LOCUS       sequence-73066-e       10666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting yellow gene and Cas9-Puro in Drosophila S2
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10666)
  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 10666)
  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-y1sgRNA-Cas9
FEATURES             Location/Qualifiers
     source          1..10666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(13..88)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(109..508)
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     promoter        509..2950
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5c promoter"
     CDS             2994..3059
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3066..3086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3111..7211
                     /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             3111..7211
                     /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             7212..7259
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7275..7328
                     /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             7353..7952
                     /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(7353..7372)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7849..7869
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7960..8094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7984..8003)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8038..8057
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(8716..9304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8796..8815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9475..10266,10267..10335))
                     /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     10098..10117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10336..10440)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10508..10526
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 agatctaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
       61 tttaacttgc tatttctagc tctaaaaccg gttccagtgt ccaaaaccga agtattgagg
      121 aaaacatacc tatataaatg atcaacatca ggaaagagca gttgagaatt ataagaattg
      181 gcaaatggtc ttaagaaccc tctgcttaag attttcaaaa tttcctttag aatcaaagtg
      241 tcctattgtt cgtttgttga aaacacagtt cgatttattg actataataa attgatagtt
      301 ttaaatatag aggcacgact aagagagttt ggttttgttt tggttaggga ccaaaaaaag
      361 tatacataac gaataaaaaa ggatttaagt gcaaatggta aaaaagtgtc gagtttttct
      421 tgagttgatt gtgctgtaat gagactctgc attcggcatt tgactcggct tttcctactc
      481 gtgccgtatt tcaggctgca agtcgaacag atctcgctgc ctgttatgtg gcccacaaac
      541 caagacacgt tttatggcca ttaaagctgg ctgatcgtcg ccaaacacca aatacataat
      601 gaatatgtac acattcgaga aagaagcgat caaagaagcg tcttcgggcg gagtaggaga
      661 atgcggagga gaaggagaac gagctgatct agtatctctc cacaatccaa tgccaactga
      721 ccaactggcc atattcggag caatttgaag ccaatttcca tcgcctggcg atcgctccat
      781 tcttggctat atgtttttca ccgttacccg gggccatttt caaagactcg tcggcaagat
      841 aagattgtgt cactcgctgt ctctcttcat ttgtcgaaga atgctgagga atttcgcgat
      901 gacgtcggcg agtattttga agaatgagaa taatttgtat ttatacgaaa atcagttagt
      961 ggaattttct acaaaaacat gttatctata gataattttg ttgcaaaata tgttgactat
     1021 gacaaagatt gtatgtatat acctttaatg tattctcatt ttcttatgta tttataatgg
     1081 caatgatgat actgatgata ttttaagatg atgccagacc acaggctgat ttctgcgtct
     1141 tttgccgaac gcagtgcatg tgcggttgtt gttttttgga atagtttcaa ttttcggact
     1201 gtccgctttg atttcagttt cttggcttat tcaaaaagca aagtaaagcc aaaaaagcga
     1261 gatggcaata ccaaatgcgg caaaacggta gtggaaggaa aggggtgcgg ggcagcggaa
     1321 ggaagggtgg ggcggggcgt ggcggggtct gtggctgggc gcgacgtcac cgacgttgga
     1381 gccactcctt tgaccatgtg tgcgtgtgtg tattattcgt gtctcgccac tcgccggttg
     1441 tttttttctt tttatctcgc tctctctagc gccatctcgt acgcatgctc aacgcaccgc
     1501 atgttgccgt gtcctttatg cgtcattttg gctcgaaata ggcaattatt taaacaaaga
     1561 ttagtcaacg aaaacgctaa aataaataag tctacaatat ggttacttat tgccatgtgt
     1621 gtgcagccaa cgatagcaac aaaagcaaca acacagtggc tttccctctt tcactttttg
     1681 tttgcaagcg cgtgcgagca agacggcacg accggcaaac gcaattacgc tgacaaagag
     1741 cagacgaagt tttggccgaa aaacatcaag gcgcctgata cgaatgcatt tgcaataaca
     1801 attgcgatat ttaatattgt ttatgaagct gtttgacttc aaaacacaca aaaaaaaaaa
     1861 taaaacaaat tatttgaaag agaattagga atcggacagc ttatcgttac gggctaacag
     1921 cacaccgaga cgaaatagct tacctgacgt cacagcctct ggaagaactg ccgccaagca
     1981 gagagagaga gaaaaagagg gagagcagct tagaccgcat gtgcttgtgt gtgaggcgtc
     2041 tctctcttcg tctcctgttt gcgcaaacgc atagactgca ctgagaaaat cgattaccta
     2101 ttttttatga atgaatattt gcactattac tattcaaaac tattaagata gcaatcacat
     2161 tcaatagcca aatactatac cacctgagcg atgcaacgaa atgatcaatt tgagcaaaaa
     2221 tgctgcatat ttaggacggc atcattatag aaatgcttct tgctgtgtac ttttctctcg
     2281 tctggcagct gtttcgccgt tattgttaaa accggcttaa gttaggtgtg ttttctacga
     2341 ctagtgatgc ccctactaga agatgtgtgt tgcacaaatg tccctgaata accaatttga
     2401 agtgcagata gcagtaaacg taagctaata tgaatattat ttaactgtaa tgttttaata
     2461 tcgctggaca ttactaataa acccactata aacacatgta catatgtatg ttttggcata
     2521 caatgagtag ttggggaaaa aatgtgtaaa agcaccgtga ccatcacagc ataaagataa
     2581 ccagctgaag tatcgaatat gagtaacccc caaattgaat cacatgccgc aactgatagg
     2641 acccatggaa gtacactctt catggcgata tacaagacac acacaagcac gaacacccag
     2701 ttgcggagga aattctccgt aaatgaaaac ccaatcggcg aacaattcat acccatatat
     2761 ggtaaaagtt ttgaacgcga cttgagagcg gagagcattg cggctgataa ggttttagcg
     2821 ctaagcgggc tttataaaac gggctgcggg accagttttc atatcactac cgtttgagtt
     2881 cttgtgctgt gtggatactc ctcccgacac aaagccgctc catcagccag cagtcgtcta
     2941 atccagagac cccggatcgg ggtacctact agtccagtgt ggtggaattc atggactata
     3001 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     3061 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     3121 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     3181 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     3241 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     3301 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     3361 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     3421 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     3481 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     3541 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     3601 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     3661 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     3721 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     3781 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     3841 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     3901 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     3961 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     4021 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     4081 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     4141 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     4201 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     4261 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     4321 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     4381 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     4441 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     4501 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     4561 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     4621 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     4681 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     4741 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     4801 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     4861 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     4921 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     4981 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     5041 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     5101 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     5161 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     5221 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     5281 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     5341 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     5401 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     5461 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     5521 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     5581 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     5641 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     5701 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     5761 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     5821 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     5881 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     5941 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     6001 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     6061 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     6121 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     6181 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     6241 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     6301 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     6361 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     6421 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     6481 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     6541 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     6601 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     6661 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     6721 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     6781 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     6841 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     6901 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     6961 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     7021 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     7081 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     7141 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     7201 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     7261 gatcaggcaa gcttgagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc
     7321 ccggccctgc tagcggtagc ggcagcggta gcatgaccga gtacaagccc acggtgcgcc
     7381 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     7441 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     7501 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     7561 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     7621 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     7681 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     7741 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     7801 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     7861 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     7921 cctggtgcat gacccgcaag cccggtgcct gactcgaggg atccagacat gataagatac
     7981 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     8041 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     8101 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     8161 aagtaaaacc tctacaaatg tggtatggct gattatgatc agtcgaccga tgcccttgag
     8221 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     8281 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat
     8341 tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt
     8401 cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg
     8461 cgagaagcag gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc
     8521 gttcgcgacg cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat
     8581 cgggatgccc gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca
     8641 gcttcaagga tcgctcgcgg ctcttaccag ccagcaaaag gccaggaacc gtaaaaaggc
     8701 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8761 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8821 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8881 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     8941 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     9001 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     9061 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     9121 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     9181 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     9241 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     9301 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     9361 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     9421 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     9481 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     9541 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     9601 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     9661 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     9721 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     9781 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     9841 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     9901 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     9961 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    10021 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    10081 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    10141 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    10201 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    10261 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    10321 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    10381 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    10441 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
    10501 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa ggaattaatt ctatattcta
    10561 aaaacacaaa tgatacttct aaaaaaaaat catgaatggc atcaactctg aatcaaatct
    10621 ttgcagatgc acctacttct catttccact gtcacatcat ttttcc
//
