LOCUS       sequence-362514-       11330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid contains hCas9 and two gRNAs that can excise the 
            genomic area around the mouse mdx mutation in dystrophin's exon 23.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11330)
  AUTHORS   Domenig SA, Bundschuh N, Lenardic A, Ghosh A, Kim I, Qabrati X, 
            D'Hulst G, Bar-Nur O
  TITLE     CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors 
            restores dystrophin expression in a mouse model of muscular 
            dystrophy.
  JOURNAL   Stem Cell Reports. 2022 Feb 8;17(2):321-336. doi: 
            10.1016/j.stemcr.2021.12.003. Epub 2022 Jan 6.
  PUBMED    34995499
REFERENCE   2  (bases 1 to 11330)
  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"; doi: 
            "10.1016/j.stemcr.2021.12.003"; journalName: "Stem Cell Reports"; 
            date: "2022-02-8- 8"; volume: "17"; issue: "2"; pages: "321-336"
COMMENT     Sequence Label: pRP[2CRISPR]-mCherry/Hygro-hCas9-U6>{mdx
            left}...
FEATURES             Location/Qualifiers
     source          1..11330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          117..344
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      356..365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             365..430
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             437..457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             482..4582
                     /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             482..4582
                     /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             4583..4630
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4658..4675)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4664..4871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        4886..5265
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5266..5469
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5420..5440
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5496..5505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5502..6209
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5647..5665)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             6210..7235
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6210..7235
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    7248..7369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7259..7278)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7313..7332
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   7469..7609
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7469..7598
                     /label=AAV2 ITR
     rep_origin      7684..8139
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7771..7790)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7981..8002
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8156..8175)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8275..8297
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8335..8353)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8421..8525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8526..8594,8595..9386)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8744..8763)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9557..10145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10046..10065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10207..10455
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        10477..10552
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        10567..10815
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        10837..10912
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        10932..11217
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
       61 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      121 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      181 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      241 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
      301 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
      361 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
      421 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      481 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
      541 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
      601 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
      661 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
      721 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
      781 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
      841 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
      901 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
      961 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1021 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1081 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     1141 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     1201 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     1261 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     1321 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     1381 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     1441 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     1501 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     1561 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     1621 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     1681 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     1741 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     1801 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     1861 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     1921 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     1981 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2041 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     2101 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     2161 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     2221 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     2281 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     2341 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     2401 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     2461 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     2521 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     2581 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     2641 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     2701 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     2761 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     2821 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     2881 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     2941 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3001 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     3061 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     3121 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     3181 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     3241 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     3301 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     3361 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     3421 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     3481 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     3541 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     3601 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     3661 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     3721 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     3781 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     3841 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     3901 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     3961 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4021 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4081 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     4141 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     4201 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     4261 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     4321 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     4381 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     4441 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     4501 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     4561 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     4621 aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     4681 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4741 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4801 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     4861 catgctgggg agcggccgcc gcgttgacat tgattattga ctagttatta atagtaatca
     4921 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     4981 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5041 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5101 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     5161 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     5221 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     5281 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     5341 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     5401 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     5461 agcagagctc tctggctaac tagagaaccc actgcgccac catggtgagc aagggcgagg
     5521 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     5581 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     5641 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     5701 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     5761 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     5821 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     5881 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aaaaccatgg
     5941 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     6001 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     6061 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     6121 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     6181 ccaccggcgg catggacgag ctgtacaaga tgaaaaagcc tgaactcacc gcgacgtctg
     6241 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     6301 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     6361 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     6421 cgctcccgat tccggaagtg cttgacattg gggaatttag cgagagcctg acctattgca
     6481 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     6541 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     6601 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     6661 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     6721 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     6781 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     6841 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     6901 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     6961 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     7021 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     7081 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     7141 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     7201 gacgccccag cactcgtccg agggcaaagg aataggagca gacatgataa gatacattga
     7261 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     7321 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     7381 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     7441 aaacctctac aaatgtggta gcggccgcag gaacccctag tgatggagtt ggccactccc
     7501 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     7561 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     7621 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     7681 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7741 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7801 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7861 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     7921 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7981 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     8041 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     8101 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     8161 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     8221 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     8281 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     8341 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     8401 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     8461 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     8521 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     8581 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     8641 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     8701 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     8761 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     8821 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     8881 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     8941 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     9001 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     9061 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     9121 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     9181 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     9241 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     9301 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     9361 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     9421 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     9481 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     9541 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     9601 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     9661 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     9721 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     9781 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     9841 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     9901 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     9961 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    10021 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    10081 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    10141 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    10201 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
    10261 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
    10321 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
    10381 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
    10441 aaaggacgaa acaccgtctt tgaaagagca ataaaagttt tagagctaga aatagcaagt
    10501 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgg
    10561 ccggccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
    10621 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
    10681 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
    10741 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
    10801 aaaggacgaa acaccgattt caggtaagcc gaggttgttt tagagctaga aatagcaagt
    10861 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
    10921 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
    10981 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
    11041 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
    11101 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
    11161 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
    11221 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
    11281 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
//
