LOCUS       Exported                8840 bp ds-DNA     circular SYN 06-AUG-2017
DEFINITION  SpCas9n (D10A Nickase mutant) with a cloning backbone for 2 custom 
            gRNAs which can be cloned in via a one-step reaction. For generation
            of DSBs with no off-target cuts.
ACCESSION   .
VERSION     .
KEYWORDS    pDG335
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8840)
  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..8840
                     /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..1280
                     /label=HA
                     /note="HA"
                     /note="color: #ff99cc; direction: RIGHT"
     misc_signal     1284..1304
                     /label=SV40 NLS
                     /note="SV40 NLS"
                     /note="color: #cc99ff; direction: RIGHT"
     gene            1314..5414
                     /label=hSpCas9n
                     /note="hSpCas9n"
                     /note="color: #ffef86; direction: RIGHT"
     polyA_signal    5478..5688
                     /label=BGH polyA
                     /note="BGH polyA"
                     /note="color: #85dae9; direction: RIGHT"
     misc_RNA        complement(5753..5828)
                     /label=chimeric guide RNA scaffold
                     /note="gRNA scaffold"
                     /note="color: #d59687; direction: LEFT"
     promoter        complement(5846..6094)
                     /label=U6
                     /note="U6 promoter"
                     /note="color: #f58a5e; direction: LEFT"
     repeat_region   6103..6243
                     /label=AAV2 ITR
                     /note="AAV2 ITR"
                     /note="color: #ffff99"
     rep_origin      6318..6773
                     /direction=RIGHT
                     /label=F1 Ori
                     /note="F1 Ori"
                     /note="color: #85dae9"
     promoter        7055..7159
                     /label=AmpR promoter
                     /note="AmpR promoter"
                     /note="color: #b7e6d7; direction: RIGHT"
     gene            7160..8020
                     /label=AmpR
                     /note="AmpR"
                     /note="color: #84b0dc; direction: RIGHT"
     rep_origin      8115..8797
                     /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 atgtacccat
     1261 acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga
     1321 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1381 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1441 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1501 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1561 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1621 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1681 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1741 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1801 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1861 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1921 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1981 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc
     2041 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2101 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2161 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2221 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2281 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2341 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2401 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2461 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2521 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2581 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2641 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2701 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2761 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2821 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2881 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2941 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3001 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3061 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3121 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3181 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3241 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3301 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3361 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3421 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3481 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3541 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3601 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3661 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3721 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3781 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3841 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3901 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3961 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4021 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4081 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4141 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4201 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4261 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4321 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4381 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4441 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4501 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4561 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4621 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4681 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4741 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4801 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4861 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4921 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4981 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5041 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5101 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5161 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5221 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5281 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5341 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5401 agctgggagg cgacagcccc aagaagaaga gaaaggtgga ggccagctaa gaattcctag
     5461 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5521 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5581 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5641 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgctcta
     5701 gaactagtgg atcccccggg ctgcaggaat tcgataatcc tgcaggaaaa aagcaccgac
     5761 tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc
     5821 tctaaaacgt cttctcgaag accccggtgt ttcgtccttt ccacaagata tataaagcca
     5881 agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt
     5941 ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata
     6001 tctttgtgtt tacagtcaaa ttaattccaa ttatctctct aacagccttg tatcgtatat
     6061 gcaaatatga aggaatcatg ggaaataggc cctcgcggcc gcaggaaccc ctagtgatgg
     6121 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     6181 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     6241 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     6301 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     6361 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     6421 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6481 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     6541 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6601 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg
     6661 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6721 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     6781 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     6841 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     6901 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6961 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     7021 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     7081 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     7141 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     7201 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     7261 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     7321 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     7381 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     7441 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     7501 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7561 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7621 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7681 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7741 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7801 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7861 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7921 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7981 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     8041 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     8101 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     8161 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     8221 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     8281 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     8341 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     8401 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     8461 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     8521 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     8581 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     8641 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     8701 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8761 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8821 ctggcctttt gctcacatgt
//
