LOCUS       sequence-376571-       16462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gypsyPRE-Hsp70Bb-Cas9-T2A-eGFP; heat-shock inducible Cas9-T2A-eGFP 
            expression regulated by upstream Polycomb response elements with one
            gypsy insulator element. Step 2 in cloning process..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16462)
  AUTHORS   Warsinger-Pepe N, Chang C, Desroberts CR, Akbari OS
  TITLE     Polycomb response elements reduce leaky expression of Cas9 under 
            temperature-inducible Hsp70Bb promoter in Drosophila melanogaster.
  JOURNAL   G3 (Bethesda). 2023 Apr 11;13(4):jkad024. doi: 
            10.1093/g3journal/jkad024.
  PUBMED    36705519
REFERENCE   2  (bases 1 to 16462)
  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: "G3 
            (Bethesda)."; date: "2023-04-11"; volume: "13(4):jkad024. doi"; 
            pages: " 10.1093/g3journal/jkad024"
COMMENT     Sequence Label: PRE-Hsp70BbCas9_1.2
FEATURES             Location/Qualifiers
     source          1..16462
                     /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    4886..5271
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        7971..8209
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     8156..8177
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             8223..8288
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8295..8315
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8340..12440
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8340..12440
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             12441..12488
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12489..12542
                     /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             join(12543..12545,12546..12548,12549..13262)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTSKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(12849..12868)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     13196..13217
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    13986..14055
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(14274..14292)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(14313..14329)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14313..14329)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14326..14348)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14337..14353
                     /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(14361..14367,14368..14385,14386..14391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14406..14427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14544..14561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14715..15303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14795..14814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15474..16265,16266..16334))
                     /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     16097..16116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16335..16439)
                     /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 cggggttaga 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 cggatccctc cgttgtatgt tgtagcgtct
     4861 gtgaagcgtc ctactcctaa ggtacaattg atcggctaaa tggtatggca agaaaaggta
     4921 tgcaatataa taatctttta ttgggtatgc aacgaaaatt tgtttcgtca acgtatgcaa
     4981 tattttttat taaaagaggg tatgcaatgt attttattaa aaacgggtat gcaatataat
     5041 aatcttttat tgggtatgca acgaaaattt gtttcgtcaa agtatgcaat attttttatt
     5101 aaaagagggt atgcaatgta ttttattaaa aacgggtatg caataaaaaa ttatttggtt
     5161 tctctaaaaa gtatgcagca cttatttttt gataaggtat gcaacaaaat tttactttgc
     5221 cgaaaatatg caatgttttt gcgaataaat tcaacgcaca cttattacgt gaggtaccct
     5281 actagtgcca ttgctacagg catcgttgcc cgacattttg ccctccggtt cccaacgatc
     5341 aactgcagag gcagcgactg cgccgcgact gcggagagag ggagagatac ggttagcctt
     5401 ctcgctcgga tcgctctcgc tttacggcgc agccattatg gtgcgcgtag tcttatctgt
     5461 atctcgctct tacgcacgtc agacttggaa tagccctctc tctttttgag ttatcggcac
     5521 tttggttctg ccgttatggc ggcatcgtgg tgtcacgctc tatataaaca tggaccgcag
     5581 tttgagctag tgccattgct acaggcatcg ttgcccgaca ttttgccctc cggttcccaa
     5641 cgatcaactg cagaggcagc gactgcgccg cgactgcgga gagagggaga gatacggtta
     5701 gccttctcgc tcggatcgct ctcgctttac ggcgcagcca ttatggtgcg cgtagtctta
     5761 tctgtatctc gctcttacgc acgtcagact tggaatagcc ctctctcttt ttgagttatc
     5821 ggcactttgg ttctgccgtt atggcggcat cgtggtgtca cgctctatat aaacatggac
     5881 cgcagtttga gctagtgcca ttgctacagg catcgttgcc cgacattttg ccctccggtt
     5941 cccaacgatc aactgcagag gcagcgactg cgccgcgact gcggagagag ggagagatac
     6001 ggttagcctt ctcgctcgga tcgctctcgc tttacggcgc agccattatg gtgcgcgtag
     6061 tcttatctgt atctcgctct tacgcacgtc agacttggaa tagccctctc tctttttgag
     6121 ttatcggcac tttggttctg ccgttatggc ggcatcgtgg tgtcacgctc tatataaaca
     6181 tggaccgcag tttgagctag tgccattgct acaggcatcg ttgcccgaca ttttgccctc
     6241 cggttcccaa cgatcaactg cagaggcagc gactgcgccg cgactgcgga gagagggaga
     6301 gatacggtta gccttctcgc tcggatcgct ctcgctttac ggcgcagcca ttatggtgcg
     6361 cgtagtctta tctgtatctc gctcttacgc acgtcagact tggaatagcc ctctctcttt
     6421 ttgagttatc ggcactttgg ttctgccgtt atggcggcat cgtggtgtca cgctctatat
     6481 aaacatggac cgcagtttga gctagtgcca ttgctacagg catcgttgcc cgacattttg
     6541 ccctccggtt cccaacgatc aactgcagag gcagcgactg cgccgcgact gcggagagag
     6601 ggagagatac ggttagcctt ctcgctcgga tcgctctcgc tttacggcgc agccattatg
     6661 gtgcgcgtag tcttatctgt atctcgctct tacgcacgtc agacttggaa tagccctctc
     6721 tctttttgag ttatcggcac tttggttctg ccgttatggc ggcatcgtgg tgtcacgctc
     6781 tatataaaca tggaccgcag tttgagctag tgccattgct acaggcatcg ttgcccgaca
     6841 ttttgccctc cggttcccaa cgatcaactg cagaggcagc gactgcgccg cgactgcgga
     6901 gagagggaga gatacggtta gccttctcgc tcggatcgct ctcgctttac ggcgcagcca
     6961 ttatggtgcg cgtagtctta tctgtatctc gctcttacgc acgtcagact tggaatagcc
     7021 ctctctcttt ttgagttatc ggcactttgg ttctgccgtt atggcggcat cgtggtgtca
     7081 cgctctatat aaacatggac cgcagtttga gctagtgcca ttgctacagg catcgttgcc
     7141 cgacattttg ccctccggtt cccaacgatc aactgcagag gcagcgactg cgccgcgact
     7201 gcggagagag ggagagatac ggttagcctt ctcgctcgga tcgctctcgc tttacggcgc
     7261 agccattatg gtgcgcgtag tcttatctgt atctcgctct tacgcacgtc agacttggaa
     7321 tagccctctc tctttttgag ttatcggcac tttggttctg ccgttatggc ggcatcgtgg
     7381 tgtcacgctc tatataaaca tggaccgcag tttgagctag tgccattgct acaggcatcg
     7441 ttgcccgaca ttttgccctc cggttcccaa cgatcaactg cagaggcagc gactgcgccg
     7501 cgactgcgga gagagggaga gatacggtta gccttctcgc tcggatcgct ctcgctttac
     7561 ggcgcagcca ttatggtgcg cgtagtctta tctgtatctc gctcttacgc acgtcagact
     7621 tggaatagcc ctctctcttt ttgagttatc ggcactttgg ttctgccgtt atggcggcat
     7681 cgtggtgtca cgctctatat aaacatggac cgcagtttga gcatgacttg aagcacaact
     7741 tacctaagcc atgacagcct cctgtcgtgt atcactgaca gctcgactct ctacgatatc
     7801 taagctgatc tcgagtcgag aaatttctct ggccgttatt cgttattctc tcttttcttt
     7861 ttgggtctct ccctctctgc actaatgctc tctcactctg tcacacagta aacggcatac
     7921 tgctctcgtt ggttcgagag agcgcgcctc gaatgttcgc gaaaagagcg ccggagtata
     7981 aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc
     8041 gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca atctgcagta
     8101 aagtgcaagt taaagtgaat caattaaaag taaccagcaa ccaagtaaat caactgcaac
     8161 tactgaaatc tgccaagaag taattattga atacaagaag agaactctgt ttaaacaaaa
     8221 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
     8281 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
     8341 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     8401 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     8461 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     8521 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     8581 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     8641 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     8701 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     8761 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     8821 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     8881 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     8941 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     9001 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     9061 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     9121 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     9181 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     9241 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     9301 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     9361 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     9421 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     9481 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     9541 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     9601 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     9661 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     9721 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     9781 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     9841 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     9901 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     9961 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
    10021 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
    10081 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
    10141 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
    10201 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
    10261 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
    10321 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
    10381 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
    10441 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
    10501 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
    10561 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
    10621 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
    10681 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
    10741 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
    10801 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
    10861 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
    10921 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
    10981 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
    11041 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
    11101 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
    11161 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
    11221 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
    11281 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
    11341 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
    11401 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
    11461 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
    11521 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
    11581 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
    11641 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
    11701 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
    11761 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
    11821 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
    11881 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
    11941 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
    12001 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
    12061 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
    12121 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
    12181 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
    12241 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
    12301 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
    12361 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
    12421 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
    12481 agaaaaagga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggcc
    12541 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
    12601 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
    12661 acggcaagct gaccctgaag ttcatctgca ccaccagcaa gctgcccgtg ccctggccca
    12721 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
    12781 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
    12841 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
    12901 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
    12961 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
    13021 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
    13081 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
    13141 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
    13201 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
    13261 aattaattaa ctagaatgaa tcgtttttaa aataacaaat caattgtttt ataatattcg
    13321 tacgattctt tgattatgta ataaaatgtg atcattagga agattacgaa aaatataaaa
    13381 aatatgagtt ctgtgtgtat aacaaatgct gtaaacgcca caattgtgtt tgttgcaaat
    13441 aaacccatga ttatttgatt aaaattgttg ttttctttgt tcatagacaa tagtgtgttt
    13501 tgcctaaacg tgtactgcat aaactccatg cgagtgtata gcgagctagt ggctaacgct
    13561 tgccccacca aagtagattc gtcaaaatcc tcaatttcat caccctcctc caagtttaac
    13621 atttggccgt cggaattaac ttctaaagat gccacataat ctaataaatg aaatagagat
    13681 tcaaacgtgg cgtcatcgtc cgtttcgacc atttccgaaa agaactcggg cataaactct
    13741 atgatttctc tggacgtggt gttgtcgaaa ctctcaaagt acgcagtcag gaacgtgcgc
    13801 gacatgtcgt cgggaaactc gcgcggaaac atgttgttgt aaccgaacgg gtcccatagc
    13861 gccaaaacca aatctgccag cgtcaataga atgagcacga tgccgacaat ggagctggct
    13921 tggatagcga ttcgagttaa cggccggagg cctgatccac tagtgtcgac gatgtaggtc
    13981 acggtctcga agccgcggtg cgggtgccag ggcgtgccct tgggctcccc gggcgcgtac
    14041 tccacctcac ccatctggtc catcatgatg aacgggtcga ggtggcggta gttgatcccg
    14101 gcgaacgcgc ggcgcaccgg gaagccctcg ccctcgaaac cgctgggcgc ggtggtcacg
    14161 gtgagcacgg gacgtgcgac ggcgtcggcg ggtgcggata cgcggggcag cgtcagcggg
    14221 ttctcgacgg tcacggcggg catgtcgaca ctagttctag ccagcttttg ttccctttag
    14281 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
    14341 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
    14401 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
    14461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
    14521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    14581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    14641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    14701 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    14761 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    14821 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    14881 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    14941 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    15001 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    15061 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    15121 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    15181 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    15241 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    15301 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    15361 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    15421 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    15481 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    15541 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    15601 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    15661 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    15721 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    15781 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    15841 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    15901 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    15961 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    16021 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    16081 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    16141 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    16201 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    16261 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    16321 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    16381 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    16441 acatttcccc gaaaagtgcc ac
//
