LOCUS       pX459V2.0-HypaCas9-mRub 9955 bp ds-DNA     circular     09-MAR-2022
DEFINITION  .
SOURCE      synthetic DNA construct                                            
  ORGANISM  synthetic DNA construct                                            
FEATURES             Location/Qualifiers
     promoter        1..241
                     /label="U6 promoter"
                     /ApEinfo_revcolor=#346ee0
                     /ApEinfo_fwdcolor=#346ee0
     misc_feature    251..270
                     /label="sgRNA_ECT2_3"
                     /ApEinfo_revcolor=#75c6a9
                     /ApEinfo_fwdcolor=#75c6a9
     misc_RNA        271..346
                     /label="g RNA scaffold"
                     /ApEinfo_revcolor=#ff9ccd
                     /ApEinfo_fwdcolor=#ff9ccd
     enhancer        443..728
                     /label="CMV enhancer"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     promoter        730..1007
                     /label="chicken beta-actin promoter"
                     /ApEinfo_revcolor=#346ee0
                     /ApEinfo_fwdcolor=#346ee0
     intron          1008..1236
                     /label="hybrid intron"
                     /ApEinfo_revcolor=#eb606c
                     /ApEinfo_fwdcolor=#eb606c
     CDS             1254..7217
                     /label="Translation 1254-7217"
     CDS             1257..1322
                     /label="3xFLAG"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     CDS             1329..1349
                     /label="SV40 NLS"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     CDS             1374..5474
                     /label="Cas9"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     misc_feature    2640..2657
                     /label="Cas9FW1"
                     /ApEinfo_revcolor=#ff00ce
                     /ApEinfo_fwdcolor=#ff00ce
     misc_feature    3419..3489
                     /label="HypaCas9FW"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    complement(3419..3489)
                     /label="HypaCas9RV"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    3444..3446
                     /label="N692A"
                     /ApEinfo_revcolor=#ff0000
                     /ApEinfo_fwdcolor=#ff0000
     misc_feature    3450..3452
                     /label="M694A"
                     /ApEinfo_revcolor=#ff0007
                     /ApEinfo_fwdcolor=#ff0007
     misc_feature    3453..3455
                     /label="Q695A"
                     /ApEinfo_revcolor=#ff0000
                     /ApEinfo_fwdcolor=#ff0000
     misc_feature    3462..3464
                     /label="H698A"
                     /ApEinfo_revcolor=#ff000e
                     /ApEinfo_fwdcolor=#ff000e
     misc_feature    4322..4339
                     /label="Cas9RV2"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     CDS             5475..5522
                     /label="nucleoplasmin NLS"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     CDS             5538..5594
                     /label="Translation 5538-5594"
     misc_feature    5538..5594
                     /label="P2A"
     misc_feature    5595..6305
                     /label="mRuby2"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     CDS             6315..6368
                     /label="T2A"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     CDS             6369..6968
                     /label="PuroR"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     polyA_signal    7002..7209
                     /label="bGH poly(A) signal"
                     /ApEinfo_revcolor=#ff3eee
                     /ApEinfo_fwdcolor=#ff3eee
     repeat_region   7218..7347
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     repeat_region   7218..7358
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     rep_origin      7433..7888
                     /label="f1 ori"
                     /ApEinfo_revcolor=#b4abac
                     /ApEinfo_fwdcolor=#b4abac
     promoter        8170..8274
                     /label="AmpR promoter"
                     /ApEinfo_revcolor=#346ee0
                     /ApEinfo_fwdcolor=#346ee0
     CDS             8275..9135
                     /label="AmpR"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     rep_origin      9306..9894
                     /label="ori"
                     /ApEinfo_revcolor=#b4abac
                     /ApEinfo_fwdcolor=#b4abac
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 TATTAACATC CACTACTGGG gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
     1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact
     1261 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata
     1321 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     1381 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1441 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1501 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1561 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1621 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1681 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1741 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1801 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1861 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1921 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1981 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     2041 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     2101 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2161 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2221 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2281 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2341 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2401 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2461 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2521 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2581 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2641 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2701 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2761 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2821 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2881 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2941 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     3001 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3061 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3121 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3181 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3241 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3301 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3361 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3421 agtccgacgg cttcgccaac agaGCcttcG CgGCgctgat cGCcgacgac agcctgacct
     3481 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3541 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3601 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3661 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3721 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3781 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3841 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3901 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3961 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     4021 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4081 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4141 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4201 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4261 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4321 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4381 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4441 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4501 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4561 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4621 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4681 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4741 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4801 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4861 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4921 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4981 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     5041 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5101 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5161 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5221 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5281 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5341 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5401 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5461 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     5521 aggaattcgg cagtggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     5581 agaaccctgg acctatggtg tctaagggcg aagagctgat caaggaaaat atgcgtatga
     5641 aggtggtcat ggaaggttcg gtcaacggcc accaattcaa atgcacaggt gaaggagaag
     5701 gcaatccgta catgggaact caaaccatga ggatcaaagt catcgaggga ggacccctgc
     5761 catttgcctt tgacattctt gccacgtcgt tcatgtatgg cagccgtact tttatcaagt
     5821 acccgaaagg cattcctgat ttctttaaac agtcctttcc tgagggtttt acttgggaaa
     5881 gagttacgag atacgaagat ggtggagtcg tcaccgtcat gcaggacacc agccttgagg
     5941 atggctgtct cgtttaccac gtccaagtca gaggggtaaa ctttccctcc aatggtcccg
     6001 tgatgcagaa gaaAaccaag ggttgggagc ctaatacaga gatgatgtat ccagcagatg
     6061 gtggtctgag gggatacact catatggcac tgaaagttga tggtggtggc catctgtctt
     6121 gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta
     6181 tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag
     6241 tacaacgcga acacgcagtt gccaagttcg ccgggcttgg tggtgggatg gacgagctgt
     6301 acaagggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     6361 ctggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     6421 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     6481 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     6541 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     6601 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     6661 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     6721 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg
     6781 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     6841 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     6901 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     6961 gtgcctgaga attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     7021 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     7081 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     7141 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     7201 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     7261 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     7321 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     7381 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     7441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     7501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     7561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     7621 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     7681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     7741 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     7801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7861 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     7921 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     7981 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     8041 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     8101 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     8161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     8221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     8281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     8341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     8401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     8461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     8521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     8581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     8641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     8701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     8761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     8821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     8881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     8941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     9001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     9061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     9121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     9181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     9241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     9301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     9361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     9421 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     9481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     9541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     9601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     9661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     9721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     9781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     9841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     9901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//