LOCUS       Akoh-pspCAS9-2Ap        9175 bp    DNA     linear       02-OCT-2018
Created: Friday, July 25, 2014 12:06
FEATURES             Location/Qualifiers
     misc_feature    173..194
                     /note="sequencing primer"
     misc_feature    252..265
                     /note="cloning site"
     CDS             1251..6188
                     /note="CAS9 2a Puro"
                     /translation="MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAADKKY
                     SIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATR
                     LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN
                     IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
                     DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL
                     FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN
                     LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD
                     QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP
                     HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS
                     EETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVYNELTKV
                     KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED
                     RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLF
                     DDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDS
                     LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI
                     VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ
                     NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHVAQ
                     ILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
                     VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG
                     ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG
                     NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI
                     LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST
                     KEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKEFGSGEGRGSLLT
                     CGDVEENPGPMTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTE
                     LQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ
                     QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    1251..5525
                     /note="SpCas9"
     misc_feature    5526..5588
                     /note="2A"
     misc_feature    5589..6188
                     /note="Puro"
     CDS             5526..5588
                     /note=""
                     /translation="GSGEGRGSLLTCGDVEENPGP"
     promoter        10..262
                     /note="U6"
     misc_feature    2724..2727
                     /note="ApaI"
     misc_feature    2856..2858
                     /note="N497A"
     misc_feature    2728..4157
                     /note="VP12 insertion"
     misc_feature    3348..3350
                     /note="R661A"
     misc_feature    3450..3452
                     /note="Q695A"
     misc_feature    4143..4145
                     /note="Q926A"
     misc_feature    2728..2729
                     /note="P"
     misc_feature    2487..2507
                     /note="For primer"
     misc_feature    3440..3460
                     /note="Rev primer"
     source          1..9175
                     /dnas_title="Akoh-pspCAS9-2Apuro (VP12-px459)"
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 GTGGGCCccc tggcccgagg gaactctcgg ttcgcatgga
     2761 tgacaagaaa gtccgaagaa acgattactc cctggaattt tgaggaagtt gtcgataaag
     2821 gtgcgtcagc tcaatcgttc atcgagagga tgaccgcctt tgacaagaat ttaccgaacg
     2881 aaaaagtatt gcctaagcac agtttacttt acgagtattt cacagtgtac aatgaactca
     2941 cgaaagttaa gtatgtcact gagggcatgc gtaaacccgc ctttctaagc ggagaacaga
     3001 agaaagcaat agtagatctg ttattcaaga ccaaccgcaa agtgacagtt aagcaattga
     3061 aagaggacta ctttaagaaa attgaatgct tcgattctgt cgagatctcc ggggtagaag
     3121 atcgatttaa tgcgtcactt ggtacgtatc atgacctcct aaagataatt aaagataagg
     3181 acttcctgga taacgaagag aatgaagata tcttagaaga tatagtgttg actcttaccc
     3241 tctttgaaga tcgggaaatg attgaggaaa gactaaaaac atacgctcac ctgttcgacg
     3301 ataaggttat gaaacagtta aagaggcgtc gctatacggg ctggggagcc ttgtcgcgga
     3361 aacttatcaa cgggataaga gacaagcaaa gtggtaaaac tattctcgat tttctaaaga
     3421 gcgacggctt cgccaatagg aactttatgg ccctgatcca tgatgactct ttaaccttca
     3481 aagaggatat acaaaaggca caggtttccg gacaagggga ctcattgcac gaacatattg
     3541 cgaatcttgc tggttcgcca gccatcaaaa agggcatact ccagacagtc aaagtagtgg
     3601 atgagctagt taaggtcatg ggacgtcaca aaccggaaaa cattgtaatc gagatggcac
     3661 gcgaaaatca aacgactcag aaggggcaaa aaaacagtcg agagcggatg aagagaatag
     3721 aagagggtat taaagaactg ggcagccaga tcttaaagga gcatcctgtg gaaaataccc
     3781 aattgcagaa cgagaaactt tacctctatt acctacaaaa tggaagggac atgtatgttg
     3841 atcaggaact ggacataaac cgtttatctg attacgacgt cgatcacatt gtaccccaat
     3901 cctttttgaa ggacgattca atcgacaata aagtgcttac acgctcggat aagaaccgag
     3961 ggaaaagtga caatgttcca agcgaggaag tcgtaaagaa aatgaagaac tattggcggc
     4021 agctcctaaa tgcgaaactg ataacgcaaa gaaagttcga taacttaact aaagctgaga
     4081 ggggtggctt gtctgaactt gacaaggccg gatttattaa acgtcagctc gtggaaaccc
     4141 gcgccatcac aaagcatGTG 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 tcggcgtctc gcccgaccac cagggcaagg
     6001 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     6061 ccttcctgga gacctccgcg ccccacaacc 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 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     6481 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     6541 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     6601 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     6661 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6721 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6781 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6841 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     6901 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6961 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     7021 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7081 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     7141 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     7201 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     7261 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     7321 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     7381 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     7441 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     7501 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     7561 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     7621 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     7681 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     7741 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     7801 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     7861 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     7921 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     7981 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     8041 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     8101 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     8161 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     8221 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     8281 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     8341 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     8401 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     8461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     8521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     8581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     8641 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     8701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     8761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     8821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     8881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     8941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     9001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     9061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     9121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//

