LOCUS       sequence-301651-       13622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The attB plasmid harboring the Hsp70Bb-Cas9-T2A-eGFP-p10 and a 
            mini-white marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13622)
  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 13622)
  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: 1137B=Hsp70Bb-Cas9
FEATURES             Location/Qualifiers
     source          1..13622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    565..598
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        904..1142
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     1089..1110
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             1156..1221
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1228..1248
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1273..5373
                     /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             1273..5373
                     /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             5374..5421
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5422..5475
                     /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(5476..5478,5479..5481,5482..6195)
                     /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(5782..5801)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6129..6150
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    6919..6988
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(7207..7225)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7246..7262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7246..7262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7259..7281)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7270..7286
                     /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(7294..7300,7301..7318,7319..7324))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7339..7360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7477..7494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7648..8236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7728..7747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8407..9198,9199..9267))
                     /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     9030..9049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9268..9372)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(9398..9853)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9535..9556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9747..9766
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9980..10002
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9994..10011
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9995..10011
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10018..10037
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10018..10036
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10070..10088)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(10540..10611,11013..11286,11361..12015,12077..12392,
                     12596..12727,12798..13412)
                     /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"
ORIGIN
        1 ataccttaca tcaaaaactc gtttatcgct acataaaaca ccgggatata ttttttatat
       61 acatactttt caaatcgcgc gccctcttca taattcacct ccaccacacc acgtttcgta
      121 gttgctcttt cgctgtctcc cacccgctct ccgcaacaca ttcacctttt gttcgacgac
      181 cttggagcga ctgtcgttag ttccgcgcga ttcggttcgc tcaaatggtt ccgagtggtt
      241 catttcgtct caatagaaat tagtaataaa tatttgtatg tacaatttat ttgctccaat
      301 atatttgtat atatttccct cacagctata tttattctaa tttaatatta tgacttttta
      361 aggtaatttt ttgtgacctg ttcggagtga ttagcgttac aatttgaact gaaagtgaca
      421 tccagtgttt gttccttgtg tagatgcatc tcaaaaaaat ggtgggcata atagtgttgt
      481 ttatatatat caaaaataac aactataata ataagaatac atttaattta gaaaatgctt
      541 ggatttcact ggaactaggc tagcataact tcgtataatg tatgctatac gaagttatgc
      601 tagcggatcc ctccgttgta tgttgtagcg tctgtgaagc gtcctactcc taagcatgac
      661 ttgaagcaca acttacctaa gccatgacag cctcctgtcg tgtatcactg acagctcgac
      721 tctctacgat atctaagctg atctcgagtc gagaaatttc tctggccgtt attcgttatt
      781 ctctcttttc tttttgggtc tctccctctc tgcactaatg ctctctcact ctgtcacaca
      841 gtaaacggca tactgctctc gttggttcga gagagcgcgc ctcgaatgtt cgcgaaaaga
      901 gcgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt caaacaagca
      961 aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg aacaagctaa
     1021 acaatctgca gtaaagtgca agttaaagtg aatcaattaa aagtaaccag caaccaagta
     1081 aatcaactgc aactactgaa atctgccaag aagtaattat tgaatacaag aagagaactc
     1141 tgtttaaaca aaatggacta taaggaccac gacggagact acaaggatca tgatattgat
     1201 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     1261 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     1321 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     1381 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     1441 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     1501 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     1561 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     1621 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     1681 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     1741 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     1801 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     1861 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1921 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1981 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     2041 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     2101 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     2161 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     2221 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     2281 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     2341 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     2401 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     2461 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     2521 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     2581 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     2641 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     2701 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     2761 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     2821 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     2881 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2941 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     3001 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     3061 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     3121 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     3181 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     3241 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     3301 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     3361 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     3421 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     3481 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     3541 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     3601 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     3661 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     3721 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     3781 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     3841 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3901 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3961 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     4021 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     4081 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     4141 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     4201 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     4261 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     4321 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     4381 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     4441 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     4501 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     4561 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     4621 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     4681 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     4741 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     4801 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     4861 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4921 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4981 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     5041 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     5101 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     5161 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     5221 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     5281 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     5341 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     5401 ggccaggcaa aaaagaaaaa ggagggcaga ggaagtcttc taacatgcgg tgacgtggag
     5461 gagaatcccg gccctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     5521 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     5581 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccag caagctgccc
     5641 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     5701 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     5761 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     5821 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     5881 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     5941 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     6001 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     6061 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     6121 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     6181 gagctgtaca agtaattaat taactagaat gaatcgtttt taaaataaca aatcaattgt
     6241 tttataatat tcgtacgatt ctttgattat gtaataaaat gtgatcatta ggaagattac
     6301 gaaaaatata aaaaatatga gttctgtgtg tataacaaat gctgtaaacg ccacaattgt
     6361 gtttgttgca aataaaccca tgattatttg attaaaattg ttgttttctt tgttcataga
     6421 caatagtgtg ttttgcctaa acgtgtactg cataaactcc atgcgagtgt atagcgagct
     6481 agtggctaac gcttgcccca ccaaagtaga ttcgtcaaaa tcctcaattt catcaccctc
     6541 ctccaagttt aacatttggc cgtcggaatt aacttctaaa gatgccacat aatctaataa
     6601 atgaaataga gattcaaacg tggcgtcatc gtccgtttcg accatttccg aaaagaactc
     6661 gggcataaac tctatgattt ctctggacgt ggtgttgtcg aaactctcaa agtacgcagt
     6721 caggaacgtg cgcgacatgt cgtcgggaaa ctcgcgcgga aacatgttgt tgtaaccgaa
     6781 cgggtcccat agcgccaaaa ccaaatctgc cagcgtcaat agaatgagca cgatgccgac
     6841 aatggagctg gcttggatag cgattcgagt taacggccgg aggcctgatc cactagtgtc
     6901 gacgatgtag gtcacggtct cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc
     6961 cccgggcgcg tactccacct cacccatctg gtccatcatg atgaacgggt cgaggtggcg
     7021 gtagttgatc ccggcgaacg cgcggcgcac cgggaagccc tcgccctcga aaccgctggg
     7081 cgcggtggtc acggtgagca cgggacgtgc gacggcgtcg gcgggtgcgg atacgcgggg
     7141 cagcgtcagc gggttctcga cggtcacggc gggcatgtcg acactagttc tagccagctt
     7201 ttgttccctt tagtgagggt taatttcgag cttggcgtaa tcatggtcat agctgtttcc
     7261 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     7321 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     7381 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     7441 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     7501 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     7561 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     7621 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     7681 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     7741 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     7801 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     7861 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     7921 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     7981 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     8041 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     8101 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     8161 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     8221 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     8281 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     8341 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     8401 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     8461 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     8521 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     8581 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     8641 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     8701 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     8761 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     8821 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     8881 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     8941 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     9001 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     9061 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     9121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     9181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     9241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     9301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     9361 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     9421 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     9481 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     9541 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     9601 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     9661 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     9721 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     9781 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     9841 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     9901 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     9961 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa
    10021 tacgactcac tatagggcga attgggtacg taccgggccc ctagtatgta tgtaagttaa
    10081 taaaacccat ttttgcggaa agtagataaa aaaaacattt ttttttttta ctgcactgga
    10141 tatcattgaa cttatctgat cagttttaaa tttacttcga tccaagggta tttgatgtac
    10201 caggttcttt cgattacctc tcactcaaaa tgacattcca ctcaaagtca gcgctgtttg
    10261 cctccttctc tgtccacaga aatatcgccg tctctttcgc cgctgcgtcc gctatctctt
    10321 tcgccaccgt ttgtagcgtt acgtagcgtc aatgtccgcc ttcagttgca ttttgtcagc
    10381 ggtttcgtga cgaagctcca agcggtttac gccatcaatt aaacacaaag tgctgtgcca
    10441 aaactcctct cgcttcttat ttttgtttgt tttttgagtg attggggtgg tgattggttt
    10501 tgggtgggta agcaggggaa agtgtgaaaa atcccggcaa tgggccaaga ggatcaggag
    10561 ctattaattc gcggaggcag caaacaccca tctgccgagc atctgaacaa tgtgagtagt
    10621 acatgtgcat acatcttaag ttcacttgat ctataggaac tgcgattgca acatcaaatt
    10681 gtctgcggcg tgagaactgc gacccacaaa aatcccaaac cgcaattgca caaacaaata
    10741 gtgacacgaa acagattatt ctggtagctg ttctcgctat ataagacaat ttttgagatc
    10801 atatcatgat caagacatct aaaggcattc attttcgact atattctttt ttacaaaaaa
    10861 tataacaacc agatatttta agctgatcct agatgcacaa aaaataaata aaagtataaa
    10921 cctacttcgt aggatacttc ggggtacttt ttgttcgggg ttagatgagc ataacgcttg
    10981 tagttgatat ttgagatccc ctatcattgc agggtgacag cggagcggct tcgcagagct
    11041 gcattaacca gggcttcggg caggccaaaa actacggcac gctccggcca cccagtccgc
    11101 cggaggactc cggttcaggg agcggccaac tagccgagaa cctcacctat gcctggcaca
    11161 atatggacat ctttggggcg gtcaatcagc cgggctccgg atggcggcag ctggtcaacc
    11221 ggacacgcgg actattctgc aacgagcgac acataccggc gcccaggaaa catttgctca
    11281 agaacggtga gtttctattc gcagtcggct gatctgtgtg aaatcttaat aaagggtcca
    11341 attaccaatt tgaaactcag tttgcggcgt ggcctatccg ggcgaacttt tggccgtgat
    11401 gggcagttcc ggtgccggaa agacgaccct gctgaatgcc cttgcctttc gatcgccgca
    11461 gggcatccaa gtatcgccat ccgggatgcg actgctcaat ggccaacctg tggacgccaa
    11521 ggagatgcag gccaggtgcg cctatgtcca gcaggatgac ctctttatcg gctccctaac
    11581 ggccagggaa cacctgattt tccaagccat ggtgcggatg ccacgacatc tgacctatcg
    11641 gcagcgagtg gcccgcgtgg atcaggtgat ccaggagctt tcgctcagca aatgtcagca
    11701 cacgatcatc ggtgtgcccg gcagggtgaa aggtctgtcc ggcggagaaa ggaagcgtct
    11761 ggcattcgcc tccgaggctc taaccgatcc gccgcttctg atctgcgatg agcccacctc
    11821 cggactggac tcctttaccg cccacagcgt cgtccaggtg ctgaagaagc tgtcgcagaa
    11881 gggcaagacc gtcatcctga ccattcatca gccgtcttcc gagctgtttg agctctttga
    11941 caagatcctt ctgatggccg agggcagggt agctttcttg ggcactccca gcgaagccgt
    12001 cgacttcttt tcctagtgag ttcgatgtgt ttattaaggg tatctagtat tacataacat
    12061 ctcaactcct atccagcgtg ggtgcccagt gtcctaccaa ctacaatccg gcggactttt
    12121 acgtacaggt gttggccgtt gtgcccggac gggagatcga gtcccgtgat cggatcgcca
    12181 agatatgcga caattttgcc attagcaaag tagcccggga tatggagcag ttgttggcca
    12241 ccaaaaatct ggagaagcca ctggagcagc cggagaatgg gtacacctac aaggccacct
    12301 ggttcatgca gttccgggcg gtcctgtggc gatcctggct gtcggtgctc aaggaaccac
    12361 tcctcgtaaa agtgcgactt attcagacaa cggtgagtgg ttccagtgga aacaaatgat
    12421 ataacgctta caattcttgg aaacaaattc gctagatttt agatagaatt gcctgattcc
    12481 acacccttct tagttttttt caatgagatg tatagtttat agttttgcag aagataaata
    12541 aatttcattt aactcgcgaa tattaatgag atgcgagtaa cattttaatt tgcagatggt
    12601 tgccatcttg attggcctca tctttttggg ccaacaactc acgcaagtgg gtgtgatgaa
    12661 tatcaacgga gccatcttcc tcttcctgac caacatgacc tttcaaaacg tctttgccac
    12721 gataaatgta agtcatgttt agaatacatt tgcatttcaa taatttacta actttctaat
    12781 gaatcgattc gatttaggtg ttcacctcag agctgccagt ttttatgagg gaggcccgaa
    12841 gtcgacttta tcgctgtgac acatactttc tgggcaaaac gattgccgaa ttgccgcttt
    12901 ttctcacagt gccactggtc ttcacggcga ttgcctatcc gatgatcgga ctgcgggccg
    12961 gagtgctgca cttcttcaac tgcctggcgc tggtcactct ggtggccaat gtgtcaacgt
    13021 ccttcggata tctaatatcc tgcgccagct cctcgacctc gatggcgctg tctgtgggtc
    13081 cgccggttat cataccattc ctgctctttg gcggcttctt cttgaactcg ggctcggtgc
    13141 cagtatacct caaatggttg tcgtacctct catggttccg ttacgccaac gagggtctgc
    13201 tgattaacca atgggcggac gtggagccgg gcgaaattag ctgcacatcg tcgaacacca
    13261 cgtgccccag ttcgggcaag gtcatcctgg agacgcttaa cttctccgcc gccgatctgc
    13321 cgctggacta cgtgggtctg gccattctca tcgtgagctt ccgggtgctc gcatatctgg
    13381 ctctaagact tcgggcccga cgcaaggagt agccgacata tatccgaaat aactgcttgt
    13441 tttttttttt taccattatt accatcgtgt ttactgttta ttgccccctc aaaaagctaa
    13501 tgtaattata tttgtgccaa taaaaacaag atatgaccta tagaatacaa gtatttcccc
    13561 ttcgaacatc cccacaagta gactttggat ttgtcttcta accaaaagac ttacacacct
    13621 gc
//
