LOCUS 140708_SpSelfcut 7679 bp DNA linear 25-JUL-2017 FEATURES Location/Qualifiers misc_feature 7366..7385 /label=Protospacer /note="modified AAVS1-T1 protospacer" misc_feature 7386..7462 /label=SpCas9 gRNA scaffold terminator 7463..7468 /note="U6 terminator" variation 7413..7413 /mod_base=A > C variation 7414..7414 /mod_base=A > C gene 590..1624 /note="Hygromycin" PCR_primer complement(1602..1621) /label=PB-Cas9-Hyg_n8647_R /note="Ordered sequence: PB-Cas9-Hyg_n8647_R: CTAAGCGTAATCTGGAACATCGTATGGGTA TTCCTTTGCCCTCGGACGAG" gene 3141..4001 /note="Amp" promoter 7061..7302 /note="U6 Promoter" PCR_primer complement(7419..7440) /note="SP-Hyg-LV_R1" PCR_primer 7229..7251 /note="Sp-Hyg-LV_F1" primer_bind 7232..7251 /label=pLKO.1 5' /note="Human U6 promoter, forward primer" variation 7387..7387 /mod_base=T > G variation 7388..7388 /mod_base=T > G misc_feature 7310..7310 /note="TSS" source 1..7679 /dnas_title="140708_SpSelfcuttingGuide_d1" ORIGIN 1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 481 gaccgaatca ccgacctctc tccccagggg gatccaccgg atcaagcttg ccagctgggg 541 cgccctctgg taaggttggg aagccctgca gatctgatcc cggggggcaA TGGATAGATC 601 CGGAAAGCCT GAACTCACCG CGACGTCTGT CGAGAAGTTT CTGATCGAAA AGTTCGACAG 661 CGTCTCCGAC CTGATGCAGC TCTCGGAGGG CGAAGAATCT CGTGCTTTCA GCTTCGATGT 721 AGGAGGGCGT GGATATGTCC TGCGGGTAAA TAGCTGCGCC GATGGTTTCT ACAAAGATCG 781 TTATGTTTAT CGGCACTTTG CATCGGCCGC GCTCCCGATT CCGGAAGTGC TTGACATTGG 841 GGAATTTAGC GAGAGCCTGA CCTATTGCAT CTCCCGCCGT GCACAGGGTG TCACGTTGCA 901 AGACCTGCCT GAAACCGAAC TGCCCGCTGT TCTGCAGCCG GTCGCGGAGG CCATGGATGC 961 GATCGCTGCG GCCGATCTTA GCCAGACGAG CGGGTTCGGC CCATTCGGAC CGCAAGGAAT 1021 CGGTCAATAC ACTACATGGC GTGATTTCAT ATGCGCGATT GCTGATCCCC ATGTGTATCA 1081 CTGGCAAACT GTGATGGACG ACACCGTCAG TGCGTCCGTC GCGCAGGCTC TCGATGAGCT 1141 GATGCTTTGG GCCGAGGACT GCCCCGAAGT CCGGCACCTC GTGCACGCGG ATTTCGGCTC 1201 CAACAATGTC CTGACGGACA ATGGCCGCAT AACAGCGGTC ATTGACTGGA GCGAGGCGAT 1261 GTTCGGGGAT TCCCAATACG AGGTCGCCAA CATCTTCTTC TGGAGGCCGT GGTTGGCTTG 1321 TATGGAGCAG CAGACGCGCT ACTTCGAGCG GAGGCATCCG GAGCTTGCAG GATCGCCGCG 1381 GCTCCGGGCG TATATGCTCC GCATTGGTCT TGACCAACTC TATCAGAGCT TGGTTGACGG 1441 CAATTTCGAT GATGCAGCTT GGGCGCAGGG TCGATGCGAC GCAATCGTCC GATCCGGAGC 1501 CGGGACTGTC GGGCGTACAC AAATCGCCCG CAGAAGCGCG GCCGTCTGGA CCGATGGCTG 1561 TGTAGAAGTA CTCGCCGATA GTGGAAACCG ACGCCCCAGC ACTCGTCCGA GGGCAAAGGA 1621 ATAGAGTAGA TGCCGACCGG GATCTATCGA TACCGTCGAC CTCGAGGGGG GGCCcggtac 1681 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg 1741 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg 1801 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 1861 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 1921 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 1981 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag 2041 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 2101 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 2161 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc 2221 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 2281 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt 2341 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg 2401 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2461 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2521 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc 2581 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 2641 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 2701 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 2761 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 2821 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 2881 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 2941 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 3001 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 3061 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 3121 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 3181 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 3241 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 3301 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 3361 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 3421 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 3481 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 3541 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 3601 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 3661 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 3721 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 3781 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 3841 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 3901 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 3961 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 4021 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 4081 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 4141 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 4201 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 4261 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 4321 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 4381 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 4441 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 4501 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 4561 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 4621 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 4681 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 4741 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 4801 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 4861 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 4921 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 4981 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 5041 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 5101 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 5161 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc 5221 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta 5281 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg 5341 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac 5401 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc 5461 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 5521 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 5581 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg 5641 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact 5701 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa 5761 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 5821 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat 5881 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 5941 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 6001 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 6061 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 6121 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat 6181 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg 6241 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat 6301 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 6361 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 6421 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 6481 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat 6541 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag 6601 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat 6661 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa 6721 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat 6781 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt 6841 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt 6901 atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga 6961 agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta 7021 tcgatcacga gactagcctc gagcggccgc ccccttcacc gagggcctat ttcccatgat 7081 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 7141 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 7201 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 7261 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccG GTAGACGCTG 7321 TGACAGAGCC AACACGCAGT CTCGGTCACA CTGATGCAGC TAGTATGCAC CTCCACCCCA 7381 CAGTGGGGTT AGAGCTAGAA ATAGCAAGTT AACCTAAGGC TAGTCCGTTA TCAACTTGAA 7441 AAAGTGGCAC CGAGTCGGTG CTTTTTTTGA ATTCtcgacc tcgagacaaa tggcagtatt 7501 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt 7561 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca 7621 aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc tcgaggggg //