LOCUS pZac-SMVP-NLS 7465 bp DNA circular 03-AUG-2016 Addgene 80944 Wei Leong Chew George Church lab FEATURES Location/Qualifiers repeat_region 2012..2141 /dnas_title="ITR" /vntifkey="34" /label=ITR repeat_region complement(6536..6665) /dnas_title="ITR" /vntifkey="34" /label=ITR promoter 2470..3022 /label=CMV_immearly_promoter misc_feature 2525..2812 /label=CAG_enhancer misc_feature 2308..3049 /note="CMV IE" protein_bind 2314..2327 /note="NF1" protein_bind 2361..2374 /note="NF1" protein_bind 2415..2428 /note="NF1" misc_feature complement(2201..2284) /note="SV40_enhancer" protein_bind 2218..2228 /note="NFkB2" promoter 2968..3049 /label=CMV_promoter misc_feature 2979..2999 /label=CMV_fwd_primer TATA_signal 3014..3019 /note="" misc_feature 3120..3252 /note="chim_int" gene 3288..5474 /note="hCas9" misc_feature 3294..3317 /note="SV40 NLS" misc_feature 5472..5474 /note="" misc_feature 5475..5780 /note="N-intein" misc_feature complement(5794..5813) /label=EBV_rev_primer 3'UTR 5794..5995 /note="SV40 pA" polyA_signal 5893..5898 /note="" promoter 6066..6384 /note="U6 promoter" misc_feature 6399..6481 /note="Sp gRNA scaffold" misc_feature 6385..6398 /note="gRNA Sp" source 1..7465 /dnas_title="pZac-SMVP-NLS SphCas9- Rma Int-N trun - SV40pA - U6 -Sp gRNA (Pd-l1 binding P2) (Addgene 80944)" ORIGIN 1 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 61 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aatagtggta 121 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 181 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 241 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 301 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 361 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 421 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 481 acgacgagcg tgacaccacg atgcctgtag taatggtaac aacgttgcgc aaactattaa 541 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 601 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 661 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 721 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 781 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1141 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 1321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 1381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 1441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 1501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 1561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 1621 tttgctgcgg ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 1681 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 1741 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 1801 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 1861 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 1921 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 1981 atgaccatga ttacgccaga tttaattaag gctgcgcgct cgctcgctca ctgaggccgc 2041 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc 2101 gcgcagagag ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc 2161 gccatgctac ttatctacCT GCgGccGcCA TTATGGAATG TGTGTCAGTT AGGGTGTGGA 2221 AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA 2281 ACCAgtcccg gtctcctccc atgcatgtca atattggcca ttagccatat tattcattgg 2341 ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa 2401 tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac 2461 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 2521 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 2581 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 2641 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 2701 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 2761 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 2821 catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 2881 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 2941 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 3001 acggtgggag gtctatataa gcagaggtcg tttagtgaac cgtcagatca ctagtagctt 3061 tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgctcgac tgatcacagg 3121 taagtatcaa ggttacaaga caggtttaag gaggccaata gaaactgggc ttgtcgagac 3181 agagaagatt cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct 3241 ttctctccac aggGGTACCG AAGCCGCTAG CGCTACCGGT CGCCACCATG GCCCCAAAGA 3301 AGAAGCGGAA GGTCGGTATC CACGGAGTCC CAGCAGCCGA CAAGAAGTAC TCCATTGGGC 3361 TCGATATCGG CACAAACAGC GTCGGCTGGG CCGTCATTAC GGACGAGTAC AAGGTGCCGA 3421 GCAAAAAATT CAAAGTTCTG GGCAATACCG ATCGCCACAG CATAAAGAAG AACCTCATTG 3481 GCGCCCTCCT GTTCGACTCC GGGGAaACGG CCGAAGCCAC GCGGCTCAAA AGAACAGCAC 3541 GGCGCAGATA TACCCGCAGA AAGAATCGGA TCTGCTACCT GCAGGAGATC TTTAGTAATG 3601 AGATGGCTAA GGTGGATGAC TCTTTCTTCC ATAGGCTGGA GGAGTCCTTT TTGGTGGAGG 3661 AGGATAAAAA GCACGAGCGC CACCCAATCT TTGGCAATAT CGTGGACGAG GTGGCGTACC 3721 ATGAAAAGTA CCCAACCATA TATCATCTGA GGAAGAAGCT TGTAGACAGT ACTGATAAGG 3781 CTGACTTGCG GTTGATCTAT CTCGCGCTGG CGCATATGAT CAAATTTCGG GGACACTTCC 3841 TCATCGAGGG GGACCTGAAC CCAGACAACA GCGATGTCGA CAAACTCTTT ATCCAACTGG 3901 TTCAGACTTA CAATCAGCTT TTCGAAGAGA ACCCGATCAA CGCATCCGGA GTTGACGCCA 3961 AAGCAATCCT GAGCGCTAGG CTGTCCAAAT CCCGGCGGCT CGAAAACCTC ATCGCACAGC 4021 TCCCTGGGGA GAAGAAGAAC GGCCTGTTTG GTAATCTTAT CGCCCTGTCA CTCGGGCTGA 4081 CCCCCAACTT TAAATCTAAC TTCGACCTGG CCGAAGATGC CAAGCTTCAA CTGAGCAAAG 4141 ACACCTACGA TGATGATCTC GACAATCTGC TGGCCCAGAT CGGCGACCAG TACGCAGACC 4201 TTTTTTTGGC GGCAAAGAAC CTGTCAGACG CCATTCTGCT GAGTGATATT CTGCGAGTGA 4261 ACACGGAGAT CACCAAAGCT CCGCTGAGCG CTAGTATGAT CAAGCGCTAT GATGAGCACC 4321 ACCAAGACTT GACTTTGCTG AAGGCCCTTG TCAGACAGCA ACTGCCTGAG AAGTACAAGG 4381 AAATTTTCTT CGATCAGTCT AAAAATGGCT ACGCCGGATA CATTGACGGC GGAGCAAGCC 4441 AGGAGGAATT TTACAAATTT ATTAAGCCCA TCTTGGAAAA AATGGACGGC ACCGAGGAGC 4501 TGCTGGTAAA GCTTAACAGA GAAGATCTGT TGCGCAAACA GCGCACTTTC GACAATGGAA 4561 GCATCCCCCA CCAGATTCAC CTGGGCGAAC TGCACGCTAT CCTCAGGCGG CAAGAGGATT 4621 TCTACCCCTT TTTGAAAGAT AACAGGGAAA AGATTGAGAA AATCCTCACA TTTCGGATAC 4681 CCTACTATGT AGGCCCCCTC GCCCGGGGAA ATTCCAGATT CGCGTGGATG ACTCGCAAAT 4741 CAGAAGAgAC CATCACTCCC TGGAACTTCG AGGAAGTCGT GGATAAGGGG GCCTCTGCCC 4801 AGTCCTTCAT CGAAAGGATG ACTAACTTTG ATAAAAATCT GCCTAACGAA AAGGTGCTTC 4861 CTAAACACTC TCTGCTGTAC GAGTACTTCA CAGTTTATAA CGAGCTCACC AAGGTCAAAT 4921 ACGTCACAGA AGGGATGAGA AAGCCAGCAT TCCTGTCTGG AGAGCAGAAG AAAGCTATCG 4981 TGGACCTCCT CTTCAAGACG AACCGGAAAG TTACCGTGAA ACAGCTCAAA GAAGACTATT 5041 TCAAAAAGAT TGAATGTTTC GACTCTGTTG AAATCAGCGG AGTGGAGGAT CGCTTCAACG 5101 CATCCCTGGG AACGTATCAC GATCTCCTGA AAATCATTAA AGACAAGGAC TTCCTGGACA 5161 ATGAGGAGAA CGAGGACATT CTTGAGGACA TTGTCCTCAC CCTTACGTTG TTTGAAGATA 5221 GGGAGATGAT TGAAGAACGC TTGAAAACTT ACGCTCATCT CTTCGACGAC AAAGTCATGA 5281 AACAGCTCAA GAGGCGCCGA TATACAGGAT GGGGGCGGCT GTCAAGAAAA CTGATCAATG 5341 GGATCCGAGA CAAGCAGAGT GGAAAGACAA TCCTGGATTT TCTTAAGTCC GATGGATTTG 5401 CCAACCGGAA CTTCATGCAG TTGATCCATG ATGACTCTCT CACCTTTAAG GAGGACATCC 5461 AGAAAGCACA AGTTtgtctg gctggcgata ctctcattac cctggccgat ggacgacgag 5521 tgcctattag agaactggtg tcacagcaga atttttccgt gtgggctctg aatcctcaga 5581 cttaccgcct ggagagggct agagtgagta gagctttctg taccggcatc aaacctgtgt 5641 accgcctcac cactagactg gggagatcca ttagggccac tgccaaccac cgatttctca 5701 cacctcaggg ctggaaacga gtcgatgaac tccagcctgg agattacctg gctctgccta 5761 ggagaatccc tactgcctcc tgaTCTCGAG CTcgatgagt ttggacaaac cacaactaga 5821 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 5881 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 5941 cagggggagg tgtgggaggt tttttaaaGC AAGTAAAACC TCTACAAATG TGGTActtaa 6001 gagggggaga ccaaagggcg agacgttaag gcctcacgtg acatgtgagc aaaaggccag 6061 caaaaTGTAC AAAAAAGCAG GCTTTAAAGG AACCAATTCA GTCGACTGGA TCCGGTACCA 6121 AGGTCGGGCA GGAAGAGGGC CTATTTCCCA TGATTCCTTC ATATTTGCAT ATACGATACA 6181 AGGCTGTTAG AGAGATAATT AGAATTAATT TGACTGTAAA CACAAAGATA TTAGTACAAA 6241 ATACGTGACG TAGAAAGTAA TAATTTCTTG GGTAGTTTGC AGTTTTAAAA TTATGTTTTA 6301 AAATGGACTA TCATATGCTT ACCGTAACTT GAAAGTATTT CGATTTCTTG GCTTTATATA 6361 TCTTGTGGAA AGGACGAAAC ACCGCTAAAG TCATCCGCGT TTTAGAGCTA GAAATAGCAA 6421 GTTAAAATAA GGCTAGTCCG TTATCAACTT GAAAAAGTGG CACCGAGTCG GTGCTTTTTT 6481 TtCTAGAggG CGGCCGCAgt agataagtag catggcgggt taatcattaa ctacaaggaa 6541 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 6601 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 6661 cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6721 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6781 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 6841 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 6901 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 6961 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 7021 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 7081 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 7141 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 7201 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7261 aacgcgaatt ttaacaaaat attaacgttt ataatttcag gtggcatctt tcggggaaat 7321 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 7381 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 7441 catttccgtg tcgcccttat tccct //