAAV-Promoter- RmaIntC-Cas9C-synpA

<400>  65
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg       60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaaac      120

gttgacattg attattgact agccgctagc aggactcacg gggatttcca agtctccacc      180

ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc      240

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata      300

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatccg gactctaagg      360

taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttctctct      420

tttagattcc aacctttgga actgaattcg ccgccaccat ggcggcggcg tgcccggaac      480

tgcgtcagct ggcgcagagc gatgtgtatt gggatccgat tgtgagcatt gaaccggatg      540

gcgtggaaga agtgtttgat ctgaccgtgc cgggcccgca taactttgtg gcgaacgata      600

ttattgcgca taactctggc cagggggaca gtcttcacga gcacatcgct aatcttgcag      660

gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat gaactcgtca      720

aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga gagaaccaaa      780

ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa gagggtataa      840

aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag cttcagaatg      900

agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat caggaactgg      960

acatcaatcg gctctccgac tacgacgtgg atcatatcgt gccccagtct tttctcaaag     1020

atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg aagagtgata     1080

acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg     1140

ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga ggtggcctgt     1200

ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca     1260

agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa aatgacaaac     1320

tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat ttcagaaagg     1380

actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat gatgcctacc     1440

tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagcttgaa tctgaatttg     1500

tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa     1560

taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt ttcaagaccg     1620

agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca aacggagaaa     1680

caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccggaag gtcctgtcca     1740

tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc tccaaggaaa     1800

gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat tgggacccca     1860

agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt gtggccaaag     1920

tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc atcacaatca     1980

tggagcgatc aagcttcgaa aaaaacccca tcgactttct cgaggcgaaa ggatataaag     2040

aggtcaaaaa agacctcatc attaagcttc ccaagtactc tctctttgag cttgaaaacg     2100

gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag ctggcactgc     2160

cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc aaagggtctc     2220

ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac cttgatgaga     2280

tcatcgagca aataagcgaa ttctccaaaa gagtgatcct cgccgacgct aacctcgata     2340

aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag gcagaaaaca     2400

ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag tacttcgaca     2460

ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc acactgattc     2520

atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc ggtggagaca     2580

gcagggctga ccccaagaag aagaggaagg tgtgacaata aaatatcttt attttcatta     2640

catctgtgtg ttggtttttt gtgttagcta gcgcgtagat aagtagcatg gcgggttaat     2700

cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc     2760

gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc     2820

agtgagcgag cgagcgcgcc ag                                              2842


<210>  66
<211>  1947
<212>  DNA
<213>  Artificial

<220>
<223>  scAAV-Promoter-GFP-SV40pA-U6-gRNA

<400>  66
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg       60

acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaaac      120

gttgacattg attattgact agccgctagc aggactcacg gggatttcca agtctccacc      180

ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc      240

gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata      300

taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatccg gactctaagg      360

taaatataaa atttttaagt gtataatgtg ttaaactact gattctaatt gtttctctct      420

tttagattcc aacctttgga actgaattcc gcgggcccgg gatccaccgg tcgccaccat      480

ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg      540

cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg      600

caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct      660

cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca      720

gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt      780

caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt      840

gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa      900

gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg      960

catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga     1020

ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta     1080

cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct     1140

gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag     1200

cggccgctag gcctcacctg cgatctcgat gctttatttg tgaaatttgt gatgctattg     1260

ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt     1320

ttatgtttca ggttcagggg gaggtgtggg aggtttttta aactagttgt acaaaaaagc     1380

aggctttaaa ggaaccaatt cagtcgactg gatccggtac caaggtcggg caggaagagg     1440

gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa     1500

ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt     1560

aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc     1620

ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa     1680

acaccggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga     1740

aaaagtggca ccgagtcggt gctttttttt agctagcgcg tagataagta gcatggcggg     1800

ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct     1860

cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg     1920

gcctcagtga gcgagcgagc gcgccag                                         1947


