LOCUS       sequence-356959-       12840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""neutrophil specific cas9 expression in zebrafish with green 
            lens"".
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12840)
  AUTHORS   Wang Y, Hsu AY, Walton EM, Park SJ, Syahirah R, Wang T, Zhou W, Ding
            C, Lemke AP, Zhang G, Tobin DM, Deng Q
  TITLE     A robust and flexible CRISPR/Cas9-based system for 
            neutrophil-specific gene inactivation in zebrafish.
  JOURNAL   J Cell Sci. 2021 Apr 15;134(8). pii: 237799. doi: 
            10.1242/jcs.258574. Epub 2021 Apr 22.
  PUBMED    33722979
REFERENCE   2  (bases 1 to 12840)
  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: "J Cell 
            Sci."; date: "2021-04-15"; volume: "134(8). pii: 237799. doi"; 
            pages: " 10.1242/jcs.258574. Epub 2021 Apr 22"
COMMENT     Sequence Label: Tol2-LyzC-Cas9-pA-Cry-GFP
FEATURES             Location/Qualifiers
     source          1..12840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     polyA_signal    1056..1177
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(1300..2013,2014..2016,2017..2019))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1303..2013,2014..2016,2017..2019))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1345..1366)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1694..1713
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      2016..2025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2667..2683
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2703..2723
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    5126..5150
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     5152..5168
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5178..5197
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5178..5196
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5232..5241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5250..5270
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5277..9377
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             9290..9298
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             9393..9413
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(9467..9485)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(9514..9538)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    9540..9674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(9737..9757)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(9765..9781)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    9797..9931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(10370..10557,10558..10569)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(10603..10619)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10603..10619)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(10656..10674)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10695..10711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10708..10730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10719..10735
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(10743..10749,10750..10767,10768..10773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10788..10809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10926..10943)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11097..11685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11177..11196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11856..12647,12648..12716))
                     /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     12479..12498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12717..12821)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 atgctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatt ggccttaaga tacattgatg agtttggaca
     1081 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     1141 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     1201 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     1261 atgtggtatg gctgattatg atctagagtc gcggccgctt tacttgtaca gctcgtccat
     1321 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     1381 gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac
     1441 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc
     1501 ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat
     1561 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
     1621 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
     1681 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
     1741 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
     1801 gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag
     1861 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
     1921 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
     1981 caccaccccg gtgaacagct cctcgccctt gctcaccatg gtggcgaccg gtaatgtcag
     2041 acctggtaac tccttactgt aacttattgc acctgatgaa caaaagatgt gaactgaacg
     2101 caagtaggag gctaatagag gctagtacag tacaaagatc cccaaatgat gtgtgccggc
     2161 cttagtggag gcagacccct ggttatatac ctgtgctgca gaaagagggg cttacaaatg
     2221 gatctctctg gaatggcatt atctcatggt gggaacagtg ttgtcagcag aatcagcggg
     2281 gtggactctg acctgagaat gacagtctag cactgaatgg ctcagacaat actcttgtgg
     2341 ccaactatca ccggaaaata agtgcctgcc aaaacaaacc agaccagatg aagctacatg
     2401 gctgtagaac gtcagtattt aatgtgttgt ggccacttgg ttcaccagat ttaagagcta
     2461 atttttttaa atatcacaga taatcaatcg cttttttttt tcttgactga aaagcttaga
     2521 ctggaaaatt ctttgaaaaa gtttgcacta cagaaaataa atacataaca tcctgcactg
     2581 aatgcacact attaatagaa aagagagcat tattgtagga aatgtattgt atatgtacat
     2641 tctgtgcatt tcacaatttt ctcgagcagg aaacagctat gaccatgatt acgccaagct
     2701 atcaactttg tatagaaaag ttggctccga attcgccctt aaaactcgag agtttgactt
     2761 gggttgatta tagcaaatag tggaacacaa acagattttg aaatattctt gcaaatttat
     2821 ttattaaatc cattcaaata ttgtcatttt ttgtactgtg taatttatat tgatttgctc
     2881 atacaatatt tacatttcgc ttaaacaata cactattatt tatatttaaa aatagctata
     2941 tataaatcaa atgtatagct cttttttaca caattaatca aagctaaaat atttgtcaca
     3001 caaatacagt aagccacttc acaatgatat cttcaaaatc tgtgcttgac tgaatcattt
     3061 catttattgt tttgtaattc acattatacc ttacgttctc caggcacagt ttgaattgtt
     3121 gataaatgaa gataatacat ctgatttaat tagtctcctt tgtttataat aactttaatc
     3181 taatttgata gtaataattc cagcaataca tccattacaa taaaaatcgc ttttttattt
     3241 tgcacattag tttgccggta tttgtttaaa gtttcaccat acttttaaat catgtgctat
     3301 ttgaaaaggg ctgttatgac tggaattaaa tgtttttttt tatatcagtg ttttagattg
     3361 aagtacaata tttataggta aaattcacca gcatatgctt aaagatagcc atgctccagg
     3421 tccttgtgtc cttctaaggt ctgtttgtgg tttaagctgt ttgttgacac acgcttgtct
     3481 tctccatttt tctgtgaagt tattgtcagt ttattgcaca ctgggttgca ttaagctgca
     3541 gttttttcac aacagttttc cacattgtaa cagtcagtct ccaaattatt ccatcagtcc
     3601 tcaaatttag tcatttgagg ccatgaaaac acttaaaact acatgaaggt agtttggcat
     3661 taggttttag acttgttctt gtttggcata ttgcaacctg tgtaaaaatg atttgatttt
     3721 tatttttttt tttttttttt cagaaagaaa cggtccaaaa agagttatgt gaacatttat
     3781 ccatgtgttt aatgtttttt aaattaaaaa aaaacatttt tattaaacca cttgtgacta
     3841 aacctttttt agttattcta ttttgtgacg agtctgaatg gccaaagtac tcttgaatcc
     3901 aaaatcttta tagtaaaatg tttaatctta cttgcagtgt tgggtataat ttgtaacaaa
     3961 gttgaattcc atatctgctg agtgaaaaag taaagtaaga gattgctgta aatatttagg
     4021 ttatttattt attaatacaa tatagaaagt aacaaaacac aatgtaaaaa atcaataaag
     4081 aaagtaaata ttaaaagtaa tgataaacta atgatgtaaa caatattagc aacaaagttt
     4141 gtacaattca gggagggtaa agaatttcac ctaatagtca aactctgtat taaataatat
     4201 aaaatctttg ttcttagtct gaagaggaga ctgtgagaga aagcttactg gttgttaaat
     4261 tagaaaattt gtaacttgca gattaaaatg atttaaataa ttttataaag gccatgtttg
     4321 aaacaaatgt aaaaacataa taaatgttaa gaatttcaga tattgagatt ttacaattta
     4381 aaaaaagcaa taaagtaata gcagagacct acactcttaa agtaaattac ccaactctgc
     4441 ttactagtaa cttttggaat agttactagt atccattgaa caaatactag aatctaatta
     4501 aattgtgtga gtgtccaatg aaaagattct tgtatttaaa acagtaccag tatctatata
     4561 tgtacaaaat ttagtgctag caaaatgcaa ataaatacta ctgcatataa tggattgcat
     4621 tgtaaatcga ctcgccataa tacatttatt tcctattttt acttttaaca aaaaataagt
     4681 tcactgagta tttcatgtca ttaaatcgca ttaaacctct tgcatgcttt atgtacacta
     4741 gtgcagttcg ctaaatgacc accggtgtca ctaataacac gatcgtttct gaattctgca
     4801 ttaaacgcca tttatacgtt gctcttatta gtaaacaatt tctccatcat gagctcgacc
     4861 agtattgcat atttcatcct cttaaatcca gagttgtttc ttttaatcac tttctcttca
     4921 tgataaaagc agtttttggt tttcagaagt ttacaacaaa cccagtcagt tattgcctca
     4981 gcaaacactg gagcaactgc acacttgtga aaagcttctt gcgaaacaca aacctttctc
     5041 tggacttata aataaaggca ttcagcagct ccaccataca cctccactgg tcttgataaa
     5101 gggatccttt aagggcgaat tcgacccaag tttgtacaaa aaagcaggct tgtaaaacga
     5161 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc
     5221 tcgaggtcga cgccaccatg gcttctccac ctaagaagaa gagaaaggtg ggaagcatgg
     5281 ataagaagta tagcatcggc ctggatattg gaactaactc cgtgggttgg gcagtgatta
     5341 cagacgacta caaggtccct agcaagaaat ttaaggtgct gggtaacacc gacaggcaca
     5401 gcatcaagaa aaatctgatt ggagccctgc tgttcggttc tggagagact gccgaagcaa
     5461 cacgcctgaa aagaacagca agaaggcgct ataccagaag gaagaataga atctgttacc
     5521 tgcaggagat tttctctaac gaaatggcta aggtggacga ttcattcttt cataggctgg
     5581 aggaaagttt cctggtcgag gaagataaga aacacgagcg ccatcctatc tttggaaaca
     5641 ttgtggacga ggtcgcctat cacgaaaaat acccaaccat ctatcatctg cgcaagaaac
     5701 tggctgactc tactgataaa gccgacctga gactgatcta tctggctctg gcccacatga
     5761 ttaagttcag gggtcatttt ctgatcgagg gcgatctgaa ccccgacaat tccgatgtgg
     5821 acaagctgtt catccagctg gtccagattt acaatcagct gtttgaggaa aaccctatta
     5881 atgcttccag agtggacgca aaagctatcc tgtcagccag gctgtccaag tcacgcagac
     5941 tggagaacct gattgcacag ctgcccggag aaaagaggaa cggtctgttt ggaaatctga
     6001 tcgctctgag tctgggcctg actcctaact tcaaaagcaa ttttgatctg gctgaggacg
     6061 ccaaactgca gctgtcaaag gacacatatg acgatgacct ggataacctg ctggcacaga
     6121 tcggagatca gtacgctgac ctgttcctgg ctgccaaaaa tctgtccgac gcaatcctgc
     6181 tgtcagatat tctgagagtg aacagcgaga ttacaaaagc acctctgagt gccagcatga
     6241 tcaagagata tgacgagcac catcaggatc tgaccctgct gaaggctctg gtcaggcagc
     6301 agctgccaga gaagtacaag gaaattttct ttgatcagtc caagaacggc tacgccggtt
     6361 atatcgacgg aggcgcatca caggaggaat tctacaagtt tatcaaacct attctggaga
     6421 agatggacgg aactgaggaa ctgctggtga aactgaatag agaggacctg ctgaggaagc
     6481 agcgcacatt tgataacggt tccatcccac accagattca tctgggagag ctgcacgcta
     6541 tcctgaggcg ccaggaagac ttctacccct ttctgaaaga taaccgcgag aagatcgaaa
     6601 aaattctgac cttcagaatc ccttactatg tgggtccact ggctcgcgga aacagcagat
     6661 ttgcctggat gactcgcaaa tccgaggaaa ccattactcc ttggaacttc gaggaagtgg
     6721 tcgataaggg cgcctctgca cagtccttca tcgagagaat gactaatttt gacaaaaacc
     6781 tgcccaatga gaaagtgctg cctaagcact ccctgctgta cgagtatttc actgtctata
     6841 acgaactgac aaaggtgaaa tacgtcaccg agggcatgag aaagccagcc ttcctgtcag
     6901 gagagcagaa gaaagcaatc gtggatctgc tgtttaaaac caacaggaaa gtgactgtca
     6961 agcagctgaa ggaggactac ttcaagaaaa ttgaatgctt cgattccgtg gagatcagcg
     7021 gagtcgaaga cagatttaac gcaagcctgg gcgcttacca cgatctgctg aagatcatta
     7081 aggataaaga cttcctggac aacgaggaaa atgaggatat cctggaagac attgtgctga
     7141 cactgaccct gtttgaggac agaggaatga tcgaggaaag actgaaaacc tatgctcatc
     7201 tgttcgatga caaggtgatg aaacagctga agagaaggcg ctacactggc tggggtagac
     7261 tgagcaggaa gctgatcaac ggcattaggg ataaacagtc aggaaagaca atcctggact
     7321 ttctgaaaag tgatggcttc gccaaccgca attttatgca gctgattcac gatgacagtc
     7381 tgaccttcaa agaggacatc cagaaggctc aggtgtctgg acagggccac tccctgcatg
     7441 agcagattgc aaacctggct ggaagcccag ccatcaagaa aggcattctg cagacagtga
     7501 aaatcgtcga tgagctggtg aaagtcatgg gccataagcc cgaaaacatc gtgattgaga
     7561 tggctcgcga aaatcagaca acccagaagg gtcagaagaa cagtagagag aggatgaaaa
     7621 gaatcgagga aggcattaag gagctgggta gccagatcct gaaagagcac ccagtggaaa
     7681 acacacagct gcagaatgag aagctgtatc tgtactatct gcagaatgga agagatatgt
     7741 acgtggacca ggagctggat attaacaggc tgtctgatta cgacgtggat catatcgtcc
     7801 cccagagttt catcaaagat gacagcattg acaacaaggt gctgaccagg tccgacaaaa
     7861 acagaggaaa atcagataat gtccctagtg aggaagtggt caagaaaatg aagaactact
     7921 ggagacagct gctgaatgcc aaactgatca ctcagaggaa gtttgataac ctgacaaaag
     7981 cagagcgcgg tggactgtca gaactggaca aagctggatt catcaagagg cagctggtgg
     8041 aaacacgcca gatcactaaa cacgtcgcac agattctgga tagtcgcatg aacacaaagt
     8101 acgatgagaa tgacaaactg atcagagaag tgaaggtcat taccctgaag agtaaactgg
     8161 tcagcgactt taggaaagat ttccagtttt ataaggtccg cgagattaac aattatcacc
     8221 atgcccatga cgcatacctg aacgccgtgg tcggtaccgc actgatcaag aaatacccaa
     8281 aactggagag cgaattcgtg tacggagact ataaggtgta cgatgtcaga aaaatgatcg
     8341 ccaagtccga gcaggaaatt ggaaaagcta ctgccaagta tttcttttac tcaaacatca
     8401 tgaatttctt taagacagag atcaccctgg ccaatggaga aatccgcaaa aggcccctga
     8461 ttgagacaaa cggagagaca ggcgaaatcg tgtgggacaa aggcagagat tttgcaaccg
     8521 tgaggaaggt cctgagcatg cctcaagtga atatcgtcaa gaaaactgag gtgcagacag
     8581 gcggtttctc aaaagaaagt attctgccaa aacgcaactc tgataagctg atcgctagaa
     8641 agaaagactg ggaccctaag aagtatggag gctttgactc tcccactgtg gcatactccg
     8701 tcctggtggt cgctaaggtg gagaagggca aaagcaagaa actgaaatct gtcaaggagc
     8761 tgctgggtat cacaattatg gagagaagct ctttcgagaa gaacccaatc gattttctgg
     8821 aggccaaagg ttataaggaa gtgaagaaag acctgatcat taaactgccc aagtacagtc
     8881 tgtttgagct ggaaaacggc aggaaacgca tgctggcaag cgctggagag ctgcagaaag
     8941 gcaatgaact ggccctgcct tctaagtacg tgaacttcct gtatctggca agccactacg
     9001 agaagctgaa aggatctcca gaggataacg aacagaaaca gctgtttgtg gagcagcaca
     9061 agcattatct ggacgagatc attgaacaga ttagcgagtt ctctaaaaga gtgatcctgg
     9121 ccgacgcaaa tctggataag gtcctgtctg cttacaacaa acacagagat aagcccatca
     9181 gggagcaggc cgaaaatatc attcatctgt tcactctgac aaacctgggc gcacctgcag
     9241 ctttcaagta cttcgacact acaatcgata gaaagaggta cacctccact aaggaggtgc
     9301 tggacgctac actgatccat cagagtatta ccggcctgta cgaaacaagg attgacctgt
     9361 ctcagctggg tggcgactca ccggtgagat ctcctaagaa gaagagaaag gtgtgaccgc
     9421 ggaatctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt
     9481 taattgcgcg cttggcgtaa tcatggtcat agcacccagc tttcttgtac aaagtggggg
     9541 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     9601 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     9661 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     9721 tgtgggaggt tttttccaac tttattatac atagttgata attcactggc cgtcgtttta
     9781 cggtaccatc gatgatgatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     9841 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     9901 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     9961 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
    10021 tatggctgat tatgatcctc tagatcagat ctgcgaagat acggccacgg gtgctcttga
    10081 tcctgtggct gattttggac tgtgctgctc gcagctgctg atgaatcaca tacttcctcc
    10141 attttcttcc actgattgac tgttataatt tccctaattt ccaggtcaag gtgctgtgca
    10201 ttgtggtaat agatgtgaca tgacgtcact tccaaaggac caatgaacat gtctgaccaa
    10261 tttcatataa tgtgaaaacg attttcatag gcagaataaa taacatttaa attaaactgg
    10321 gcatcagcgc aattcaattg gtttggtaat agcaagggaa aatagaatga agtgatctcc
    10381 aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt
    10441 ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag
    10501 taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt
    10561 acacctctgg ttcttgaccc cctaccttca gcaagcccag cagatccact agttctagag
    10621 cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc
    10681 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
    10741 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
    10801 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
    10861 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
    10921 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
    10981 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
    11041 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
    11101 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
    11161 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
    11221 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
    11281 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
    11341 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
    11401 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
    11461 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
    11521 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
    11581 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
    11641 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
    11701 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaaggcat tttggtcatg
    11761 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
    11821 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
    11881 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
    11941 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
    12001 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
    12061 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
    12121 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
    12181 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
    12241 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
    12301 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
    12361 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
    12421 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
    12481 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
    12541 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
    12601 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
    12661 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
    12721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
    12781 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
//
