LOCUS       sequence-376570-       16069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PRE-Hsp70Bb-Cas9-T2A-eGFP; heat-shock inducible Cas9-T2A-eGFP 
            expression regulated by upstream Polycomb response elements. Step 1 
            in cloning process.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16069)
  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 16069)
  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_1.0
FEATURES             Location/Qualifiers
     source          1..16069
                     /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)."
     promoter        7578..7816
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     7763..7784
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             7830..7895
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7902..7922
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7947..12047
                     /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             7947..12047
                     /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             12048..12095
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12096..12149
                     /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(12150..12152,12153..12155,12156..12869)
                     /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(12456..12475)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     12803..12824
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    13593..13662
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(13881..13899)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(13920..13936)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13920..13936)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13933..13955)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13944..13960
                     /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(13968..13974,13975..13992,13993..13998))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14013..14034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14151..14168)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14322..14910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14402..14421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15081..15872,15873..15941))
                     /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     15704..15723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15942..16046)
                     /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 ggtacctact agtgccattg ctacaggcat cgttgcccga
     4921 cattttgccc tccggttccc aacgatcaac tgcagaggca gcgactgcgc cgcgactgcg
     4981 gagagaggga gagatacggt tagccttctc gctcggatcg ctctcgcttt acggcgcagc
     5041 cattatggtg cgcgtagtct tatctgtatc tcgctcttac gcacgtcaga cttggaatag
     5101 ccctctctct ttttgagtta tcggcacttt ggttctgccg ttatggcggc atcgtggtgt
     5161 cacgctctat ataaacatgg accgcagttt gagctagtgc cattgctaca ggcatcgttg
     5221 cccgacattt tgccctccgg ttcccaacga tcaactgcag aggcagcgac tgcgccgcga
     5281 ctgcggagag agggagagat acggttagcc ttctcgctcg gatcgctctc gctttacggc
     5341 gcagccatta tggtgcgcgt agtcttatct gtatctcgct cttacgcacg tcagacttgg
     5401 aatagccctc tctctttttg agttatcggc actttggttc tgccgttatg gcggcatcgt
     5461 ggtgtcacgc tctatataaa catggaccgc agtttgagct agtgccattg ctacaggcat
     5521 cgttgcccga cattttgccc tccggttccc aacgatcaac tgcagaggca gcgactgcgc
     5581 cgcgactgcg gagagaggga gagatacggt tagccttctc gctcggatcg ctctcgcttt
     5641 acggcgcagc cattatggtg cgcgtagtct tatctgtatc tcgctcttac gcacgtcaga
     5701 cttggaatag ccctctctct ttttgagtta tcggcacttt ggttctgccg ttatggcggc
     5761 atcgtggtgt cacgctctat ataaacatgg accgcagttt gagctagtgc cattgctaca
     5821 ggcatcgttg cccgacattt tgccctccgg ttcccaacga tcaactgcag aggcagcgac
     5881 tgcgccgcga ctgcggagag agggagagat acggttagcc ttctcgctcg gatcgctctc
     5941 gctttacggc gcagccatta tggtgcgcgt agtcttatct gtatctcgct cttacgcacg
     6001 tcagacttgg aatagccctc tctctttttg agttatcggc actttggttc tgccgttatg
     6061 gcggcatcgt ggtgtcacgc tctatataaa catggaccgc agtttgagct agtgccattg
     6121 ctacaggcat cgttgcccga cattttgccc tccggttccc aacgatcaac tgcagaggca
     6181 gcgactgcgc cgcgactgcg gagagaggga gagatacggt tagccttctc gctcggatcg
     6241 ctctcgcttt acggcgcagc cattatggtg cgcgtagtct tatctgtatc tcgctcttac
     6301 gcacgtcaga cttggaatag ccctctctct ttttgagtta tcggcacttt ggttctgccg
     6361 ttatggcggc atcgtggtgt cacgctctat ataaacatgg accgcagttt gagctagtgc
     6421 cattgctaca ggcatcgttg cccgacattt tgccctccgg ttcccaacga tcaactgcag
     6481 aggcagcgac tgcgccgcga ctgcggagag agggagagat acggttagcc ttctcgctcg
     6541 gatcgctctc gctttacggc gcagccatta tggtgcgcgt agtcttatct gtatctcgct
     6601 cttacgcacg tcagacttgg aatagccctc tctctttttg agttatcggc actttggttc
     6661 tgccgttatg gcggcatcgt ggtgtcacgc tctatataaa catggaccgc agtttgagct
     6721 agtgccattg ctacaggcat cgttgcccga cattttgccc tccggttccc aacgatcaac
     6781 tgcagaggca gcgactgcgc cgcgactgcg gagagaggga gagatacggt tagccttctc
     6841 gctcggatcg ctctcgcttt acggcgcagc cattatggtg cgcgtagtct tatctgtatc
     6901 tcgctcttac gcacgtcaga cttggaatag ccctctctct ttttgagtta tcggcacttt
     6961 ggttctgccg ttatggcggc atcgtggtgt cacgctctat ataaacatgg accgcagttt
     7021 gagctagtgc cattgctaca ggcatcgttg cccgacattt tgccctccgg ttcccaacga
     7081 tcaactgcag aggcagcgac tgcgccgcga ctgcggagag agggagagat acggttagcc
     7141 ttctcgctcg gatcgctctc gctttacggc gcagccatta tggtgcgcgt agtcttatct
     7201 gtatctcgct cttacgcacg tcagacttgg aatagccctc tctctttttg agttatcggc
     7261 actttggttc tgccgttatg gcggcatcgt ggtgtcacgc tctatataaa catggaccgc
     7321 agtttgagca tgacttgaag cacaacttac ctaagccatg acagcctcct gtcgtgtatc
     7381 actgacagct cgactctcta cgatatctaa gctgatctcg agtcgagaaa tttctctggc
     7441 cgttattcgt tattctctct tttctttttg ggtctctccc tctctgcact aatgctctct
     7501 cactctgtca cacagtaaac ggcatactgc tctcgttggt tcgagagagc gcgcctcgaa
     7561 tgttcgcgaa aagagcgccg gagtataaat agaggcgctt cgtctacgga gcgacaattc
     7621 aattcaaaca agcaaagtga acacgtcgct aagcgaaagc taagcaaata aacaagcgca
     7681 gctgaacaag ctaaacaatc tgcagtaaag tgcaagttaa agtgaatcaa ttaaaagtaa
     7741 ccagcaacca agtaaatcaa ctgcaactac tgaaatctgc caagaagtaa ttattgaata
     7801 caagaagaga actctgttta aacaaaatgg actataagga ccacgacgga gactacaagg
     7861 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
     7921 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gacatcggca
     7981 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     8041 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     8101 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     8161 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     8221 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     8281 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     8341 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     8401 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     8461 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     8521 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     8581 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     8641 agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca
     8701 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     8761 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     8821 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     8881 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     8941 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     9001 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     9061 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     9121 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     9181 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     9241 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     9301 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     9361 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     9421 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     9481 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     9541 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     9601 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     9661 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     9721 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     9781 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     9841 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     9901 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     9961 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
    10021 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
    10081 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
    10141 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
    10201 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
    10261 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
    10321 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
    10381 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
    10441 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
    10501 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
    10561 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
    10621 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
    10681 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
    10741 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
    10801 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
    10861 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
    10921 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
    10981 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
    11041 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
    11101 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
    11161 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
    11221 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
    11281 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
    11341 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
    11401 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
    11461 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
    11521 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
    11581 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
    11641 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
    11701 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
    11761 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
    11821 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
    11881 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
    11941 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
    12001 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
    12061 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaggg cagaggaagt cttctaacat
    12121 gcggtgacgt ggaggagaat cccggcccta tggtgagcaa gggcgaggag ctgttcaccg
    12181 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
    12241 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
    12301 ccagcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
    12361 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
    12421 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
    12481 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
    12541 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
    12601 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
    12661 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
    12721 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
    12781 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
    12841 ctctcggcat ggacgagctg tacaagtaat taattaacta gaatgaatcg tttttaaaat
    12901 aacaaatcaa ttgttttata atattcgtac gattctttga ttatgtaata aaatgtgatc
    12961 attaggaaga ttacgaaaaa tataaaaaat atgagttctg tgtgtataac aaatgctgta
    13021 aacgccacaa ttgtgtttgt tgcaaataaa cccatgatta tttgattaaa attgttgttt
    13081 tctttgttca tagacaatag tgtgttttgc ctaaacgtgt actgcataaa ctccatgcga
    13141 gtgtatagcg agctagtggc taacgcttgc cccaccaaag tagattcgtc aaaatcctca
    13201 atttcatcac cctcctccaa gtttaacatt tggccgtcgg aattaacttc taaagatgcc
    13261 acataatcta ataaatgaaa tagagattca aacgtggcgt catcgtccgt ttcgaccatt
    13321 tccgaaaaga actcgggcat aaactctatg atttctctgg acgtggtgtt gtcgaaactc
    13381 tcaaagtacg cagtcaggaa cgtgcgcgac atgtcgtcgg gaaactcgcg cggaaacatg
    13441 ttgttgtaac cgaacgggtc ccatagcgcc aaaaccaaat ctgccagcgt caatagaatg
    13501 agcacgatgc cgacaatgga gctggcttgg atagcgattc gagttaacgg ccggaggcct
    13561 gatccactag tgtcgacgat gtaggtcacg gtctcgaagc cgcggtgcgg gtgccagggc
    13621 gtgcccttgg gctccccggg cgcgtactcc acctcaccca tctggtccat catgatgaac
    13681 gggtcgaggt ggcggtagtt gatcccggcg aacgcgcggc gcaccgggaa gccctcgccc
    13741 tcgaaaccgc tgggcgcggt ggtcacggtg agcacgggac gtgcgacggc gtcggcgggt
    13801 gcggatacgc ggggcagcgt cagcgggttc tcgacggtca cggcgggcat gtcgacacta
    13861 gttctagcca gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg
    13921 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
    13981 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
    14041 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
    14101 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
    14161 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
    14221 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
    14281 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
    14341 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
    14401 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
    14461 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
    14521 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
    14581 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
    14641 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
    14701 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
    14761 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
    14821 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
    14881 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
    14941 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
    15001 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
    15061 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
    15121 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
    15181 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
    15241 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
    15301 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
    15361 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
    15421 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
    15481 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
    15541 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
    15601 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
    15661 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
    15721 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
    15781 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
    15841 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
    15901 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
    15961 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
    16021 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
