LOCUS       .                       6310 bp    DNA     linear   UNK 01-JAN-1980
DEFINITION  .
ACCESSION   <unknown id>
VERSION     <unknown id>
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     misc_feature    1..86
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
                     /note="color: #00ccff"
                     /label="gRNA scaffold"
     terminator      87..92
                     /note="RNA polymerase III transcription terminator"
                     /note="color: #ffffff"
                     /label="polIII terminator"
     CDS             2774..5389
                     /codon_start=1
                     /product="downstream fragment of Streptococcus pyogenes
                     Cas9"
                     /transl_table=1
                     /translation="NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS
                     GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
                     IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW
                     GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD
                     SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
                     RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDY
                     DVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR
                     KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK
                     VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
                     KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
                     VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
                     GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV
                     KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP
                     EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN
                     IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG
                     D"
                     /label="Cas9-Down"
                     /note="color: #993366"
     CDS             660..1520
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="color: #ccffcc"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     rep_origin      1691..2279
                     /direction=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /note="color: #ffff00"
                     /label="ori"
     polyA_signal    5461..5937
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
                     /label="hGH poly(A) signal"
     repeat_region   153..293
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /note="color: #ffe4c4"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     repeat_region   2624..2764
                     /note="inverted terminal repeat of adeno-associated virus
                     serotype 2"
                     /note="color: #ffe4c4"
                     /rpt_type=inverted
                     /label="AAV2 ITR"
     promoter        555..659
                     /gene="bla"
                     /label="AmpR promoter"
                     /note="color: #ffffff"
     CDS             5426..5449
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
                     /note="color: #cc99b2"
     CDS             5405..5425
                     /codon_start=1
                     /locus_tag=""
                     /note=""
                     /note="color: #993366"
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /protein_id=""
                     /transl_table=1
                     /translation="PKKKRKV"
                     /label="SV40 NLS"
     promoter        6060..6308
                     /note="RNA polymerase III promoter for human U6 snRNA
                     (Domitrovich & Kunkel, 2003)"
                     /note="color: #ffffff"
                     /label="U6 promoter"
ORIGIN
        1 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
       61 ttgaaaaagt ggcaccgagt cggtgctttt tttgagcaaa aggccagcaa aaggccagga
      121 accgtaaaaa ggccgcgttg ctggcgagat ctaggaaccc ctagtgatgg agttggccac
      181 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
      241 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggcagcttg
      301 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
      361 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
      421 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gccaaagggc ctcgtgatac
      481 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
      541 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      601 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
      661 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
      721 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
      781 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
      841 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
      901 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
      961 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1021 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1081 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1141 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1201 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1261 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1321 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1381 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1441 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1501 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1561 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     1621 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1681 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1741 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1801 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     1861 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1921 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1981 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2041 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2101 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2161 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2221 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2281 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2341 tctttcctgc gttatcccct gattctgtgg ataaccgtaa gcggaagagc gcccaatacg
     2401 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2461 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     2521 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     2581 acaatttcac acaggaaaca gctatgacca tgattacgaa ttgcctgcag gcagctgcgc
     2641 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc
     2701 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
     2761 tcctatcgat atcaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag
     2821 cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga
     2881 gggcatgcgc aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct
     2941 gttcaagacc aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat
     3001 cgagtgcttc gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg
     3061 cacctaccac gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa
     3121 cgaggacatc ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat
     3181 cgaggagcgc ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa
     3241 gcgccgccgc tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga
     3301 caagcagagc ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa
     3361 cttcatgcag ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca
     3421 ggtgagcggc cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc
     3481 catcaagaag ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg
     3541 ccgccacaag cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa
     3601 gggccagaag aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg
     3661 cagccagatc ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta
     3721 cctgtactac ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg
     3781 cctgagcgac tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat
     3841 cgacaacaag gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag
     3901 cgaggaggtg gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat
     3961 cacccagcgc aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga
     4021 caaggccggc ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc
     4081 ccagatcctg gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga
     4141 ggtgaaggtg atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt
     4201 ctacaaggtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     4261 ggtgggcacc gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga
     4321 ctacaaggtg tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc
     4381 caccgccaag tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct
     4441 ggccaacggc gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat
     4501 cgtgtgggac aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt
     4561 gaacatcgtg aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc
     4621 caagcgcaac agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg
     4681 cggcttcgac agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg
     4741 caagagcaag aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag
     4801 cagcttcgag aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa
     4861 ggacctgatc atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg
     4921 catgctggcc agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta
     4981 cgtgaacttc ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa
     5041 cgagcagaag cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca
     5101 gatcagcgag ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag
     5161 cgcctacaac aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct
     5221 gttcaccctg accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga
     5281 ccgcaagcgc tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat
     5341 caccggtctg tacgagaccc gcatcgacct gagccagctg ggcggcgacg gcggctccgg
     5401 acctccaaag aaaaagagaa aagtagacta caaggacgac gatgacaagt aaggatccac
     5461 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     5521 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     5581 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     5641 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     5701 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     5761 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagatgg
     5821 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     5881 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttatc
     5941 gatagcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6001 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagag
     6061 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     6121 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     6181 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     6241 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     6301 gaaacaccgg
//
