LOCUS Exported 9584 bp ds-DNA circular SYN 06-AUG-2017 DEFINITION SpCas9 with 2A-Puro and a cloning backbone for 2 custom gRNAs which can be cloned in via a one-step reaction. For generation of double knock-outs and large deletions in a single plasmid system. ACCESSION . VERSION . KEYWORDS pDG459 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9584) AUTHORS Fatwa Adikusuma, Chandran Pfitzner and Paul Thomas TITLE Direct Submission JOURNAL Exported Sunday, Aug 6, 2017 from SnapGene Viewer 3.2.1 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9584 /organism="synthetic DNA construct" /lab_host="Streptococcus pyogenes" /mol_type="other DNA" promoter 1..249 /label=U6 /note="U6 promoter" /note="color: #f58a5e; direction: RIGHT" misc_RNA 268..343 /label=chimeric guide RNA scaffold /note="gRNA scaffold" /note="color: #d59687; direction: RIGHT" enhancer 440..725 /label=CAG enhancer /note="CAG enhancer" /note="color: #ffef86" promoter 727..1004 /label=Chicken beta-actin promoter /note="Chicken beta-actin promoter" /note="color: #ccffff; direction: RIGHT" intron 1005..1233 /label=hybrid intron /note="hybrid intron" /note="color: #b1ff67" misc_feature 1254..1319 /label=3xFLAG /note="3xFLAG" /note="color: #85dae9; direction: RIGHT" misc_signal 1326..1349 /label=SV40 NLS /note="SV40 NLS" /note="color: #cc99ff; direction: RIGHT" gene 1371..5471 /label=hSpCas9 /note="hSpCas9" /note="color: #f8d3a9; direction: RIGHT" misc_signal 5472..5519 /label=nucleoplasmin NLS /note="nucleoplasmin NLS" /note="color: #b1ff67; direction: RIGHT" misc_feature 5535..5588 /label=T2A /note="T2A" /note="color: #ff9ccd; direction: RIGHT" gene 5589..6188 /label=PuroR /note="PuroR" /note="color: #9eafd2; direction: RIGHT" polyA_signal 6219..6429 /label=BGH polyA /note="BGH polyA" /note="color: #85dae9; direction: RIGHT" misc_RNA complement(6497..6572) /label=chimeric guide RNA scaffold /note="gRNA scaffold" /note="color: #d59687; direction: LEFT" promoter complement(6590..6838) /label=U6 /note="U6 promoter" /note="color: #f58a5e; direction: LEFT" repeat_region 6847..6987 /label=AAV2 ITR /note="AAV2 ITR" /note="color: #ffff99" rep_origin 7062..7517 /direction=RIGHT /label=F1 Ori /note="F1 Ori" /note="color: #85dae9" promoter 7799..7903 /label=AmpR promoter /note="AmpR promoter" /note="color: #b7e6d7; direction: RIGHT" gene 7904..8764 /label=AmpR /note="AmpR" /note="color: #84b0dc; direction: RIGHT" rep_origin 8859..9541 /direction=RIGHT /label=ColE1 origin /note="ColE1 origin" /note="color: #9eafd2" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1381 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg 5521 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc 5581 ctggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca 5641 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 5701 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 5761 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 5821 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 5881 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 5941 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg 6001 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 6061 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 6121 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 6181 gtgcctgaga attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 6241 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 6301 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6361 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 6421 tgctggggag cggccgctct agaactagtg gatcccccgg gctgcaggaa ttcgatacga 6481 atcctgcagg aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc 6541 ttattttaac ttgctatttc tagctctaaa acgtcttctc gaagaccccg gtgtttcgtc 6601 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat 6661 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc 6721 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct 6781 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctcgc 6841 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 6901 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 6961 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 7021 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 7081 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 7141 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 7201 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 7261 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 7321 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 7381 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc 7441 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 7501 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 7561 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 7621 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 7681 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 7741 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 7801 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 7861 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 7921 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 7981 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 8041 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 8101 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 8161 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 8221 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 8281 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 8341 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 8401 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 8461 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 8521 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 8581 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc 8641 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 8701 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 8761 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 8821 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 8881 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 8941 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 9001 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 9061 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 9121 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 9181 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 9241 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 9301 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 9361 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 9421 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 9481 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 9541 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt //