LOCUS       sequence-389919-       10634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA and Cas9-Puro in Drosophila S2 cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10634)
  AUTHORS   Bassett AR, Tibbit C, Ponting CP, Liu JL
  TITLE     Mutagenesis and homologous recombination in Drosophila cell lines 
            using CRISPR/Cas9.
  JOURNAL   Biol Open. 2013 Dec 10. pii: bio.20137120v1. doi: 
            10.1242/bio.20137120.
  PUBMED    24326186
REFERENCE   2  (bases 1 to 10634)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biol Open. 
            2013 Dec 10. pii: bio.20137120v1. doi: 10.1242/bio.20137120."
COMMENT     Sequence Label: pAc-sgRNA-Cas9
FEATURES             Location/Qualifiers
     source          1..10634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(22..97)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(116..515)
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     CDS             2993..3058
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3065..3085
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3110..7210
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3110..7210
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7211..7258
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7274..7327
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             7352..7951
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7352..7371)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7848..7868
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7959..8093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7983..8002)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8037..8056
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(8694..9282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8774..8793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9453..10244,10245..10313))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     10076..10095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10314..10418)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10486..10504
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 catttttcca gatctaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac
       61 tagccttatt ttaacttgct atttctagct ctaaaacgga agagctcgct cttccgaagt
      121 attgaggaaa acatacctat ataaatgatc aacatcagga aagagcagtt gagaattata
      181 agaattggca aatggtctta agaaccctct gcttaagatt ttcaaaattt cctttagaat
      241 caaagtgtcc tattgttcgt ttgttgaaaa cacagttcga tttattgact ataataaatt
      301 gatagtttta aatatagagg cacgactaag agagtttggt tttgttttgg ttagggacca
      361 aaaaaagtat acataacgaa taaaaaagga tttaagtgca aatggtaaaa aagtgtcgag
      421 tttttcttga gttgattgtg ctgtaatgag actctgcatt cggcatttga ctcggctttt
      481 cctactcgtg ccgtatttca ggctgcaagt cgaacagatc tcgctgcctg ttatgtggcc
      541 cacaaaccaa gacacgtttt atggccatta aagctggctg atcgtcgcca aacaccaaat
      601 acataatgaa tatgtacaca ttcgagaaag aagcgatcaa agaagcgtct tcgggcggag
      661 taggagaatg cggaggagaa ggagaacgag ctgatctagt atctctccac aatccaatgc
      721 caactgacca actggccata ttcggagcaa tttgaagcca atttccatcg cctggcgatc
      781 gctccattct tggctatatg tttttcaccg ttacccgggg ccattttcaa agactcgtcg
      841 gcaagataag attgtgtcac tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt
      901 tcgcgatgac gtcggcgagt attttgaaga atgagaataa tttgtattta tacgaaaatc
      961 agttagtgga attttctaca aaaacatgtt atctatagat aattttgttg caaaatatgt
     1021 tgactatgac aaagattgta tgtatatacc tttaatgtat tctcattttc ttatgtattt
     1081 ataatggcaa tgatgatact gatgatattt taagatgatg ccagaccaaa aggcttgaat
     1141 ttctgcgtct tttgccgaac gcagtgcatg tgcaattgtt gttttttgga atattcaatt
     1201 ttcggactgt ccgctttgat ttcagtttct tggcttattc aaaaagcaaa gtaaagccaa
     1261 aaaagcgaga tggcaatacc aaatgcggca aaacggtagt ggaaggaaag gggtgcgggg
     1321 cagcggaagg aagggtgggg cggggcgtgg cggggtctgt ggctgggcgc gacgtcaccg
     1381 acgttggagc cactcctttg accatgtgtg cgtgtgtgta ttattcgtgt ctcgccactc
     1441 gccggttgtt tttttctttt tatgctgcgc tctctctagc gccatctcgc ttacgcatgc
     1501 tcaacgcacc gcatgttgcc gtttcctttt atgcgtcatt ttggctcgaa ataggcaatt
     1561 atttaaacaa agattagtca acgaaaacgc taaaataaat aagtctacaa tatggttact
     1621 tattgccatg tgtgtgcagc caacgatagc aacaaaagca acaacacagg tggctttccc
     1681 tctttcactt tttgtttgca agccgcgtgc gagcaagacg gcacgaccgg caaacgcaat
     1741 tacgctgaca aagagcagac gaagttttgg cgaaaaacat caaggcgcct gatacgaatg
     1801 catttgcaat aacaattgcg atatttaata ttgtttatga agctgtttga cttcaaaaca
     1861 cacaaaaaaa aaaataaaac aaattatttg aaagagaatt aggaatcgga cgcttatcgt
     1921 tagggtaaca acaagaaatg cttactgagt cacagcctct ggaaaactgc cgcaagccag
     1981 agagagagag aaaaagaggg agagcagctt agaccgcatg tgcttgtgtg tgaggcgtct
     2041 ctctcttcgt ctctgttgcg caaacgcata gactgcactg aaaaaatcga ttacctattt
     2101 tttatgaatg aatatttgca ctattactat tcaaaactat taagatagca atcacattca
     2161 atagccaaat actataccac ctgagcgatg caacgaaatg atcaatttga gcaaaaatgc
     2221 tgcatattta ggacggcatc attatagaaa tgcttcttgc tgtgtacttt tctctcgtct
     2281 ggcagctgtt tcgccgttat tgttaaaacc ggcttaagtt aggtgtgttt tctacgacta
     2341 gtgaatgccc tactagaaga tgtgtgttgc acaaaatgtc cctggaataa ccaatttgaa
     2401 gtgcagatag cagtaaacgt aagctaatat gaatattatt taactgtaat gttttaatat
     2461 cgctggacat tactaataaa cccactataa acacatgtac atatgtatgt tttggcatac
     2521 aatgagtagt tggggaaaaa atgtgtaaaa gcaccgtgac catcacagca taaagataac
     2581 cagctgaagt atcgaatatg agtaaccccc aaattgaatc acatgccgca actgatagga
     2641 cccatggaag tacactcttc atggcgatat acaagacaca cacaagcacg aacacccagt
     2701 tgcggaggaa attctccgta aatgaaaacc caatcggcga acaattcata cccatatatg
     2761 gtaaaagttt tgaacgcgac ttgagagcgg agagcattgc ggctgataag gttttagcgc
     2821 taagcgggct ttataaaacg ggctgcggga ccagttttca tatcactacc gtttgagttc
     2881 ttgtgctgtg tggatactcc tcccgacaca aagccgctcc atcagccagc agtcgtctaa
     2941 tccagagacc ccggatcggg gtacctacta gtccagtgtg gtggaattca tggactataa
     3001 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     3061 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     3121 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     3181 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     3241 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     3301 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     3361 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     3421 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     3481 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     3541 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     3601 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     3661 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     3721 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     3781 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     3841 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     3901 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     3961 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     4021 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     4081 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     4141 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     4201 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     4261 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     4321 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     4381 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     4441 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     4501 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     4561 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     4621 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     4681 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     4741 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     4801 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     4861 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     4921 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     4981 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     5041 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     5101 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     5161 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     5221 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     5281 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     5341 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     5401 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     5461 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     5521 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     5581 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     5641 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     5701 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     5761 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     5821 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     5881 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     5941 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     6001 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     6061 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     6121 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     6181 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     6241 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     6301 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     6361 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     6421 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     6481 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     6541 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     6601 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     6661 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     6721 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     6781 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     6841 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     6901 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     6961 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     7021 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     7081 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     7141 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     7201 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg
     7261 atcaggcaag cttgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     7321 cggccctgct agcggtagcg gcagcggtag catgaccgag tacaagccca cggtgcgcct
     7381 cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta
     7441 ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca
     7501 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     7561 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     7621 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     7681 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     7741 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     7801 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     7861 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     7921 ctggtgcatg acccgcaagc ccggtgcctg actcgaggga tccagacatg ataagataca
     7981 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     8041 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     8101 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     8161 agtaaaacct ctacaaatgt ggtatggctg attatgatca gtcgaccgat gcccttgaga
     8221 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     8281 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     8341 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     8401 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     8461 gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt cttgctggcg
     8521 ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc cggcggcatc
     8581 gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca tcagggacag
     8641 cttcaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     8701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     8761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     8821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     8881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     8941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     9001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     9061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     9121 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     9181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     9241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     9301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     9361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     9421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     9481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     9541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     9601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     9661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     9721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     9781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     9841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     9901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     9961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
    10021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
    10081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
    10141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
    10201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
    10261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
    10321 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
    10381 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
    10441 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
    10501 aggccctttc gtcttcaaga attaattcta tattctaaaa acacaaatga tacttctaaa
    10561 aaaaaatcat gaatggcatc aactctgaat caaatctttg cagatgcacc tacttctcat
    10621 ttccactgtc acat
//
