LOCUS       sequence-455548-       15621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of b-actin driven Cas9-GFP and U6-driven 4 sgRNAs in 
            zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15621)
  AUTHORS   Siniscalco AM, Perera RP, Greenslade JE, Veeravenkatasubramanian H, 
            Masters A, Doll HM, Raj B
  TITLE     Barcoding Notch signaling in the developing brain.
  JOURNAL   Development. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102.
  PUBMED    39575683
REFERENCE   2  (bases 1 to 15621)
  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: 
            "Development. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102."
COMMENT     Sequence Label: p128Tol2-Olactb:Cas9-t2A-GFP, 4xU6:sgRNA
FEATURES             Location/Qualifiers
     source          1..15621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1175..1191)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1176..1192)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1217..1235)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1237..1257)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(1277..1293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(1666..1828,1829..1840)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     2189..2210
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2373..2477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2478..2546,2547..3338)
                     /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(2696..2715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3509..4097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3998..4017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4251..4268
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4385..4406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4421..4426,4427..4444,4445..4451)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4459..4475
                     /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)."
     primer_bind     4464..4486
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4483..4499
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4520..4538
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4575..4591
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4575..4591
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(4625..4636,4637..4824)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    5263..5397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     7570..7586
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7594..7614
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    7677..7811
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    7813..7837
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        7866..7884
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(join(7921..8634,8635..8637,8638..8640))
                     /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(7924..8634,8635..8637,8638..8640))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7966..7987)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     8315..8334
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8574..8595
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(8647..8700)
                     /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             complement(8716..8736)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8752..12852)
                     /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             8831..8839
                     /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             complement(12859..12879)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      12888..12897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(12933..12951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(12961..12977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(12979..13003)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        13003..13021
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 attcttgcat ttctgatcag cagacctaac ttttaaatgt ccatttttgt tttgttttat
       61 taacccgctg aaataaaagc ttctgcttgt ggggtgaaac agttctctct cgagagttgt
      121 atgtaaataa ttctttatgt aagagtttta aatctctcat aaaaaacact ctcatatgga
      181 atgaatcgtt cttgtcgatc aaaggccaac tcactgcatt tacttattta caggcctgaa
      241 atgaaatctg taacccattc aaggtgcgtc catctaaaac acatttttat ttctatttgt
      301 cagtattctg ggagggcggg ttctggtaaa gttaagagca cactggaagg actgactgct
      361 gaaactggaa aaacacagac aggggttggc tgggaaagga tgtaatcctg cctcacagga
      421 gtgtcccttc gtagtaggct ccttacgtga ttgcaggtga atgaatgtat gtgtcagagt
      481 tccagctcat ccagctgtgt ggtaggtgat ttgcaatcac ttcttatgaa gcattatggc
      541 aagttcatac taagatagtt ctacagactt gattcaaatg ggcagatgat gttaccttct
      601 aaatttgatc tgcttttgaa taaaaaaaat acttgacttt gtttgaaggc cagaagtggg
      661 cagtaccagg atgaaggaaa taaaaaaaaa caaagaaaag aaaaccagtc ctgctagttc
      721 tctgaccatc atggattgat aagtgacttc accactttac cacaacacat cgtacaatgg
      781 gacattctta caattctttc atgatctgat cttcttccag tttgtgattt ttttttattc
      841 atgtcacata cttttactgc cgagtagttt gttttcatta acactattgc cacaccaggg
      901 ttcacctgca agtgaagcga atccttctta tcagcagtat taaatatcgt gcatttgttt
      961 tgaacaattc tcagatgagg tcagagtaaa aaaatgagcc agatcagttc accaagaaag
     1021 cactagctga cacgtaaaac aaaccagtca gaccccattc acttcaatgt agaatgacca
     1081 cccatcaaca gaaaaaaaac tgtcttggta tgttttttct ttagctctgt ggttagatat
     1141 aatattcatg accactgtaa ttgaattccc atatcgatac cgtcgacctc gagggggggc
     1201 ccggtaccca attcgcccta tagtgagtcg tattaccaac ttttctatac aaagttgata
     1261 gcttggcgta atcatggtca tagctgtttc ctgctcgagg gcccatctgg cctgtgtttc
     1321 agacaccagg gagtctctgc tcacgtttcc tgctatttgc agcctctcta tcaagactaa
     1381 tacacctctt cccgcatcgg ctgcctgtga gaggcttttc agcactgcag gattgctttt
     1441 cagccccaaa agagctaggc ttgacactaa caattttgag aatcagcttc tactgaagtt
     1501 aaatctgagg ttttacaact ttgagtagcg tgtactggca ttagattgtc tgtcttatag
     1561 tttgataatt aaatacaaac agttctaaag caggataaaa ccttgtatgc atttcattta
     1621 atgttttttg agattaaaag cttaaacaag aatctctagt tttctttctt gcttttactt
     1681 ttacttcctt aatactcaag tacaatttta atggagtact tttttacttt tactcaagta
     1741 agattctagc cagatacttt tacttttaat tgagtaaaat tttccctaag tacttgtact
     1801 ttcacttgag taaaattttt gagtactttt tacacctctg tcaagaactc ctggacaaac
     1861 ctctgacctg tgtggaacag agtggatatg ggtgtctgaa cagatattca cgtcttttgc
     1921 agatcagagg gcatttctgg tgttgtgcaa tgacctgggt ccaacaggta ggcgtactcg
     1981 tatccaagag cattgttgga caggaggtag tctccattcc tataaagagg aaggaatggc
     2041 accatgtagt agccgtcatt gtggccaatt ggtgcattgg tacgtgggta gatggaccgg
     2101 ggaggctgat gagttcttag ccagcgtggg ccatcgccct gatagacggt ttttcgccct
     2161 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2221 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2281 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2341 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2401 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2461 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2521 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2581 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2641 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2701 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2761 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     2821 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     2881 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     2941 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3001 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3061 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3121 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3181 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3241 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3301 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3361 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3421 tttttgataa tctcatgacc aaaatgcctt aacgtgagtt ttcgttccac tgagcgtcag
     3481 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3541 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3601 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3661 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3721 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3781 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3841 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3901 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3961 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4021 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4081 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4141 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4201 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4261 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     4321 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     4381 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     4441 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     4501 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctccacc
     4561 gcggtggcgg ccgctctaga actagtggat ctgctgggct tgctgaaggt agggggtcaa
     4621 gaaccagagg tgtaaagtac ttgagtaatt ttacttgatt actgtactta agtattattt
     4681 ttggggattt ttactttact tgagtacaat taaaaatcaa tacttttact tttacttaat
     4741 tacatttttt tagaaaaaaa agtacttttt actccttaca attttattta cagtcaaaaa
     4801 gtacttattt tttggagatc acttcattct attttccctt gctattacca aaccaattga
     4861 attgcgctga tgcccagttt aatttaaatg ttatttattc tgcctatgaa aatcgttttc
     4921 acattatatg aaattggtca gacatgttca ttggtccttt ggaagtgacg tcatgtcaca
     4981 tctattacca caatgcacag caccttgacc tggaaattag ggaaattata acagtcaatc
     5041 agtggaagaa aatggaggaa gtatgtgatt catcagcagc tgcgagcagc acagtccaaa
     5101 atcagccaca ggatcaagag cacccgtggc cgtatcttcg cagatctgat ctagaggatc
     5161 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5221 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcatc
     5401 atcgatacat taaatgaaat gcatcagtaa gtctctcagc gtttctccag cctcggtcat
     5461 tcagtaagac gagtttcctg ctctcggcgt cttttgttct ggtcttcaag gaggggggaa
     5521 atgttctgcg catgcctgtg ggggagggag aaggacacgt cactgaaaac gtccctgcat
     5581 cacaccgaga cacccaatca ctcaagccaa gaccagataa ttttgcatat gctttacagt
     5641 ttgaaaatta ccacggtaat ccctcacaca aactctggat ttgagatcct tcaggtttta
     5701 tcagtttgca ggtttatgtc accatgatat agggtcagac ttgatctaag ggagctgaat
     5761 aagtggttta gtcactcacc acctcccaaa aacataccca gaagtccctg gtatatatag
     5821 ctctccctcc agctcttggt tcggactgga ggacttctgg gggtttaaga gctatgctgg
     5881 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     5941 gtcggtgctt ttttagtcaa gtctctcagc gtttctccag cctcggtcat tcagtaagac
     6001 gagtttcctg ctctcggcgt cttttgttct ggtcttcaag gaggggggaa atgttctgcg
     6061 catgcctgtg ggggagggag aaggacacgt cactgaaaac gtccctgcat cacaccgaga
     6121 cacccaatca ctcaagccaa gaccagataa ttttgcatat gctttacagt ttgaaaatta
     6181 ccacggtaat ccctcacaca aactctggat ttgagatcct tcaggtttta tcagtttgca
     6241 ggtttatgtc accatgatat agggtcagac ttgatctaag ggagctgaat aagtggttta
     6301 gtcactcacc acctcccaaa aacataccca gaagtccctg gtatatatag ctctccctcc
     6361 agctcttggt tcgccctgaa gctgaaggac gggtttaaga gctatgctgg aaacagcata
     6421 gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     6481 tttttaaact cattaccctc cacgtgtctg tctgggtttt catgggctct gctctagtga
     6541 gagcagcttc cttttgtcgg agtgttttgt gctcttatcc aaggcagggg ggatctgcgc
     6601 atgcctgtgg ggggaggaga aggacacgtg aacaaaagct cctcgatgtc acacaggaag
     6661 ttcaggaact catccaatca ctctaaagaa acggcctgtt tccttcgcat acgcttacag
     6721 ctccaaaact ctacggtaaa cctacataaa ctgctggttt tcaaatttta aagaatttaa
     6781 gggtttacag gtttactact acacagtgat ttactgacac atgtaagtgt aaatgagttg
     6841 aataagtaag taagccatat accacacatg aaacacatac ccagaagtca ctggtatata
     6901 tagccgtcct ccagactccc agttcggagt tcaagtccat ctacagttta agagctatgc
     6961 tggaaacagc atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac
     7021 cgagtcggtg ctttttttaa accaatccga gagtctgtga atgtttcgat gggccgtttg
     7081 ctggcgtctg agcatgttgt cctctggtct gcggcggggg aaggtccgcg catgcctgtg
     7141 ggggaaggcg aaggacacga ggacaaaaga tcagttacgt cacagaagaa ctcatccaat
     7201 cacacaagcc taaaaccagc ttcctctgca tatgctttac agttcaaaaa tactacggta
     7261 aacctctaca aaactgctgg tttcaaactt tgaaggtttt aagcgtttgc aggtttgccc
     7321 cgaagaggtt tactgtcatg tttgaggtaa atgagttgaa taagtaggtt tatccactta
     7381 ccacatggca gaaacatacc cagaagtccc gggtttatat agcagttctc caggctccta
     7441 gttcgctgcc cggctactac tacggtttaa gagctatgct ggaaacagca tagcaagttt
     7501 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttaaa
     7561 cctggtaccg taaaacgacg gccagtgaat tatcaactat gtataataaa gttggaaaaa
     7621 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     7681 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     7741 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     7801 atgtctggat cccccacttt gtacaagaaa gctgggtgct atgaccatga ttacgccaag
     7861 cgcgcaatta accctcacta aagggaacaa aagctggagc tccaccgcgg tggcggccgc
     7921 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag
     7981 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta
     8041 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc
     8101 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc
     8161 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt
     8221 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     8281 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     8341 gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     8401 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag
     8461 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
     8521 gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     8581 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcttcgccct tgctcaccat
     8641 tctagaaggg ccgggattct cttccacgtc accgcatgtc agcagactgc ctctgccctc
     8701 tccgccacca gacctcacct ttctcttctt cttaggggat cccaccggtg agtcgccacc
     8761 cagctgagac aggtcaatcc ttgtttcgta caggccggta atactctgat ggatcagtgt
     8821 agcgtccagc acctccttag tggaggtgta cctctttcta tcgattgtag tgtcgaagta
     8881 cttgaaagct gcaggtgcgc ccaggtttgt cagagtgaac agatgaatga tattttcggc
     8941 ctgctccctg atgggcttat ctctgtgttt gttgtaagca gacaggacct tatccagatt
     9001 tgcgtcggcc aggatcactc ttttagagaa ctcgctaatc tgttcaatga tctcgtccag
     9061 ataatgcttg tgctgctcca caaacagctg tttctgttcg ttatcctctg gagatccttt
     9121 cagcttctcg tagtggcttg ccagatacag gaagttcacg tacttagaag gcagggccag
     9181 ttcattgcct ttctgcagct ctccagcgct tgccagcatg cgtttcctgc cgttttccag
     9241 ctcaaacaga ctgtacttgg gcagtttaat gatcaggtct ttcttcactt ccttataacc
     9301 tttggcctcc agaaaatcga ttgggttctt ctcgaaagag cttctctcca taattgtgat
     9361 acccagcagc tccttgacag atttcagttt cttgcttttg cccttctcca ccttagcgac
     9421 caccaggacg gagtatgcca cagtgggaga gtcaaagcct ccatacttct tagggtccca
     9481 gtctttcttt ctagcgatca gcttatcaga gttgcgtttt ggcagaatac tttcttttga
     9541 gaaaccgcct gtctgcacct cagttttctt gacgatattc acttgaggca tgctcaggac
     9601 cttcctcacg gttgcaaaat ctctgccttt gtcccacacg atttcgcctg tctctccgtt
     9661 tgtctcaatc aggggccttt tgcggatttc tccattggcc agggtgatct ctgtcttaaa
     9721 gaaattcatg atgtttgagt aaaagaaata cttggcagta gcttttccaa tttcctgctc
     9781 ggacttggcg atcatttttc tgacatcgta caccttatag tctccgtaca cgaattcgct
     9841 ctccagtttt gggtatttct tgatcagtgc ggtaccgacc acggcgttca ggtatgcgtc
     9901 atgggcatgg tgataattgt taatctcgcg gaccttataa aactggaaat ctttcctaaa
     9961 gtcgctgacc agtttactct tcagggtaat gaccttcact tctctgatca gtttgtcatt
    10021 ctcatcgtac tttgtgttca tgcgactatc cagaatctgt gcgacgtgtt tagtgatctg
    10081 gcgtgtttcc accagctgcc tcttgatgaa tccagctttg tccagttctg acagtccacc
    10141 gcgctctgct tttgtcaggt tatcaaactt cctctgagtg atcagtttgg cattcagcag
    10201 ctgtctccag tagttcttca ttttcttgac cacttcctca ctagggacat tatctgattt
    10261 tcctctgttt ttgtcggacc tggtcagcac cttgttgtca atgctgtcat ctttgatgaa
    10321 actctggggg acgatatgat ccacgtcgta atcagacagc ctgttaatat ccagctcctg
    10381 gtccacgtac atatctcttc cattctgcag atagtacaga tacagcttct cattctgcag
    10441 ctgtgtgttt tccactgggt gctctttcag gatctggcta cccagctcct taatgccttc
    10501 ctcgattctt ttcatcctct ctctactgtt cttctgaccc ttctgggttg tctgattttc
    10561 gcgagccatc tcaatcacga tgttttcggg cttatggccc atgactttca ccagctcatc
    10621 gacgattttc actgtctgca gaatgccttt cttgatggct gggcttccag ccaggtttgc
    10681 aatctgctca tgcagggagt ggccctgtcc agacacctga gccttctgga tgtcctcttt
    10741 gaaggtcaga ctgtcatcgt gaatcagctg cataaaattg cggttggcga agccatcact
    10801 tttcagaaag tccaggattg tctttcctga ctgtttatcc ctaatgccgt tgatcagctt
    10861 cctgctcagt ctaccccagc cagtgtagcg ccttctcttc agctgtttca tcaccttgtc
    10921 atcgaacaga tgagcatagg ttttcagtct ttcctcgatc attcctctgt cctcaaacag
    10981 ggtcagtgtc agcacaatgt cttccaggat atcctcattt tcctcgttgt ccaggaagtc
    11041 tttatcctta atgatcttca gcagatcgtg gtaagcgccc aggcttgcgt taaatctgtc
    11101 ttcgactccg ctgatctcca cggaatcgaa gcattcaatt ttcttgaagt agtcctcctt
    11161 cagctgcttg acagtcactt tcctgttggt tttaaacagc agatccacga ttgctttctt
    11221 ctgctctcct gacaggaagg ctggctttct catgccctcg gtgacgtatt tcacctttgt
    11281 cagttcgtta tagacagtga aatactcgta cagcagggag tgcttaggca gcactttctc
    11341 attgggcagg tttttgtcaa aattagtcat tctctcgatg aaggactgtg cagaggcgcc
    11401 cttatcgacc acttcctcga agttccaagg agtaatggtt tcctcggatt tgcgagtcat
    11461 ccaggcaaat ctgctgtttc cgcgagccag tggacccaca tagtaaggga ttctgaaggt
    11521 cagaattttt tcgatcttct cgcggttatc tttcagaaag gggtagaagt cttcctggcg
    11581 cctcaggata gcgtgcagct ctcccagatg aatctggtgt gggatggaac cgttatcaaa
    11641 tgtgcgctgc ttcctcagca ggtcctctct attcagtttc accagcagtt cctcagttcc
    11701 gtccatcttc tccagaatag gtttgataaa cttgtagaat tcctcctgtg atgcgcctcc
    11761 gtcgatataa ccggcgtagc cgttcttgga ctgatcaaag aaaatttcct tgtacttctc
    11821 tggcagctgc tgcctgacca gagccttcag cagggtcaga tcctgatggt gctcgtcata
    11881 tctcttgatc atgctggcac tcagaggtgc ttttgtaatc tcgctgttca ctctcagaat
    11941 atctgacagc aggattgcgt cggacagatt tttggcagcc aggaacaggt cagcgtactg
    12001 atctccgatc tgtgccagca ggttatccag gtcatcgtca tatgtgtcct ttgacagctg
    12061 cagtttggcg tcctcagcca gatcaaaatt gcttttgaag ttaggagtca ggcccagact
    12121 cagagcgatc agatttccaa acagaccgtt cctcttttct ccgggcagct gtgcaatcag
    12181 gttctccagt ctgcgtgact tggacagcct ggctgacagg atagcttttg cgtccactct
    12241 ggaagcatta atagggtttt cctcaaacag ctgattgtaa atctggacca gctggatgaa
    12301 cagcttgtcc acatcggaat tgtcggggtt cagatcgccc tcgatcagaa aatgacccct
    12361 gaacttaatc atgtgggcca gagccagata gatcagtctc aggtcggctt tatcagtaga
    12421 gtcagccagt ttcttgcgca gatgatagat ggttgggtat ttttcgtgat aggcgacctc
    12481 gtccacaatg tttccaaaga taggatggcg ctcgtgtttc ttatcttcct cgaccaggaa
    12541 actttcctcc agcctatgaa agaatgaatc gtccacctta gccatttcgt tagagaaaat
    12601 ctcctgcagg taacagattc tattcttcct tctggtatag cgccttcttg ctgttctttt
    12661 caggcgtgtt gcttcggcag tctctccaga accgaacagc agggctccaa tcagattttt
    12721 cttgatgctg tgcctgtcgg tgttacccag caccttaaat ttcttgctag ggaccttgta
    12781 gtcgtctgta atcactgccc aacccacgga gttagttcca atatccaggc cgatgctata
    12841 cttcttatcc atgcttccca cctttctctt cttcttaggt ggagaagcca tggtggcgtc
    12901 gacctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
    12961 actggccgtc gttttacaag cctgcttttt tgtacaaact tgaattaacc ctcactaaag
    13021 ggaacaaaag ctggagctcc accgcggtgg cggccgctct agaaatgtcg acggctaaac
    13081 tggaaaagaa caaacaaaaa aggcgttaac accaaagaca cgcccatctg catctgcttt
    13141 gcagtcagat gtcccatttc ctgtcaggga gcggcagttc cccaaagtgt ccgcttggtg
    13201 gaggtagagc gctacattcc gagcccagac aaaaggaagt ggtgctgcat tctcttgtta
    13261 ccatataagg acatagttcg gttttgagca gctttgcggc taccggcaag gcacgcggcc
    13321 aaagtacagc aagaggggag ggactttaga ctatcggacc cggcgacgaa agcctactcc
    13381 cactggttca ttacagctct actttgtccc cgttccctcc cattgttaca caagactgaa
    13441 aaatttctta atcactgcgt cataatttgt gcgaagtcgt tgctggcata aagcaccacg
    13501 acccgtcaag ctttagtagg aaattaaaac gtgtgcgagc ccatgactga tcaatcattt
    13561 cctgaaagcc acgccttttc ttgaatgaat gatttgtcat ttgccctttt cttcgccgtc
    13621 aaactacagt tacatggtca atttaatcga atacatttaa aatgctttaa gggttattac
    13681 attttataca aacaaatgta taacagtccc acccccctac atgtgaggat tcagagtcaa
    13741 gtctagaagg gcgtgttagc atgaagctag gctaatatgc taaagtaggt cacaccccgt
    13801 aacggcgcac aaagggaccc gccaggatcc agacaccctc aactcggacg ggtaacgtcc
    13861 gtagaaggac tcgaaaaatt caccccacca tgtaagatac gaatagtaat tatttagttg
    13921 cctatataaa atatacaatt aaaaccaaac tgaaaagcca tttgccggtt acaatcaatt
    13981 ggcacctgtg cgtttaagtg gctaattttg gtacaacttg attgatgcca aaacaaacaa
    14041 aattgtcaat tttaaaaaga taattaaaag tttaaccatt tcaggcctaa aaataaattt
    14101 agtggccaca aaattctgct aatggtcacc tccaattaag aattaaactg cacaattaaa
    14161 caatcgtaag gtttccaaaa catttgaatt aaaccaaata tcgtctctat cttacctttg
    14221 ggtggggggc ttttcgtttc aagggaatcg gtttcaggcg aatgatatcc cgcacaagct
    14281 gtgtgtgacg gcgctcagag tgaatttgct agccgtgggc acgtggtatt tatagtccgt
    14341 ccgttgcccg gctctttcca taaaaggtaa actttcggaa tcgcggcatg tgattggtcc
    14401 agcgtcactc actggggcac gtcgggtgca cgctgtggtg cactgagcgt agaaagaggg
    14461 aggcgataac agtaacgggc taggggaaaa taaaaggata gggattgatg tgaggcctaa
    14521 tctaaggata taatgtttct tgaacaaata ttaatacata tagagtttac aaagtttcaa
    14581 ataaatataa aatatgaacc tgtataaagc tgtcgagcaa gtccccctgc ttatctgatc
    14641 attcaaacag cagagcacag gctgctcctc cctgtgaata cttttcctct taaaacctgt
    14701 ctctcaagat ttcagtctgg aaatgcaaga tatgtttgat gcaatttgag gacaattaca
    14761 acgtggatgt tgctgtacac tctggaaagc agaatgccac ctcagatgga gtcacagacc
    14821 ctccaagcca aacacatgac tcacaaaaga cactatggac tgtaaaagtg ccctgtcaga
    14881 ggatggacaa taaccacttt tcccactaag caatggtaaa aactcaatct cactaatacc
    14941 aattgaccat gtatggcatt agtctggatt cttgttaact tctcagagct cttgctactt
    15001 cctctttccc ctacaaatga agaaaaaagg tccacataag aggacattaa ggctgacttg
    15061 catcttcttt tagtcataaa tgacctattg agtgaaaata accactgtca gacccaaccc
    15121 atacgctact atacattcac cttaatcgat taaactcaga gagagacaaa taaagtcaca
    15181 catgctctta tttcctctct gtggtttact gcaaagggtg taactttcct gactttgggt
    15241 tttataggct tagggttctt tgcttttacc ttacttgtct tccattcatt agtttcacag
    15301 tttatttaaa ctttttttgc tccagaaaca tagctgccca tacaagttaa ataacccaga
    15361 ggccatttaa acatgtcact gtctgcgaga cacaagcagc aactactttt cttcctcttg
    15421 taccacttcc tcttcctcgc tctcagttac accaattctt ttatgctaaa tgccatctcc
    15481 tcttccttaa attcattcgt ggctttaagt tttaaatggt ctttattttc atcaacacat
    15541 ttccacttta tacatggaaa cgtttcagct ttttaatcaa gtttcaaaca aaacatgctt
    15601 catttccttc tgaattagca g
//
