LOCUS       Exported                9581 bp ds-DNA     circular SYN 06-AUG-2017
DEFINITION  SpCas9n (D10A Nickase mutant) with 2A-Puro and 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    pDG462
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9581)
  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..9581
                     /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=hSpCas9n
                     /note="hSpCas9n"
                     /note="color: #ffef86; 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(6494..6569)
                     /label=chimeric guide RNA scaffold
                     /note="gRNA scaffold"
                     /note="color: #d59687; direction: LEFT"
     promoter        complement(6587..6835)
                     /label=U6
                     /note="U6 promoter"
                     /note="color: #f58a5e; direction: LEFT"
     repeat_region   6844..6984
                     /label=AAV2 ITR
                     /note="AAV2 ITR"
                     /note="color: #ffff99"
     rep_origin      7059..7514
                     /direction=RIGHT
                     /label=F1 Ori
                     /note="F1 Ori"
                     /note="color: #85dae9"
     promoter        7796..7900
                     /label=AmpR promoter
                     /note="AmpR promoter"
                     /note="color: #b7e6d7; direction: RIGHT"
     promoter        7831..7859
                     /label=AmpR_promoter
                     /note="AmpR_promoter"
                     /note="color: #ffef86"
     gene            7901..8761
                     /label=AmpR
                     /note="AmpR"
                     /note="color: #84b0dc; direction: RIGHT"
     rep_origin      8856..9538
                     /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 cctggccatc 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 ttcgataatc
     6481 ctgcaggaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
     6541 ttttaacttg ctatttctag ctctaaaacg tcttctcgaa gaccccggtg tttcgtcctt
     6601 tccacaagat atataaagcc aagaaatcga aatactttca agttacggta agcatatgat
     6661 agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat tactttctac
     6721 gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcca attatctctc
     6781 taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg ccctcgcggc
     6841 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     6901 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     6961 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     7021 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     7081 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7141 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7201 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7261 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7321 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7381 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     7441 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     7501 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7561 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7621 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7681 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     7741 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     7801 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     7861 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     7921 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     7981 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     8041 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     8101 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     8161 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     8221 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     8281 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     8341 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     8401 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     8461 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     8521 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     8581 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     8641 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     8701 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     8761 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     8821 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     8881 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     8941 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     9001 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     9061 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     9121 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     9181 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     9241 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     9301 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     9361 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     9421 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     9481 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     9541 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
