LOCUS       sequence-292051-       13159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The piggyBac plasmid harboring Hsp70Bb-Cas9-T2A-eGFP-p10 and 
            Opie2-dsRed-SV10 (marker gene).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13159)
  AUTHORS   Kandul NP, Liu J, Akbari OS
  TITLE     Temperature-Inducible Precision-Guided Sterile Insect Technique.
  JOURNAL   CRISPR J. 2021 Dec;4(6):822-835. doi: 10.1089/crispr.2021.0077. Epub
            2021 Nov 3.
  PUBMED    34735288
REFERENCE   2  (bases 1 to 13159)
  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"; doi: 
            "10.1089/crispr.2021.0077"; journalName: "CRISPR J"; date: 
            "2021-12"; volume: "4"; issue: "6"; pages: "822-835"
COMMENT     Sequence Label: 1137=Hsp70Bb-Cas9[dsRed]
FEATURES             Location/Qualifiers
     source          1..13159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   651..685
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(866..882)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(866..882)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(879..901)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    890..906
                     /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(914..920,921..938,939..944))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    959..980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1097..1114)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1268..1856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1348..1367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2027..2818,2819..2887))
                     /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     2650..2669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2888..2992)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3060..3078
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3116..3138)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3238..3257
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3451..3473
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3465..3482
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3466..3482
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4186..4248
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5282..5351
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        5701..5939
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     5886..5907
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             5953..6018
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6025..6045
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6070..10170
                     /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             6070..10170
                     /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             10171..10218
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10219..10272
                     /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(10273..10275,10276..10278,10279..10992)
                     /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(10579..10598)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10926..10947
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(12227..12229,12230..12232,12233..12907)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(12410..12430)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     12628..12647
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     12822..12845
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    13021..13142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13058..13077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13112..13131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gatctcggat ctgacaatgt tcagtgcaga gactcggcta cgcctcgtgg actttgaagt
       61 tgaccaacaa tgtttattct tacctctaat agtcctctgt ggcaaggtca agattctgtt
      121 agaagccaat gaagaacctg gttgttcaat aacattttgt tcgtctaata tttcactacc
      181 gcttgacgtt ggctgcactt catgtacctc atctataaac gcttcttctg tatcgctctg
      241 gacgtcatct tcacttacgt gatctgatat ttcactgtca gaatcctcac caacaagctc
      301 gtcatcgctt tgcagaagag cagagaggat atgctcatcg tctaaagaac tacccatttt
      361 attatatatt agtcacgata tctataacaa gaaaatatat atataataag ttatcacgta
      421 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
      481 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
      541 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
      601 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
      661 ttttacgcag actatctttc tagggttaaa aaagatttgc gctttactcg acctaaactt
      721 taaacacgtc atagaatctt cgtttgacaa aaaccacatt gtggccaagc tgtgtgacgc
      781 gacgcgcgct aaagaatggc aaaccaagtc gcgcgagcgt cgactctaga ggatccccgg
      841 gtaccgagct cgaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc
      901 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
      961 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1021 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1081 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1141 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1201 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1261 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1321 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1381 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1441 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1501 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1561 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1621 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1681 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1741 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1801 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1861 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1921 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2161 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3001 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     3061 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     3121 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3181 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     3241 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     3301 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
     3361 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3421 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3481 gtgccaagct ttgtttaaaa atataacaaa attgtgatcc cacaaaatga agtggggcaa
     3541 aatcaaataa ttaatagtgt ccgtaaactt gttggtcttc aactttttga ggaacacgtt
     3601 ggacggcaaa tccgtgacta taacacaagt tgatttaata attttagcca acacgtcggg
     3661 ctgcgtgttt tttgccgacg cgtctgtgta cacgttgatt aactggtcga ttaaactgtt
     3721 gaaataattt aatttttggt tcttctttaa atctgtgatg aaatttttta aaataacttt
     3781 aaattcttca ttggtaaaaa atgccacgtt ttgcaacttg tgagggtcta atatgaggtc
     3841 aaactcagta ggagttttat ccaaaaaaga aaacatgatt acgtctgtac acgaacgcgt
     3901 attaacgcag agtgcaaagt ataagagggt taaaaaatat attttacgca ccatatacgc
     3961 atcgggttga tatcgttaat atggatcaat ttgaacagtt gattaacgtg tctctgctca
     4021 agtctttgat caaaacgcaa atcgacgaaa atgtgtcgga caatatcaag tcgatgagcg
     4081 aaaaactaaa aaggctagaa tacgacaatc tcacagacag cgttgagata tacggtattc
     4141 acgacagcag gctgaataat aaaaaaatta gaaactatta tttaacccta gaaagataat
     4201 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
     4261 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
     4321 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
     4381 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
     4441 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
     4501 gcttaactta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
     4561 cacatatcaa tattatgctc tcgacaaata acttttttgc attttttgca cgatgcattt
     4621 gcctttcgcc ttattttaga ggggcagtaa gtacagtaag tacgtttttt cattactggc
     4681 tcttcagtac tgtcatctga tgtaccaggc acttcatttg gcaaaatatt agagatatta
     4741 tcgcgcaaat atctcttcaa agtaggagct tctaaacgct tacgcataaa cgatgacgtc
     4801 aggctcatgt aaaggtttct cataaatttt ttgcgacttt gaaccttttc tcccttgcta
     4861 ctgacattat ggctgtatat aataaaagaa tttatgcagg caatgtttat cattccgtac
     4921 aataatgcca taggccacct attcgtcttc ctactgcagg tcatcacaga acacatttgg
     4981 tctagcgtgt ccactccgcc tttagtttga ttataataca taaccatttg cggtttaccg
     5041 gtactttcgt tgatagaagc atcctcatca caagatgata ataagtatac catcttagct
     5101 ggcttcggtt tatatgagac gagagtaagg ggtccgtcaa aacaaaacat cgatgttccc
     5161 actggcctgg agcgactgtt tttcagtact tccggtatct cgcgtttgtt tgatcgcacg
     5221 gttcccacaa tggttaattc gagctcgccc ggggtcctag gtcgacgatg taggtcacgg
     5281 tctcgaagcc gcggtgcggg tgccagggcg tgcccttggg ctccccgggc gcgtactcca
     5341 cctcacccat ctggtccatc atgatgaacg ggtcgaggtg gcggtagttg atcccggcga
     5401 acgcgcggcg caccgggaag ccctcgccct cgaaaccgct gggcgcggtg gtcacggtga
     5461 gcacgggacg tgcgacggcg tcggcgggtg cggatacgcg gggcagcgtc agcgggttct
     5521 cgacggtcac ggcgggcatg tcgactcgag aaatttctct ggccgttatt cgttattctc
     5581 tcttttcttt ttgggtctct ccctctctgc actaatgctc tctcactctg tcacacagta
     5641 aacggcatac tgctctcgtt ggttcgagag agcgcgcctc gaatgttcgc gaaaagagcg
     5701 ccggagtata aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag
     5761 tgaacacgtc gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca
     5821 atctgcagta aagtgcaagt taaagtgaat caattaaaag taaccagcaa ccaagtaaat
     5881 caactgcaac tactgaaatc tgccaagaag taattattga atacaagaag agaactctgt
     5941 ttaaacaaaa tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
     6001 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
     6061 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     6121 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     6181 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     6241 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     6301 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     6361 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     6421 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     6481 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     6541 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     6601 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     6661 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     6721 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     6781 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     6841 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     6901 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     6961 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     7021 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     7081 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     7141 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     7201 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     7261 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     7321 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     7381 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     7441 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     7501 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     7561 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     7621 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     7681 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     7741 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     7801 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     7861 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     7921 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     7981 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     8041 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     8101 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     8161 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     8221 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     8281 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     8341 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     8401 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     8461 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     8521 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     8581 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     8641 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     8701 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     8761 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     8821 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     8881 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     8941 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     9001 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     9061 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     9121 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     9181 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     9241 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     9301 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     9361 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     9421 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     9481 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     9541 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     9601 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     9661 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     9721 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     9781 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     9841 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     9901 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     9961 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
    10021 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
    10081 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
    10141 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
    10201 caggcaaaaa agaaaaagga gggcagagga agtcttctaa catgcggtga cgtggaggag
    10261 aatcccggcc ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
    10321 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
    10381 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccagcaa gctgcccgtg
    10441 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
    10501 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
    10561 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
    10621 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
    10681 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
    10741 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
    10801 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
    10861 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
    10921 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
    10981 ctgtacaagt aattaattaa ctagaatgaa tcgtttttaa aataacaaat caattgtttt
    11041 ataatattcg tacgattctt tgattatgta ataaaatgtg atcattagga agattacgaa
    11101 aaatataaaa aatatgagtt ctgtgtgtat aacaaatgct gtaaacgcca caattgtgtt
    11161 tgttgcaaat aaacccatga ttatttgatt aaaattgttg ttttctttgt tcatagacaa
    11221 tagtgtgttt tgcctaaacg tgtactgcat aaactccatg cgagtgtata gcgagctagt
    11281 ggctaacgct tgccccacca aagtagattc gtcaaaatcc tcaatttcat caccctcctc
    11341 caagtttaac atttggccgt cggaattaac ttctaaagat gccacataat ctaataaatg
    11401 aaatagagat tcaaacgtgg cgtcatcgtc cgtttcgacc atttccgaaa agaactcggg
    11461 cataaactct atgatttctc tggacgtggt gttgtcgaaa ctctcaaagt acgcagtcag
    11521 gaacgtgcgc gacatgtcgt cgggaaactc gcgcggaaac atgttgttgt aaccgaacgg
    11581 gtcccatagc gccaaaacca aatctgccag cgtcaataga atgagcacga tgccgacaat
    11641 ggagctggct tggatagcga ttcgagttaa cggccggggc gcgcccatac tcggtggcct
    11701 ccccaccacc aacttttttg cactgcaaaa aaacacgctt ttgcacgcgg gcccatacat
    11761 agtacaaact ctacgtttcg tagactattt tacataaata gtctacaccg ttgtatacgc
    11821 tccaaataca ctaccacaca ttgaaccttt ttgcagtgca aaaaagtacg tgtcggcagt
    11881 cacgtaggcc ggccttatcg ggtcgcgtcc tgtcacgtac gaatcacatt atcggaccgg
    11941 acgagtgttg tcttatcgtg acaggacgcc agcttcctgt gttgctaacc gcagccggac
    12001 gcaactcctt atcggaacag gacgcgcctc catatcagcc gcgcgttatc tcatgcgcgt
    12061 gaccggacac gaggcgcccg tcccgcttat cgcgcctata aatacagccc gcaacgatct
    12121 ggtaaacaca gttgaacagc atctgttaca gcgacacaac atgagccggt ccaacaacgc
    12181 caacgcgccc acgccatcca accgccgccg caacctgtct ctggtgatgg tgcgctcctc
    12241 caagaacgtc atcaaggagt tcatgcgctt caaggtgcgc atggagggca ccgtgaacgg
    12301 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggcc acaacaccgt
    12361 gaagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca
    12421 gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc gacatccccg actacaagaa
    12481 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
    12541 ggtgaccgtg acccaggact cctccctgca ggacggctgc ttcatctaca aggtgaagtt
    12601 catcggcgtg aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
    12661 ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccacaaggc
    12721 cctgaagctg aaggacggcg gccactacct ggtggagttc aagtccatct acatggccaa
    12781 gaagcccgtg cagctgcccg gctactacta cgtggactcc aagctggaca tcacctccca
    12841 caacgaggac tacaccatcg tggagcagta cgagcgcacc gagggccgcc accacctgtt
    12901 cctgtaggac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta
    12961 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
    13021 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
    13081 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
    13141 taaagcttct aggccggcc
//
