LOCUS       sequence-376572-       16870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aka PRE-Hsp70BbCas9 or 2gypsyPRE-Hsp70Bb-Cas9-T2A-eGFP; heat-shock 
            inducible Cas9-T2A-eGFP expression regulated by upstream Polycomb 
            response elements with two gypsy insulator elements. Final product.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16870)
  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 16870)
  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_1.3
FEATURES             Location/Qualifiers
     source          1..16870
                     /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"
     protein_bind    866..1251
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     CDS             join(1553..1624,2026..2299,2374..3028,3090..3405,3609..3740,
                     3811..4425)
                     /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    5200..5233
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5294..5679
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        8379..8617
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     8564..8585
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             8631..8696
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8703..8723
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8748..12848
                     /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             8748..12848
                     /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             12849..12896
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12897..12950
                     /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(12951..12953,12954..12956,12957..13670)
                     /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(13257..13276)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     13604..13625
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    14394..14463
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(14682..14700)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(14721..14737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14721..14737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14734..14756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14745..14761
                     /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(14769..14775,14776..14793,14794..14799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14814..14835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14952..14969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(15123..15711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(15203..15222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15882..16673,16674..16742))
                     /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     16505..16524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16743..16847)
                     /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 agtcagcgcg gccgcaattg atcggctaaa tggtatggca agaaaaggta
      901 tgcaatataa taatctttta ttgggtatgc aacgaaaatt tgtttcgtca acgtatgcaa
      961 tattttttat taaaagaggg tatgcaatgt attttattaa aaacgggtat gcaatataat
     1021 aatcttttat tgggtatgca acgaaaattt gtttcgtcaa agtatgcaat attttttatt
     1081 aaaagagggt atgcaatgta ttttattaaa aacgggtatg caataaaaaa ttatttggtt
     1141 tctctaaaaa gtatgcagca cttatttttt gataaggtat gcaacaaaat tttactttgc
     1201 cgaaaatatg caatgttttt gcgaataaat tcaacgcaca cttattacgt gacctgcagg
     1261 catgcgctgt ttgcctcctt ctctgtccac agaaatatcg ccgtctcttt cgccgctgcg
     1321 tccgctatct ctttcgccac cgtttgtagc gttacgtagc gtcaatgtcc gccttcagtt
     1381 gcattttgtc agcggtttcg tgacgaagct ccaagcggtt tacgccatca attaaacaca
     1441 aagtgctgtg ccaaaactcc tctcgcttct tatttttgtt tgttttttga gtgattgggg
     1501 tggtgattgg ttttgggtgg gtaagcaggg gaaagtgtga aaaatcccgg caatgggcca
     1561 agaggatcag gagctattaa ttcgcggagg cagcaaacac ccatctgccg agcatctgaa
     1621 caatgtgagt agtacatgtg catacatctt aagttcactt gatctatagg aactgcgatt
     1681 gcaacatcaa attgtctgcg gcgtgagaac tgcgacccac aaaaatccca aaccgcaatt
     1741 gcacaaacaa atagtgacac gaaacagatt attctggtag ctgttctcgc tatataagac
     1801 aatttttgag atcatatcat gatcaagaca tctaaaggca ttcattttcg actatattct
     1861 tttttacaaa aaatataaca accagatatt ttaagctgat cctagatgca caaaaaataa
     1921 ataaaagtat aaacctactt cgtaggatac ttcggggtac tttttgttcg gggttagatg
     1981 agcataacgc ttgtagttga tatttgagat cccctatcat tgcagggtga cagcggagcg
     2041 gcttcgcaga gctgcattaa ccagggcttc gggcaggcca aaaactacgg cacgctccgg
     2101 ccacccagtc cgccggagga ctccggttca gggagcggcc aactagccga gaacctcacc
     2161 tatgcctggc acaatatgga catctttggg gcggtcaatc agccgggctc cggatggcgg
     2221 cagctggtca accggacacg cggactattc tgcaacgagc gacacatacc ggcgcccagg
     2281 aaacatttgc tcaagaacgg tgagtttcta ttcgcagtcg gctgatctgt gtgaaatctt
     2341 aataaagggt ccaattacca atttgaaact cagtttgcgg cgtggcctat ccgggcgaac
     2401 ttttggccgt gatgggcagt tccggtgccg gaaagacgac cctgctgaat gcccttgcct
     2461 ttcgatcgcc gcagggcatc caagtatcgc catccgggat gcgactgctc aatggccaac
     2521 ctgtggacgc caaggagatg caggccaggt gcgcctatgt ccagcaggat gacctcttta
     2581 tcggctccct aacggccagg gaacacctga ttttccaagc catggtgcgg atgccacgac
     2641 atctgaccta tcggcagcga gtggcccgcg tggatcaggt gatccaggag ctttcgctca
     2701 gcaaatgtca gcacacgatc atcggtgtgc ccggcagggt gaaaggtctg tccggcggag
     2761 aaaggaagcg tctggcattc gcctccgagg ctctaaccga tccgccgctt ctgatctgcg
     2821 atgagcccac ctccggactg gactccttta ccgcccacag cgtcgtccag gtgctgaaga
     2881 agctgtcgca gaagggcaag accgtcatcc tgaccattca tcagccgtct tccgagctgt
     2941 ttgagctctt tgacaagatc cttctgatgg ccgagggcag ggtagctttc ttgggcactc
     3001 ccagcgaagc cgtcgacttc ttttcctagt gagttcgatg tgtttattaa gggtatctag
     3061 tattacataa catctcaact cctatccagc gtgggtgccc agtgtcctac caactacaat
     3121 ccggcggact tttacgtaca ggtgttggcc gttgtgcccg gacgggagat cgagtcccgt
     3181 gatcggatcg ccaagatatg cgacaatttt gccattagca aagtagcccg ggatatggag
     3241 cagttgttgg ccaccaaaaa tctggagaag ccactggagc agccggagaa tgggtacacc
     3301 tacaaggcca cctggttcat gcagttccgg gcggtcctgt ggcgatcctg gctgtcggtg
     3361 ctcaaggaac cactcctcgt aaaagtgcga cttattcaga caacggtgag tggttccagt
     3421 ggaaacaaat gatataacgc ttacaattct tggaaacaaa ttcgctagat tttagataga
     3481 attgcctgat tccacaccct tcttagtttt tttcaatgag atgtatagtt tatagttttg
     3541 cagaagataa ataaatttca tttaactcgc gaatattaat gagatgcgag taacatttta
     3601 atttgcagat ggttgccatc ttgattggcc tcatcttttt gggccaacaa ctcacgcaag
     3661 tgggtgtgat gaatatcaac ggagccatct tcctcttcct gaccaacatg acctttcaaa
     3721 acgtctttgc cacgataaat gtaagtcatg tttagaatac atttgcattt caataattta
     3781 ctaactttct aatgaatcga ttcgatttag gtgttcacct cagagctgcc agtttttatg
     3841 agggaggccc gaagtcgact ttatcgctgt gacacatact ttctgggcaa aacgattgcc
     3901 gaattgccgc tttttctcac agtgccactg gtcttcacgg cgattgccta tccgatgatc
     3961 ggactgcggg ccggagtgct gcacttcttc aactgcctgg cgctggtcac tctggtggcc
     4021 aatgtgtcaa cgtccttcgg atatctaata tcctgcgcca gctcctcgac ctcgatggcg
     4081 ctgtctgtgg gtccgccggt tatcatacca ttcctgctct ttggcggctt cttcttgaac
     4141 tcgggctcgg tgccagtata cctcaaatgg ttgtcgtacc tctcatggtt ccgttacgcc
     4201 aacgagggtc tgctgattaa ccaatgggcg gacgtggagc cgggcgaaat tagctgcaca
     4261 tcgtcgaaca ccacgtgccc cagttcgggc aaggtcatcc tggagacgct taacttctcc
     4321 gccgccgatc tgccgctgga ctacgtgggt ctggccattc tcatcgtgag cttccgggtg
     4381 ctcgcatatc tggctctaag acttcgggcc cgacgcaagg agtagccgac atatatccga
     4441 aataactgct tgtttttttt ttttaccatt attaccatcg tgtttactgt ttattgcccc
     4501 ctcaaaaagc taatgtaatt atatttgtgc caataaaaac aagatatgac ctatagaata
     4561 caagtatttc cccttcgaac atccccacaa gtagactttg gatttgtctt ctaaccaaaa
     4621 gacttacaca cctgcatacc ttacatcaaa aactcgttta tcgctacata aaacaccggg
     4681 atatattttt tatatacata cttttcaaat cgcgcgccct cttcataatt cacctccacc
     4741 acaccacgtt tcgtagttgc tctttcgctg tctcccaccc gctctccgca acacattcac
     4801 cttttgttcg acgaccttgg agcgactgtc gttagttccg cgcgattcgg ttcgctcaaa
     4861 tggttccgag tggttcattt cgtctcaata gaaattagta ataaatattt gtatgtacaa
     4921 tttatttgct ccaatatatt tgtatatatt tccctcacag ctatatttat tctaatttaa
     4981 tattatgact ttttaaggta attttttgtg acctgttcgg agtgattagc gttacaattt
     5041 gaactgaaag tgacatccag tgtttgttcc ttgtgtagat gcatctcaaa aaaatggtgg
     5101 gcataatagt gttgtttata tatatcaaaa ataacaacta taataataag aatacattta
     5161 atttagaaaa tgcttggatt tcactggaac taggctagca taacttcgta taatgtatgc
     5221 tatacgaagt tatgctagcg gatccctccg ttgtatgttg tagcgtctgt gaagcgtcct
     5281 actcctaagg tacaattgat cggctaaatg gtatggcaag aaaaggtatg caatataata
     5341 atcttttatt gggtatgcaa cgaaaatttg tttcgtcaac gtatgcaata ttttttatta
     5401 aaagagggta tgcaatgtat tttattaaaa acgggtatgc aatataataa tcttttattg
     5461 ggtatgcaac gaaaatttgt ttcgtcaaag tatgcaatat tttttattaa aagagggtat
     5521 gcaatgtatt ttattaaaaa cgggtatgca ataaaaaatt atttggtttc tctaaaaagt
     5581 atgcagcact tattttttga taaggtatgc aacaaaattt tactttgccg aaaatatgca
     5641 atgtttttgc gaataaattc aacgcacact tattacgtga ggtaccctac tagtgccatt
     5701 gctacaggca tcgttgcccg acattttgcc ctccggttcc caacgatcaa ctgcagaggc
     5761 agcgactgcg ccgcgactgc ggagagaggg agagatacgg ttagccttct cgctcggatc
     5821 gctctcgctt tacggcgcag ccattatggt gcgcgtagtc ttatctgtat ctcgctctta
     5881 cgcacgtcag acttggaata gccctctctc tttttgagtt atcggcactt tggttctgcc
     5941 gttatggcgg catcgtggtg tcacgctcta tataaacatg gaccgcagtt tgagctagtg
     6001 ccattgctac aggcatcgtt gcccgacatt ttgccctccg gttcccaacg atcaactgca
     6061 gaggcagcga ctgcgccgcg actgcggaga gagggagaga tacggttagc cttctcgctc
     6121 ggatcgctct cgctttacgg cgcagccatt atggtgcgcg tagtcttatc tgtatctcgc
     6181 tcttacgcac gtcagacttg gaatagccct ctctcttttt gagttatcgg cactttggtt
     6241 ctgccgttat ggcggcatcg tggtgtcacg ctctatataa acatggaccg cagtttgagc
     6301 tagtgccatt gctacaggca tcgttgcccg acattttgcc ctccggttcc caacgatcaa
     6361 ctgcagaggc agcgactgcg ccgcgactgc ggagagaggg agagatacgg ttagccttct
     6421 cgctcggatc gctctcgctt tacggcgcag ccattatggt gcgcgtagtc ttatctgtat
     6481 ctcgctctta cgcacgtcag acttggaata gccctctctc tttttgagtt atcggcactt
     6541 tggttctgcc gttatggcgg catcgtggtg tcacgctcta tataaacatg gaccgcagtt
     6601 tgagctagtg ccattgctac aggcatcgtt gcccgacatt ttgccctccg gttcccaacg
     6661 atcaactgca gaggcagcga ctgcgccgcg actgcggaga gagggagaga tacggttagc
     6721 cttctcgctc ggatcgctct cgctttacgg cgcagccatt atggtgcgcg tagtcttatc
     6781 tgtatctcgc tcttacgcac gtcagacttg gaatagccct ctctcttttt gagttatcgg
     6841 cactttggtt ctgccgttat ggcggcatcg tggtgtcacg ctctatataa acatggaccg
     6901 cagtttgagc tagtgccatt gctacaggca tcgttgcccg acattttgcc ctccggttcc
     6961 caacgatcaa ctgcagaggc agcgactgcg ccgcgactgc ggagagaggg agagatacgg
     7021 ttagccttct cgctcggatc gctctcgctt tacggcgcag ccattatggt gcgcgtagtc
     7081 ttatctgtat ctcgctctta cgcacgtcag acttggaata gccctctctc tttttgagtt
     7141 atcggcactt tggttctgcc gttatggcgg catcgtggtg tcacgctcta tataaacatg
     7201 gaccgcagtt tgagctagtg ccattgctac aggcatcgtt gcccgacatt ttgccctccg
     7261 gttcccaacg atcaactgca gaggcagcga ctgcgccgcg actgcggaga gagggagaga
     7321 tacggttagc cttctcgctc ggatcgctct cgctttacgg cgcagccatt atggtgcgcg
     7381 tagtcttatc tgtatctcgc tcttacgcac gtcagacttg gaatagccct ctctcttttt
     7441 gagttatcgg cactttggtt ctgccgttat ggcggcatcg tggtgtcacg ctctatataa
     7501 acatggaccg cagtttgagc tagtgccatt gctacaggca tcgttgcccg acattttgcc
     7561 ctccggttcc caacgatcaa ctgcagaggc agcgactgcg ccgcgactgc ggagagaggg
     7621 agagatacgg ttagccttct cgctcggatc gctctcgctt tacggcgcag ccattatggt
     7681 gcgcgtagtc ttatctgtat ctcgctctta cgcacgtcag acttggaata gccctctctc
     7741 tttttgagtt atcggcactt tggttctgcc gttatggcgg catcgtggtg tcacgctcta
     7801 tataaacatg gaccgcagtt tgagctagtg ccattgctac aggcatcgtt gcccgacatt
     7861 ttgccctccg gttcccaacg atcaactgca gaggcagcga ctgcgccgcg actgcggaga
     7921 gagggagaga tacggttagc cttctcgctc ggatcgctct cgctttacgg cgcagccatt
     7981 atggtgcgcg tagtcttatc tgtatctcgc tcttacgcac gtcagacttg gaatagccct
     8041 ctctcttttt gagttatcgg cactttggtt ctgccgttat ggcggcatcg tggtgtcacg
     8101 ctctatataa acatggaccg cagtttgagc atgacttgaa gcacaactta cctaagccat
     8161 gacagcctcc tgtcgtgtat cactgacagc tcgactctct acgatatcta agctgatctc
     8221 gagtcgagaa atttctctgg ccgttattcg ttattctctc ttttcttttt gggtctctcc
     8281 ctctctgcac taatgctctc tcactctgtc acacagtaaa cggcatactg ctctcgttgg
     8341 ttcgagagag cgcgcctcga atgttcgcga aaagagcgcc ggagtataaa tagaggcgct
     8401 tcgtctacgg agcgacaatt caattcaaac aagcaaagtg aacacgtcgc taagcgaaag
     8461 ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat ctgcagtaaa gtgcaagtta
     8521 aagtgaatca attaaaagta accagcaacc aagtaaatca actgcaacta ctgaaatctg
     8581 ccaagaagta attattgaat acaagaagag aactctgttt aaacaaaatg gactataagg
     8641 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg
     8701 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca
     8761 gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     8821 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     8881 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     8941 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     9001 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     9061 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     9121 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     9181 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     9241 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     9301 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     9361 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     9421 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc
     9481 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     9541 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     9601 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     9661 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     9721 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     9781 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     9841 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     9901 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     9961 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
    10021 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
    10081 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
    10141 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
    10201 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
    10261 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
    10321 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
    10381 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
    10441 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
    10501 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
    10561 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
    10621 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
    10681 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
    10741 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
    10801 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
    10861 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
    10921 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
    10981 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
    11041 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
    11101 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
    11161 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
    11221 aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct
    11281 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
    11341 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
    11401 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
    11461 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
    11521 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
    11581 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
    11641 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
    11701 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
    11761 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
    11821 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
    11881 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
    11941 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
    12001 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
    12061 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
    12121 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
    12181 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
    12241 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
    12301 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
    12361 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
    12421 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
    12481 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
    12541 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
    12601 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
    12661 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
    12721 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
    12781 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
    12841 gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaaggagg
    12901 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct atggtgagca
    12961 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
    13021 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
    13081 ccctgaagtt catctgcacc accagcaagc tgcccgtgcc ctggcccacc ctcgtgacca
    13141 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
    13201 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
    13261 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
    13321 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
    13381 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
    13441 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
    13501 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
    13561 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
    13621 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa ttaattaact
    13681 agaatgaatc gtttttaaaa taacaaatca attgttttat aatattcgta cgattctttg
    13741 attatgtaat aaaatgtgat cattaggaag attacgaaaa atataaaaaa tatgagttct
    13801 gtgtgtataa caaatgctgt aaacgccaca attgtgtttg ttgcaaataa acccatgatt
    13861 atttgattaa aattgttgtt ttctttgttc atagacaata gtgtgttttg cctaaacgtg
    13921 tactgcataa actccatgcg agtgtatagc gagctagtgg ctaacgcttg ccccaccaaa
    13981 gtagattcgt caaaatcctc aatttcatca ccctcctcca agtttaacat ttggccgtcg
    14041 gaattaactt ctaaagatgc cacataatct aataaatgaa atagagattc aaacgtggcg
    14101 tcatcgtccg tttcgaccat ttccgaaaag aactcgggca taaactctat gatttctctg
    14161 gacgtggtgt tgtcgaaact ctcaaagtac gcagtcagga acgtgcgcga catgtcgtcg
    14221 ggaaactcgc gcggaaacat gttgttgtaa ccgaacgggt cccatagcgc caaaaccaaa
    14281 tctgccagcg tcaatagaat gagcacgatg ccgacaatgg agctggcttg gatagcgatt
    14341 cgagttaacg gccggaggcc tgatccacta gtgtcgacga tgtaggtcac ggtctcgaag
    14401 ccgcggtgcg ggtgccaggg cgtgcccttg ggctccccgg gcgcgtactc cacctcaccc
    14461 atctggtcca tcatgatgaa cgggtcgagg tggcggtagt tgatcccggc gaacgcgcgg
    14521 cgcaccggga agccctcgcc ctcgaaaccg ctgggcgcgg tggtcacggt gagcacggga
    14581 cgtgcgacgg cgtcggcggg tgcggatacg cggggcagcg tcagcgggtt ctcgacggtc
    14641 acggcgggca tgtcgacact agttctagcc agcttttgtt ccctttagtg agggttaatt
    14701 tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
    14761 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
    14821 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
    14881 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
    14941 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
    15001 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
    15061 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
    15121 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
    15181 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
    15241 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
    15301 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
    15361 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
    15421 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
    15481 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
    15541 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
    15601 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
    15661 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
    15721 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
    15781 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
    15841 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
    15901 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
    15961 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
    16021 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
    16081 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
    16141 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
    16201 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
    16261 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
    16321 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
    16381 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
    16441 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
    16501 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
    16561 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
    16621 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
    16681 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
    16741 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
    16801 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
    16861 aaagtgccac
//
