LOCUS       sequence-391654-        9457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct encoding SpCas9-D10A nickase and an 
            AAVS1-targeting guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9457)
  AUTHORS   Wang Q, Liu J, Janssen JM, Le Bouteiller M, Frock RL, Goncalves MAFV
  TITLE     Precise and broad scope genome editing based on high-specificity 
            Cas9 nickases.
  JOURNAL   Nucleic Acids Res. 2021 Jan 25;49(2):1173-1198. doi: 
            10.1093/nar/gkaa1236.
  PUBMED    33398349
REFERENCE   2  (bases 1 to 9457)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2021-01-25- 25"; volume: "49"; issue: 
            "2"; pages: "1173-1198"
COMMENT     Sequence Label: AV44_pCAG.Cas9-D10A.gRNA.S1
FEATURES             Location/Qualifiers
     source          1..9457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        212..460
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     212..232
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     383..402
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        481..556
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        758..1137
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1139..1414
                     /label=chicken beta-actin promoter
     intron          1415..2431
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2355..2377
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2439..2458
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2529..2538
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2535..2561,2562..2564,2565..6638)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2535..6638
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6651..6671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6726..6745)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6770..6818
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        complement(7264..7282)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7337..7355)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7423..7527
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7528..7596,7597..8388)
                     /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     complement(7746..7765)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8559..9147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9048..9067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9301..9318
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     9385..9401
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9408..9427
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9408..9426
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atgttattcc tccttattta aaagcttaag gtgcacggcc cacgtggcca ctagtacttc
       61 tcgagctctg tacatgtccg cggtcgcgac gtacgcgtat cgatggcgcc agctgcaggc
      121 ggccgccata tgcatcctag gcctattaat attccggagt atacgtagcc ggctaacgtt
      181 gactggatcc ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata
      241 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      301 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      361 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      421 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggggccacta gggacaggat
      481 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      541 ggcaccgagt cggtgctttt tttctagacc cagctttctt gtacaacaac cggtacctct
      601 agaactatag ctagcatgcg caaatttaaa gcgctgatat cgatcgcgcg cagatctgtc
      661 atgatgatca ttgcaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      721 gcgatgtacg ggccagatat actcgagcca tggattcgac attgattatt gactagttat
      781 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      841 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      901 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      961 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     1021 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1081 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
     1141 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     1201 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
     1261 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
     1321 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     1381 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
     1441 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     1501 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
     1561 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     1621 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     1681 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     1741 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     1801 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1861 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     1921 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     1981 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     2041 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     2101 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     2161 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     2221 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     2281 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     2341 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     2401 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     2461 gtctcatcat tttggcaaag aattatcgca tgcctgcaga gctctagagt ctagaggatc
     2521 gaacccttgc caccatggac aagaagtact ccattgggct cgctatcggc acaaacagcg
     2581 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
     2641 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
     2701 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
     2761 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
     2821 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
     2881 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
     2941 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
     3001 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
     3061 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
     3121 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     3181 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     3241 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     3301 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     3361 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     3421 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     3481 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
     3541 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     3601 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     3661 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     3721 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
     3781 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     3841 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     3901 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     3961 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     4021 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     4081 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     4141 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
     4201 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
     4261 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     4321 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     4381 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     4441 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
     4501 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     4561 gaaagacaat cctggatttt cttaagtccg atggatttgc caacaggaac ttcatgcagt
     4621 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     4681 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     4741 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     4801 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
     4861 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     4921 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     4981 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     5041 acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     5101 tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg
     5161 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     5221 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
     5281 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     5341 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     5401 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
     5461 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     5521 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     5581 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     5641 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     5701 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     5761 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     5821 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
     5881 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     5941 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     6001 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     6061 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     6121 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     6181 gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     6241 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
     6301 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     6361 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     6421 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     6481 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
     6541 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     6601 atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga
     6661 agaggaaggt gtgaaagggt tcgatcccta ccggttagta atgagtttaa acgggggagg
     6721 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     6781 agacagaata aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     6841 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     6901 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     6961 cggggcggca ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca
     7021 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     7081 tacacttgcc agcgccctag cgcccggggt tataattacc tcaggtcgac gtcccatgtg
     7141 caggtgctga attcgaaggc attccacgac gtggtcacca gccatggcca tgtccaactc
     7201 catcaaatca gcttgagtag ccatgcccat ggctccagcc tgtcctcgta ccagcttttg
     7261 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttccaat
     7321 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     7381 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     7441 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     7501 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     7561 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     7621 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     7681 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     7741 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     7801 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     7861 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     7921 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     7981 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     8041 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     8101 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     8161 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     8221 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     8281 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     8341 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     8401 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     8461 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     8521 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     8581 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     8641 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     8701 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     8761 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     8821 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     8881 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     8941 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     9001 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     9061 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     9121 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     9181 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     9241 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     9301 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     9361 ctgtgcggta tttcacaccg catagtaaaa cgacggccag tgaattgtaa tacgactcac
     9421 tatagggcga attggagctt ggcgtaatca tggtcat
//
