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
//
