LOCUS       sequence-308951-       12658 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Cas9 at high level.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12658)
  AUTHORS   Port F, Strein C, Stricker M, Rauscher B, Heigwer F, Zhou J, 
            Beyersdorffer C, Frei J, Hess A, Kern K, Lange L, Langner N, Malamud
            R, Pavlovic B, Radecke K, Schmitt L, Voos L, Valentini E, Boutros M
  TITLE     A large-scale resource for tissue-specific CRISPR mutagenesis in 
            Drosophila.
  JOURNAL   Elife. 2020 Feb 13;9. pii: 53865. doi: 10.7554/eLife.53865.
  PUBMED    32053108
REFERENCE   2  (bases 1 to 12658)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2020-02-13"; volume: "9. pii: 53865. doi"; pages: " 
            10.7554/eLife.53865"
COMMENT     Sequence Label: UAS-u(S)Cas9
FEATURES             Location/Qualifiers
     source          1..12658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1145..1216,1618..1891,1966..2620,2682..2997,3201..3332,
                     3403..4017)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4792..4825
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4858..4952
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     promoter        4971..5209
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     5156..5177
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             5309..9412
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5309..9412
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5309..9412
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9425..9445
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          9532..9597
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             9727..9747
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    10019..10153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10056..10075)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10110..10129
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    10182..10251
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(10470..10488)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10509..10525)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10509..10525)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10522..10544)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10533..10549
                     /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(10557..10563,10564..10581,10582..10587))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10602..10623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10740..10757)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10911..11499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10991..11010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11670..12461,12462..12530))
                     /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     12293..12312
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12531..12635)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt ttaaatttac
      781 ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact caaaatgaca
      841 ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat cgccgtctct
      901 ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta gcgtcaatgt
      961 ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg tttacgccat
     1021 caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg tttgtttttt
     1081 gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt gaaaaatccc
     1141 ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac acccatctgc
     1201 cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac ttgatctata
     1261 ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc acaaaaatcc
     1321 caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt agctgttctc
     1381 gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg cattcatttt
     1441 cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg atcctagatg
     1501 cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt actttttgtt
     1561 ctgggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc attgcagggt
     1621 gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc caaaaactac
     1681 ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg ccaactagcc
     1741 gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa tcagccgggc
     1801 tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga gcgacacata
     1861 ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt cggctgatct
     1921 gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc ggcgtggcct
     1981 atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg accctgctga
     2041 atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg atgcgactgc
     2101 tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat gtccagcagg
     2161 atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccaa gccatggtgc
     2221 ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag gtgatccagg
     2281 agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg gtgaaaggtc
     2341 tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggctctaacc gatccgccgc
     2401 ttctgatctg cgatgagccc acctccggac tggactcctt taccgcccac agcgtcgtcc
     2461 aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt catcagccgt
     2521 cttccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc agggtagctt
     2581 tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga tgtgtttatt
     2641 aagggtatct agtattacat aacatctcaa ctcctatcca gcgtgggtgc ccagtgtcct
     2701 accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc cggacgggag
     2761 atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgccattag caaagtagcc
     2821 cgggatatgg agcagttgtt ggccaccaaa aatctggaga agccactgga gcagccggag
     2881 aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct gtggcgatcc
     2941 tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca gacaacggtg
     3001 agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca aattcgctag
     3061 attttagata gaattgcctg attccacacc cttcttagtt tttttcaatg agatgtatag
     3121 tttatagttt tgcagaagat aaataaattt catttaactc gcgaatatta atgagatgcg
     3181 agtaacattt taatttgcag atggttgcca tcttgattgg cctcatcttt ttgggccaac
     3241 aactcacgca agtgggtgtg atgaatatca acggagccat cttcctcttc ctgaccaaca
     3301 tgacctttca aaacgtcttt gccacgataa atgtaagtca tgtttagaat acatttgcat
     3361 ttcaataatt tactaacttt ctaatgaatc gattcgattt aggtgttcac ctcagagctg
     3421 ccagttttta tgagggaggc ccgaagtcga ctttatcgct gtgacacata ctttctgggc
     3481 aaaacgattg ccgaattgcc gctttttctc acagtgccac tggtcttcac ggcgattgcc
     3541 tatccgatga tcggactgcg ggccggagtg ctgcacttct tcaactgcct ggcgctggtc
     3601 actctggtgg ccaatgtgtc aacgtccttc ggatatctaa tatcctgcgc cagctcctcg
     3661 acctcgatgg cgctgtctgt gggtccgccg gttatcatac cattcctgct ctttggcggc
     3721 ttcttcttga actcgggctc ggtgccagta tacctcaaat ggttgtcgta cctctcatgg
     3781 ttccgttacg ccaacgaggg tctgctgatt aaccaatggg cggacgtgga gccgggcgaa
     3841 attagctgca catcgtcgaa caccacgtgc cccagttcgg gcaaggtcat cctggagacg
     3901 cttaacttct ccgccgccga tctgccgctg gactacgtgg gtctggccat tctcatcgtg
     3961 agcttccggg tgctcgcata tctggctcta agacttcggg cccgacgcaa ggagtagccg
     4021 acatatatcc gaaataactg cttgtttttt ttttttacca ttattaccat cgtgtttact
     4081 gtttattgcc ccctcaaaaa gctaatgtaa ttatatttgt gccaataaaa acaagatatg
     4141 acctatagaa tacaagtatt tccccttcga acatccccac aagtagactt tggatttgtc
     4201 ttctaaccaa aagacttaca cacctgcata ccttacatca aaaactcgtt tatcgctaca
     4261 taaaacaccg ggatatattt tttatataca tacttttcaa atcgcgcgcc ctcttcataa
     4321 ttcacctcca ccacaccacg tttcgtagtt gctctttcgc tgtctcccac ccgctctccg
     4381 caacacattc accttttgtt cgacgacctt ggagcgactg tcgttagttc cgcgcgattc
     4441 ggttcgctca aatggttccg agtggttcat ttcgtctcaa tagaaattag taataaatat
     4501 ttgtatgtac aatttatttg ctccaatata tttgtatata tttccctcac agctatattt
     4561 attctaattt aatattatga ctttttaagg taattttttg tgacctgttc ggagtgatta
     4621 gcgttacaat ttgaactgaa agtgacatcc agtgtttgtt ccttgtgtag atgcatctca
     4681 aaaaaatggt gggcataata gtgttgttta tatatatcaa aaataacaac tataataata
     4741 agaatacatt taatttagaa aatgcttgga tttcactgga actaggctag cataacttcg
     4801 tataatgtat gctatacgaa gttatgctag cggatccaag cttgcatgcc tgcaggtcgg
     4861 agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt
     4921 actgtcctcc gagcggagta ctgtcctccg agcggagact ctagcgagcg ccggagtata
     4981 aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc
     5041 gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca atctgcagta
     5101 aagtgcaagt taaagtgaat caattaaaag taaccagcaa ccaagtaaat caactgcaac
     5161 tactgaaatc tgccaagaag taattattga atacaagaag agaactctga atagggaatt
     5221 gggaattcca acatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     5281 gtcgagctgg acggctctag ataataacat ggacaagaag tactccattg ggctcgatat
     5341 cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa
     5401 attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct
     5461 cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag
     5521 atatacccgc agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc
     5581 taaggtggat gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa
     5641 aaagcacgag cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa
     5701 gtacccaacc atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt
     5761 gcggttgatc tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga
     5821 gggggacctg aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac
     5881 ttacaatcag cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat
     5941 cctgagcgct aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg
     6001 ggagaagaag aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa
     6061 ctttaaatct aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta
     6121 cgatgatgat ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt
     6181 ggcggcaaag aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga
     6241 gatcaccaaa gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga
     6301 cttgactttg ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt
     6361 cttcgatcag tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga
     6421 attttacaaa tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt
     6481 aaagcttaac agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc
     6541 ccaccagatt cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc
     6601 ctttttgaaa gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta
     6661 tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga
     6721 gaccatcact ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt
     6781 catcgaaagg atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca
     6841 ctctctgctg tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac
     6901 agaagggatg agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct
     6961 cctcttcaag acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa
     7021 gattgaatgt ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct
     7081 gggaacgtat cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga
     7141 gaacgaggac attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat
     7201 gattgaagaa cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct
     7261 caagaggcgc cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg
     7321 agacaagcag agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg
     7381 gaacttcatg cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc
     7441 acaagtttct ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc
     7501 agctatcaaa aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat
     7561 gggaaggcat aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca
     7621 gaagggacag aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact
     7681 ggggtcccaa atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct
     7741 ctacctgtac tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa
     7801 tcggctctcc gactacgacg tggatcatat cgtgccccag tcttttctca aagatgattc
     7861 tattgataat aaagtgttga caagatccga taaaaataga gggaagagtg ataacgtccc
     7921 ctcagaagaa gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact
     7981 gatcacacaa cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt
     8041 ggataaagcc ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt
     8101 ggcccaaatt ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg
     8161 agaggtgaaa gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca
     8221 gttttataag gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc
     8281 agtggtaggc actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg
     8341 agactataaa gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa
     8401 ggccaccgct aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac
     8461 actggccaat ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga
     8521 aatcgtgtgg gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca
     8581 ggtgaacatc gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct
     8641 cccgaaaagg aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata
     8701 cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa
     8761 agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg
     8821 atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa
     8881 aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa
     8941 acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa
     9001 atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga
     9061 taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga
     9121 gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct
     9181 ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa acattatcca
     9241 cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat
     9301 agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc
     9361 aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc
     9421 tgaccccaag aagaagagga aggtgtgagc tagcggatct ttgtgaagga accttacttc
     9481 tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa ggtaaatata
     9541 aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt
     9601 ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg
     9661 ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct
     9721 actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta
     9781 agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc
     9841 acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattt gatgtatagt
     9901 gccttgacta gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     9961 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
    10021 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
    10081 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
    10141 tcatgtctgg atccactagt gtcgacgatg taggtcacgg tctcgaagcc gcggtgcggg
    10201 tgccagggcg tgcccttggg ctccccgggc gcgtactcca cctcacccat ctggtccatc
    10261 atgatgaacg ggtcgaggtg gcggtagttg atcccggcga acgcgcggcg caccgggaag
    10321 ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga gcacgggacg tgcgacggcg
    10381 tcggcgggtg cggatacgcg gggcagcgtc agcgggttct cgacggtcac ggcgggcatg
    10441 tcgacactag ttctagccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg
    10501 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
    10561 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
    10621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
    10681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
    10741 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
    10801 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
    10861 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
    10921 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
    10981 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
    11041 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
    11101 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
    11161 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
    11221 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
    11281 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
    11341 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
    11401 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
    11461 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
    11521 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
    11581 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
    11641 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
    11701 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
    11761 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
    11821 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
    11881 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
    11941 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
    12001 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
    12061 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
    12121 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
    12181 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
    12241 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
    12301 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
    12361 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
    12421 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
    12481 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
    12541 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
    12601 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
