LOCUS       sequence-301649-       14904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The attB plasmid with the mini-white harboring two U6.3-gRNAs 
            targeting Dmel tra and bTub genes, and Hsp70Bb-Cas9-T2A-eGFP-p10..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14904)
  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 14904)
  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: 1098E=TI-pgSIT[tra,bTub,Hasp70Bb-Cas9]
FEATURES             Location/Qualifiers
     source          1..14904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        13..88
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        562..800
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     747..768
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             814..879
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             886..906
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             931..5031
                     /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             931..5031
                     /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             5032..5079
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5080..5133
                     /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(5134..5136,5137..5139,5140..5853)
                     /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(5440..5459)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5787..5808
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    6577..6646
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(6865..6883)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6904..6920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6904..6920)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6917..6939)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6928..6944
                     /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(6952..6958,6959..6976,6977..6982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6997..7018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7135..7152)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7306..7894)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7386..7405)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8065..8856,8857..8925))
                     /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     8688..8707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8926..9030)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(9056..9511)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9193..9214)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9405..9424
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9638..9660
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9652..9669
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9653..9669
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9676..9695
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9676..9694
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9728..9746)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(10198..10269,10671..10944,11019..11673,11735..12050,
                     12254..12385,12456..13070)
                     /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    13845..13878
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        13897..14317
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        14338..14413
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        14476..14896
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
ORIGIN
        1 tgcatcagct gggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
       61 acttgaaaaa gtggcaccga gtcggtgctt ttttttttgc ctacctggag cctgagagtt
      121 gttcaataaa ataaaaatgt ttcgtttttt tgctttcgcc agtatttatt atttttcatc
      181 aatatgtatt caatttggta tgtatttagt aattgtaata tatagacaat ggttttccgt
      241 tgacgtacat acatctgacg tgtgtttatt tagacataat agttatgttt tcacatcttt
      301 ttaatgttcg cttaatgcgt atgcattcta gacaattgtg ctcggcaaca gtatatttgt
      361 ggtgtgccaa ccaacaacct gcaggagctc cagcttttgt ctcgagtcga gaaatttctc
      421 tggccgttat tcgttattct ctcttttctt tttgggtctc tccctctctg cactaatgct
      481 ctctcactct gtcacacagt aaacggcata ctgctctcgt tggttcgaga gagcgcgcct
      541 cgaatgttcg cgaaaagagc gccggagtat aaatagaggc gcttcgtcta cggagcgaca
      601 attcaattca aacaagcaaa gtgaacacgt cgctaagcga aagctaagca aataaacaag
      661 cgcagctgaa caagctaaac aatctgcagt aaagtgcaag ttaaagtgaa tcaattaaaa
      721 gtaaccagca accaagtaaa tcaactgcaa ctactgaaat ctgccaagaa gtaattattg
      781 aatacaagaa gagaactctg tttaaacaaa atggactata aggaccacga cggagactac
      841 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
      901 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
      961 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     1021 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     1081 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     1141 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     1201 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     1261 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     1321 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     1381 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     1441 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     1501 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     1561 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     1621 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     1681 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     1741 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     1801 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     1861 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     1921 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     1981 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     2041 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     2101 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     2161 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     2221 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     2281 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     2341 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     2401 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     2461 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     2521 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     2581 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     2641 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     2701 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     2761 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     2821 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     2881 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     2941 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     3001 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     3061 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     3121 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     3181 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     3241 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     3301 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     3361 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     3421 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     3481 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     3541 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     3601 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     3661 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     3721 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     3781 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     3841 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     3901 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     3961 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     4021 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     4081 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     4141 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     4201 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     4261 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     4321 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     4381 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     4441 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     4501 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     4561 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     4621 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     4681 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     4741 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     4801 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     4861 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     4921 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     4981 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     5041 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg agggcagagg aagtcttcta
     5101 acatgcggtg acgtggagga gaatcccggc cctatggtga gcaagggcga ggagctgttc
     5161 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     5221 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     5281 accaccagca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     5341 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     5401 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     5461 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     5521 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5581 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5641 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5701 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     5761 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5821 atcactctcg gcatggacga gctgtacaag taattaatta actagaatga atcgttttta
     5881 aaataacaaa tcaattgttt tataatattc gtacgattct ttgattatgt aataaaatgt
     5941 gatcattagg aagattacga aaaatataaa aaatatgagt tctgtgtgta taacaaatgc
     6001 tgtaaacgcc acaattgtgt ttgttgcaaa taaacccatg attatttgat taaaattgtt
     6061 gttttctttg ttcatagaca atagtgtgtt ttgcctaaac gtgtactgca taaactccat
     6121 gcgagtgtat agcgagctag tggctaacgc ttgccccacc aaagtagatt cgtcaaaatc
     6181 ctcaatttca tcaccctcct ccaagtttaa catttggccg tcggaattaa cttctaaaga
     6241 tgccacataa tctaataaat gaaatagaga ttcaaacgtg gcgtcatcgt ccgtttcgac
     6301 catttccgaa aagaactcgg gcataaactc tatgatttct ctggacgtgg tgttgtcgaa
     6361 actctcaaag tacgcagtca ggaacgtgcg cgacatgtcg tcgggaaact cgcgcggaaa
     6421 catgttgttg taaccgaacg ggtcccatag cgccaaaacc aaatctgcca gcgtcaatag
     6481 aatgagcacg atgccgacaa tggagctggc ttggatagcg attcgagtta acggccggag
     6541 gcctgatcca ctagtgtcga cgatgtaggt cacggtctcg aagccgcggt gcgggtgcca
     6601 gggcgtgccc ttgggctccc cgggcgcgta ctccacctca cccatctggt ccatcatgat
     6661 gaacgggtcg aggtggcggt agttgatccc ggcgaacgcg cggcgcaccg ggaagccctc
     6721 gccctcgaaa ccgctgggcg cggtggtcac ggtgagcacg ggacgtgcga cggcgtcggc
     6781 gggtgcggat acgcggggca gcgtcagcgg gttctcgacg gtcacggcgg gcatgtcgac
     6841 actagttcta gccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
     6901 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6961 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     7021 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     7081 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     7141 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     7201 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     7261 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     7321 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     7381 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     7441 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     7501 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     7561 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     7621 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7681 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7741 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7801 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7861 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7921 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7981 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     8041 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     8101 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     8161 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     8221 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     8281 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     8341 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     8401 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     8461 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     8521 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     8581 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8641 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8701 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8761 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8821 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8881 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8941 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     9001 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     9061 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     9121 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     9181 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     9241 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     9301 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     9361 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     9421 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     9481 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     9541 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     9601 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     9661 acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacgta ccgggcccct
     9721 agtatgtatg taagttaata aaacccattt ttgcggaaag tagataaaaa aaacattttt
     9781 tttttttact gcactggata tcattgaact tatctgatca gttttaaatt tacttcgatc
     9841 caagggtatt tgatgtacca ggttctttcg attacctctc actcaaaatg acattccact
     9901 caaagtcagc gctgtttgcc tccttctctg tccacagaaa tatcgccgtc tctttcgccg
     9961 ctgcgtccgc tatctctttc gccaccgttt gtagcgttac gtagcgtcaa tgtccgcctt
    10021 cagttgcatt ttgtcagcgg tttcgtgacg aagctccaag cggtttacgc catcaattaa
    10081 acacaaagtg ctgtgccaaa actcctctcg cttcttattt ttgtttgttt tttgagtgat
    10141 tggggtggtg attggttttg ggtgggtaag caggggaaag tgtgaaaaat cccggcaatg
    10201 ggccaagagg atcaggagct attaattcgc ggaggcagca aacacccatc tgccgagcat
    10261 ctgaacaatg tgagtagtac atgtgcatac atcttaagtt cacttgatct ataggaactg
    10321 cgattgcaac atcaaattgt ctgcggcgtg agaactgcga cccacaaaaa tcccaaaccg
    10381 caattgcaca aacaaatagt gacacgaaac agattattct ggtagctgtt ctcgctatat
    10441 aagacaattt ttgagatcat atcatgatca agacatctaa aggcattcat tttcgactat
    10501 attctttttt acaaaaaata taacaaccag atattttaag ctgatcctag atgcacaaaa
    10561 aataaataaa agtataaacc tacttcgtag gatacttcgg ggtacttttt gttcggggtt
    10621 agatgagcat aacgcttgta gttgatattt gagatcccct atcattgcag ggtgacagcg
    10681 gagcggcttc gcagagctgc attaaccagg gcttcgggca ggccaaaaac tacggcacgc
    10741 tccggccacc cagtccgccg gaggactccg gttcagggag cggccaacta gccgagaacc
    10801 tcacctatgc ctggcacaat atggacatct ttggggcggt caatcagccg ggctccggat
    10861 ggcggcagct ggtcaaccgg acacgcggac tattctgcaa cgagcgacac ataccggcgc
    10921 ccaggaaaca tttgctcaag aacggtgagt ttctattcgc agtcggctga tctgtgtgaa
    10981 atcttaataa agggtccaat taccaatttg aaactcagtt tgcggcgtgg cctatccggg
    11041 cgaacttttg gccgtgatgg gcagttccgg tgccggaaag acgaccctgc tgaatgccct
    11101 tgcctttcga tcgccgcagg gcatccaagt atcgccatcc gggatgcgac tgctcaatgg
    11161 ccaacctgtg gacgccaagg agatgcaggc caggtgcgcc tatgtccagc aggatgacct
    11221 ctttatcggc tccctaacgg ccagggaaca cctgattttc caagccatgg tgcggatgcc
    11281 acgacatctg acctatcggc agcgagtggc ccgcgtggat caggtgatcc aggagctttc
    11341 gctcagcaaa tgtcagcaca cgatcatcgg tgtgcccggc agggtgaaag gtctgtccgg
    11401 cggagaaagg aagcgtctgg cattcgcctc cgaggctcta accgatccgc cgcttctgat
    11461 ctgcgatgag cccacctccg gactggactc ctttaccgcc cacagcgtcg tccaggtgct
    11521 gaagaagctg tcgcagaagg gcaagaccgt catcctgacc attcatcagc cgtcttccga
    11581 gctgtttgag ctctttgaca agatccttct gatggccgag ggcagggtag ctttcttggg
    11641 cactcccagc gaagccgtcg acttcttttc ctagtgagtt cgatgtgttt attaagggta
    11701 tctagtatta cataacatct caactcctat ccagcgtggg tgcccagtgt cctaccaact
    11761 acaatccggc ggacttttac gtacaggtgt tggccgttgt gcccggacgg gagatcgagt
    11821 cccgtgatcg gatcgccaag atatgcgaca attttgccat tagcaaagta gcccgggata
    11881 tggagcagtt gttggccacc aaaaatctgg agaagccact ggagcagccg gagaatgggt
    11941 acacctacaa ggccacctgg ttcatgcagt tccgggcggt cctgtggcga tcctggctgt
    12001 cggtgctcaa ggaaccactc ctcgtaaaag tgcgacttat tcagacaacg gtgagtggtt
    12061 ccagtggaaa caaatgatat aacgcttaca attcttggaa acaaattcgc tagattttag
    12121 atagaattgc ctgattccac acccttctta gtttttttca atgagatgta tagtttatag
    12181 ttttgcagaa gataaataaa tttcatttaa ctcgcgaata ttaatgagat gcgagtaaca
    12241 ttttaatttg cagatggttg ccatcttgat tggcctcatc tttttgggcc aacaactcac
    12301 gcaagtgggt gtgatgaata tcaacggagc catcttcctc ttcctgacca acatgacctt
    12361 tcaaaacgtc tttgccacga taaatgtaag tcatgtttag aatacatttg catttcaata
    12421 atttactaac tttctaatga atcgattcga tttaggtgtt cacctcagag ctgccagttt
    12481 ttatgaggga ggcccgaagt cgactttatc gctgtgacac atactttctg ggcaaaacga
    12541 ttgccgaatt gccgcttttt ctcacagtgc cactggtctt cacggcgatt gcctatccga
    12601 tgatcggact gcgggccgga gtgctgcact tcttcaactg cctggcgctg gtcactctgg
    12661 tggccaatgt gtcaacgtcc ttcggatatc taatatcctg cgccagctcc tcgacctcga
    12721 tggcgctgtc tgtgggtccg ccggttatca taccattcct gctctttggc ggcttcttct
    12781 tgaactcggg ctcggtgcca gtatacctca aatggttgtc gtacctctca tggttccgtt
    12841 acgccaacga gggtctgctg attaaccaat gggcggacgt ggagccgggc gaaattagct
    12901 gcacatcgtc gaacaccacg tgccccagtt cgggcaaggt catcctggag acgcttaact
    12961 tctccgccgc cgatctgccg ctggactacg tgggtctggc cattctcatc gtgagcttcc
    13021 gggtgctcgc atatctggct ctaagacttc gggcccgacg caaggagtag ccgacatata
    13081 tccgaaataa ctgcttgttt ttttttttta ccattattac catcgtgttt actgtttatt
    13141 gccccctcaa aaagctaatg taattatatt tgtgccaata aaaacaagat atgacctata
    13201 gaatacaagt atttcccctt cgaacatccc cacaagtaga ctttggattt gtcttctaac
    13261 caaaagactt acacacctgc ataccttaca tcaaaaactc gtttatcgct acataaaaca
    13321 ccgggatata ttttttatat acatactttt caaatcgcgc gccctcttca taattcacct
    13381 ccaccacacc acgtttcgta gttgctcttt cgctgtctcc cacccgctct ccgcaacaca
    13441 ttcacctttt gttcgacgac cttggagcga ctgtcgttag ttccgcgcga ttcggttcgc
    13501 tcaaatggtt ccgagtggtt catttcgtct caatagaaat tagtaataaa tatttgtatg
    13561 tacaatttat ttgctccaat atatttgtat atatttccct cacagctata tttattctaa
    13621 tttaatatta tgacttttta aggtaatttt ttgtgacctg ttcggagtga ttagcgttac
    13681 aatttgaact gaaagtgaca tccagtgttt gttccttgtg tagatgcatc tcaaaaaaat
    13741 ggtgggcata atagtgttgt ttatatatat caaaaataac aactataata ataagaatac
    13801 atttaattta gaaaatgctt ggatttcact ggaactaggc tagcataact tcgtataatg
    13861 tatgctatac gaagttatgc tagcggatcc gaattctttt ttgctcacct gtgattgctc
    13921 ctactcaaat acaaaaacat caaattttct gtcaataaag catatttatt tatatttatt
    13981 ttacaggaaa gaattccttt taaagtgtat tttaacctat aatgaaaaac gattaaaaaa
    14041 aatacataaa ataattcgaa aatttttgaa tagcccaggt tgataaaaat tcatttcata
    14101 cgttttataa cttatgcccc taagtatttt ttgaccatag tgtttcaatt ctacattaat
    14161 tttacagagt agaatgaaac gccacctact cagccaagag gcgaaaaggt tagctcgcca
    14221 agcagagagg gcgccagtgc tcactacttt ttataattct caacttcttt ttccagactc
    14281 agttcgtata tatagaccta ttttcaattt aacgtcggat tccgtacttt gcagacggtt
    14341 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
    14401 accgagtcgg tgcttttttt tttggtaccg ggaattggga attgggcaat atttaaatgg
    14461 cggcgcgccg aattcttttt tgctcacctg tgattgctcc tactcaaata caaaaacatc
    14521 aaattttctg tcaataaagc atatttattt atatttattt tacaggaaag aattcctttt
    14581 aaagtgtatt ttaacctata atgaaaaacg attaaaaaaa atacataaaa taattcgaaa
    14641 atttttgaat agcccaggtt gataaaaatt catttcatac gttttataac ttatgcccct
    14701 aagtattttt tgaccatagt gtttcaattc tacattaatt ttacagagta gaatgaaacg
    14761 ccacctactc agccaagagg cgaaaaggtt agctcgccaa gcagagaggg cgccagtgct
    14821 cactactttt tataattctc aacttctttt tccagactca gttcgtatat atagacctat
    14881 tttcaattta acgtcgcctg agtg
//
