LOCUS       sequence-292053-       14921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The attB plasmid with the mini-white harboring two U6.3-gRNAs 
            targeting Dmel sxl 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 14921)
  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 14921)
  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: 1098A=TI-pgSIT[sxl,bTub,Hasp70Bb-Cas9]
FEATURES             Location/Qualifiers
     source          1..14921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward 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"
     gene            666..4802
                     /gene="white"
                     /label=mini-white
                     /note="This modified version of the white gene lacks part 
                     of the first intron."
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1145..1216,1618..1891,1966..2620,2682..2997,3218..3349,
                     3420..4034)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This modified version of the white gene lacks part 
                     of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4809..4842
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4861..5281
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5302..5377
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5440..5860
                     /label=dU6-3 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-3 
                     snRNA (Port et al., 2014)"
     misc_RNA        5881..5956
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6430..6668
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     6615..6636
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             6682..6747
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6754..6774
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6799..10899
                     /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             6799..10899
                     /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             10900..10947
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10948..11001
                     /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(11002..11004,11005..11007,11008..11721)
                     /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(11308..11327)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     11655..11676
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    12445..12514
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(12733..12751)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12772..12788)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12772..12788)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12785..12807)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12796..12812
                     /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(12820..12826,12827..12844,12845..12850))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12865..12886
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13003..13020)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13174..13762)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13254..13273)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13933..14724,14725..14793))
                     /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     14556..14575
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14794..14898)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt ttaaatttac
      781 ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact caaaatgaca
      841 ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat cgccgtctct
      901 ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta gcgtcaatgt
      961 ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg tttacgccat
     1021 caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg tttgtttttt
     1081 gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt gaaaaatccc
     1141 ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac acccatctgc
     1201 cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac ttgatctata
     1261 ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc acaaaaatcc
     1321 caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt agctgttctc
     1381 gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg cattcatttt
     1441 cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg atcctagatg
     1501 cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt actttttgtt
     1561 cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc attgcagggt
     1621 gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc caaaaactac
     1681 ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg ccaactagcc
     1741 gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa tcagccgggc
     1801 tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga gcgacacata
     1861 ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt cggctgatct
     1921 gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc ggcgtggcct
     1981 atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg accctgctga
     2041 atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg atgcgactgc
     2101 tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat gtccagcagg
     2161 atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccag gccatggtgc
     2221 ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag gtgatccagg
     2281 agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg gtgaaaggtc
     2341 tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggcactaacc gatccgccgc
     2401 ttctgatctg cgatgagccc acctccggac tggactcatt taccgcccac agcgtcgtcc
     2461 aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt catcagccgt
     2521 cttccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc agggtagctt
     2581 tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga tgtgtttatt
     2641 aagggtatct agcattacat tacatctcaa ctcctatcca gcgtgggtgc ccagtgtcct
     2701 accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc cggacgggag
     2761 atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgctattag caaagtagcc
     2821 cgggatatgg agcagttgtt ggccaccaaa aatttggaga agccactgga gcagccggag
     2881 aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct gtggcgatcc
     2941 tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca gacaacggtg
     3001 agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca aattcgctag
     3061 attttagtta gaattgcctg attccacacc cttcttagtt tttttcaatg agatgtatag
     3121 tttatagttt tgcagaaaat aaataaattt catttaactc gcgaacatgt tgaagatatg
     3181 aatattaatg agatgcgagt aacattttaa tttgcagatg gttgccatct tgattggcct
     3241 catctttttg ggccaacaac tcacgcaagt gggcgtgatg aatatcaacg gagccatctt
     3301 cctcttcctg accaacatga cctttcaaaa cgtctttgcc acgataaatg taagtcttgt
     3361 ttagaataca tttgcatatt aataatttac taactttcta atgaatcgat tcgatttagg
     3421 tgttcacctc agagctgcca gtttttatga gggaggcccg aagtcgactt tatcgctgtg
     3481 acacatactt tctgggcaaa acgattgccg aattaccgct ttttctcaca gtgccactgg
     3541 tcttcacggc gattgcctat ccgatgatcg gactgcgggc cggagtgctg cacttcttca
     3601 actgcctggc gctggtcact ctggtggcca atgtgtcaac gtccttcgga tatctaatat
     3661 cctgcgccag ctcctcgacc tcgatggcgc tgtctgtggg tccgccggtt atcataccat
     3721 tcctgctctt tggcggcttc ttcttgaact cgggctcggt gccagtatac ctcaaatggt
     3781 tgtcgtacct ctcatggttc cgttacgcca acgagggtct gctgattaac caatgggcgg
     3841 acgtggagcc gggcgaaatt agctgcacat cgtcgaacac cacgtgcccc agttcgggca
     3901 aggtcatcct ggagacgctt aacttctccg ccgccgatct gccgctggac tacgtgggtc
     3961 tggccattct catcgtgagc ttccgggtgc tcgcatatct ggctctaaga cttcgggccc
     4021 gacgcaagga gtagccgaca tatatccgaa ataactgctt gttttttttt tttaccatta
     4081 ttaccatcgt gtttactgtt tattgccccc tcaaaaagct aatgtaatta tatttgtgcc
     4141 aataaaaaca agatatgacc tatagaatac aagtatttcc ccttcgaaca tccccacaag
     4201 tagactttgg atttgtcttc taaccaaaag acttacacac ctgcatacct tacatcaaaa
     4261 actcgtttat cgctacataa aacaccggga tatatttttt atatacatac ttttcaaatc
     4321 gcgcgccctc ttcataattc acctccacca caccacgttt cgtagttgct ctttcgctgt
     4381 ctcccacccg ctctccgcaa cacattcacc ttttgttcga cgaccttgga gcgactgtcg
     4441 ttagttccgc gcgattcggt tcgctcaaat ggttccgagt ggttcatttc gtctcaatag
     4501 aaattagtaa taaatatttg tatgtacaat ttatttgctc caatatattt gtatatattt
     4561 ccctcacagc tatatttatt ctaatttaat attatgactt tttaaggtaa ttttttgtga
     4621 cctgttcgga gtgattagcg ttacaatttg aactgaaagt gacatccagt gtttgttcct
     4681 tgtgtagatg catctcaaaa aaatggtggg cataatagtg ttgtttatat atatcaaaaa
     4741 taacaactat aataataaga atacatttaa tttagaaaat gcttggattt cactggaact
     4801 aggctagcat aacttcgtat aatgtatgct atacgaagtt atgctagcgg atccgaattc
     4861 ttttttgctc acctgtgatt gctcctactc aaatacaaaa acatcaaatt ttctgtcaat
     4921 aaagcatatt tatttatatt tattttacag gaaagaattc cttttaaagt gtattttaac
     4981 ctataatgaa aaacgattaa aaaaaataca taaaataatt cgaaaatttt tgaatagccc
     5041 aggttgataa aaattcattt catacgtttt ataacttatg cccctaagta ttttttgacc
     5101 atagtgtttc aattctacat taattttaca gagtagaatg aaacgccacc tactcagcca
     5161 agaggcgaaa aggttagctc gccaagcaga gagggcgcca gtgctcacta ctttttataa
     5221 ttctcaactt ctttttccag actcagttcg tatatataga cctattttca atttaacgtc
     5281 ggattgtcaa ctacttgccc cgttttagag ctagaaatag caagttaaaa taaggctagt
     5341 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttttttggt accgggaatt
     5401 gggaattggg caatatttaa atggcggcgc gccgaattct tttttgctca cctgtgattg
     5461 ctcctactca aatacaaaaa catcaaattt tctgtcaata aagcatattt atttatattt
     5521 attttacagg aaagaattcc ttttaaagtg tattttaacc tataatgaaa aacgattaaa
     5581 aaaaatacat aaaataattc gaaaattttt gaatagccca ggttgataaa aattcatttc
     5641 atacgtttta taacttatgc ccctaagtat tttttgacca tagtgtttca attctacatt
     5701 aattttacag agtagaatga aacgccacct actcagccaa gaggcgaaaa ggttagctcg
     5761 ccaagcagag agggcgccag tgctcactac tttttataat tctcaacttc tttttccaga
     5821 ctcagttcgt atatatagac ctattttcaa tttaacgtcg cctgagtgtg catcagctgg
     5881 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     5941 ggcaccgagt cggtgctttt ttttttgcct acctggagcc tgagagttgt tcaataaaat
     6001 aaaaatgttt cgtttttttg ctttcgccag tatttattat ttttcatcaa tatgtattca
     6061 atttggtatg tatttagtaa ttgtaatata tagacaatgg ttttccgttg acgtacatac
     6121 atctgacgtg tgtttattta gacataatag ttatgttttc acatcttttt aatgttcgct
     6181 taatgcgtat gcattctaga caattgtgct cggcaacagt atatttgtgg tgtgccaacc
     6241 aacaacctgc aggagctcca gcttttgtct cgagtcgaga aatttctctg gccgttattc
     6301 gttattctct cttttctttt tgggtctctc cctctctgca ctaatgctct ctcactctgt
     6361 cacacagtaa acggcatact gctctcgttg gttcgagaga gcgcgcctcg aatgttcgcg
     6421 aaaagagcgc cggagtataa atagaggcgc ttcgtctacg gagcgacaat tcaattcaaa
     6481 caagcaaagt gaacacgtcg ctaagcgaaa gctaagcaaa taaacaagcg cagctgaaca
     6541 agctaaacaa tctgcagtaa agtgcaagtt aaagtgaatc aattaaaagt aaccagcaac
     6601 caagtaaatc aactgcaact actgaaatct gccaagaagt aattattgaa tacaagaaga
     6661 gaactctgtt taaacaaaat ggactataag gaccacgacg gagactacaa ggatcatgat
     6721 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
     6781 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct
     6841 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     6901 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     6961 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     7021 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     7081 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     7141 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     7201 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     7261 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     7321 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     7381 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     7441 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     7501 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     7561 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     7621 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     7681 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     7741 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     7801 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     7861 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     7921 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     7981 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     8041 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     8101 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     8161 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     8221 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     8281 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     8341 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     8401 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     8461 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     8521 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     8581 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     8641 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     8701 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     8761 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     8821 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     8881 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     8941 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     9001 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     9061 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     9121 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     9181 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     9241 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     9301 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     9361 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     9421 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     9481 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     9541 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     9601 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     9661 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     9721 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     9781 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     9841 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     9901 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     9961 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
    10021 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
    10081 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
    10141 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
    10201 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
    10261 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
    10321 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
    10381 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
    10441 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
    10501 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
    10561 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
    10621 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
    10681 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
    10741 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
    10801 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
    10861 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa
    10921 aaggccggcc aggcaaaaaa gaaaaaggag ggcagaggaa gtcttctaac atgcggtgac
    10981 gtggaggaga atcccggccc tatggtgagc aagggcgagg agctgttcac cggggtggtg
    11041 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
    11101 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccagcaag
    11161 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
    11221 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
    11281 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
    11341 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
    11401 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
    11461 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
    11521 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
    11581 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
    11641 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
    11701 atggacgagc tgtacaagta attaattaac tagaatgaat cgtttttaaa ataacaaatc
    11761 aattgtttta taatattcgt acgattcttt gattatgtaa taaaatgtga tcattaggaa
    11821 gattacgaaa aatataaaaa atatgagttc tgtgtgtata acaaatgctg taaacgccac
    11881 aattgtgttt gttgcaaata aacccatgat tatttgatta aaattgttgt tttctttgtt
    11941 catagacaat agtgtgtttt gcctaaacgt gtactgcata aactccatgc gagtgtatag
    12001 cgagctagtg gctaacgctt gccccaccaa agtagattcg tcaaaatcct caatttcatc
    12061 accctcctcc aagtttaaca tttggccgtc ggaattaact tctaaagatg ccacataatc
    12121 taataaatga aatagagatt caaacgtggc gtcatcgtcc gtttcgacca tttccgaaaa
    12181 gaactcgggc ataaactcta tgatttctct ggacgtggtg ttgtcgaaac tctcaaagta
    12241 cgcagtcagg aacgtgcgcg acatgtcgtc gggaaactcg cgcggaaaca tgttgttgta
    12301 accgaacggg tcccatagcg ccaaaaccaa atctgccagc gtcaatagaa tgagcacgat
    12361 gccgacaatg gagctggctt ggatagcgat tcgagttaac ggccggaggc ctgatccact
    12421 agtgtcgacg atgtaggtca cggtctcgaa gccgcggtgc gggtgccagg gcgtgccctt
    12481 gggctccccg ggcgcgtact ccacctcacc catctggtcc atcatgatga acgggtcgag
    12541 gtggcggtag ttgatcccgg cgaacgcgcg gcgcaccggg aagccctcgc cctcgaaacc
    12601 gctgggcgcg gtggtcacgg tgagcacggg acgtgcgacg gcgtcggcgg gtgcggatac
    12661 gcggggcagc gtcagcgggt tctcgacggt cacggcgggc atgtcgacac tagttctagc
    12721 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
    12781 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    12841 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    12901 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
    12961 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
    13021 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
    13081 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
    13141 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    13201 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    13261 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    13321 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    13381 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    13441 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    13501 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    13561 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
    13621 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    13681 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    13741 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    13801 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    13861 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    13921 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    13981 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    14041 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    14101 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    14161 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    14221 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    14281 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    14341 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    14401 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    14461 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    14521 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    14581 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    14641 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    14701 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    14761 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    14821 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    14881 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
