LOCUS       sequence-314881-       13250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid supports Cas9 expression only in males, Opie2-dsRed tagged, 
            and can be integrated with pBac or phC31..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13250)
  AUTHORS   Kandul NP, Liu J, Bennett JB, Marshall JM, Akbari OS
  TITLE     A confinable home and rescue gene drive for population modification.
  JOURNAL   Elife. 2021 Mar 5;10. pii: 65939. doi: 10.7554/eLife.65939.
  PUBMED    33666174
REFERENCE   2  (bases 1 to 13250)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2021-03-5"; volume: "10. pii: 65939. doi"; pages: " 
            10.7554/eLife.65939"
COMMENT     Sequence Label: 874V=beta-Tub-NLS-hSpCas9-T2A-GFP, Opie2-dsRed-SV40
FEATURES             Location/Qualifiers
     source          1..13250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..66
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             73..93
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             118..4218
                     /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             118..4218
                     /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             4219..4266
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4267..4320
                     /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(4321..4323,4324..4326,4327..5040)
                     /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(4627..4646)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4974..4995
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(6275..6277,6278..6280,6281..6955)
                     /codon_start=1
                     /product="wild-type DsRed"
                     /label=DsRed1
                     /note="mammalian codon-optimized"
                     /translation="MVRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTV
                     KLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     KALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRH
                     HLFL"
     primer_bind     complement(6458..6478)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     6676..6695
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6870..6893
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    7069..7190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7106..7125)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7160..7179
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7858..7892
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8073..8089)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8073..8089)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8086..8108)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8097..8113
                     /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(8121..8127,8128..8145,8146..8151))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8166..8187
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8304..8321)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8475..9063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8555..8574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9234..10025,10026..10094))
                     /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     9857..9876
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10095..10199)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10267..10285
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(10323..10345)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     10445..10464
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10658..10680
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10672..10689
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10673..10689
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   11393..11455
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    12489..12558
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
ORIGIN
        1 gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac
       61 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
      121 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
      181 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
      241 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
      301 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
      361 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
      421 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
      481 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
      541 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
      601 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
      661 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
      721 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
      781 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
      841 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
      901 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
      961 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     1021 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     1081 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     1141 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     1201 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     1261 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     1321 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     1381 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     1441 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     1501 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     1561 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     1621 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     1681 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     1741 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     1801 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     1861 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     1921 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     1981 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     2041 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     2101 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     2161 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     2221 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     2281 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     2341 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     2401 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     2461 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     2521 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     2581 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     2641 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     2701 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     2761 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     2821 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     2881 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     2941 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     3001 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     3061 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     3121 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     3181 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     3241 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     3301 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     3361 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     3421 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     3481 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     3541 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     3601 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     3661 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     3721 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     3781 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     3841 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     3901 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     3961 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     4021 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     4081 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     4141 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     4201 tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     4261 aaaaaggagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     4321 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4381 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4441 ggcaagctga ccctgaagtt catctgcacc accagcaagc tgcccgtgcc ctggcccacc
     4501 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4561 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4621 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4681 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4741 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4801 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4861 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4921 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4981 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     5041 ttaattaact agaatgaatc gtttttaaaa taacaaatca attgttttat aatattcgta
     5101 cgattctttg attatgtaat aaaatgtgat cattaggaag attacgaaaa atataaaaaa
     5161 tatgagttct gtgtgtataa caaatgctgt aaacgccaca attgtgtttg ttgcaaataa
     5221 acccatgatt atttgattaa aattgttgtt ttctttgttc atagacaata gtgtgttttg
     5281 cctaaacgtg tactgcataa actccatgcg agtgtatagc gagctagtgg ctaacgcttg
     5341 ccccaccaaa gtagattcgt caaaatcctc aatttcatca ccctcctcca agtttaacat
     5401 ttggccgtcg gaattaactt ctaaagatgc cacataatct aataaatgaa atagagattc
     5461 aaacgtggcg tcatcgtccg tttcgaccat ttccgaaaag aactcgggca taaactctat
     5521 gatttctctg gacgtggtgt tgtcgaaact ctcaaagtac gcagtcagga acgtgcgcga
     5581 catgtcgtcg ggaaactcgc gcggaaacat gttgttgtaa ccgaacgggt cccatagcgc
     5641 caaaaccaaa tctgccagcg tcaatagaat gagcacgatg ccgacaatgg agctggcttg
     5701 gatagcgatt cgagttaacg gccggggcgc gcccatactc ggtggcctcc ccaccaccaa
     5761 cttttttgca ctgcaaaaaa acacgctttt gcacgcgggc ccatacatag tacaaactct
     5821 acgtttcgta gactatttta cataaatagt ctacaccgtt gtatacgctc caaatacact
     5881 accacacatt gaaccttttt gcagtgcaaa aaagtacgtg tcggcagtca cgtaggccgg
     5941 ccttatcggg tcgcgtcctg tcacgtacga atcacattat cggaccggac gagtgttgtc
     6001 ttatcgtgac aggacgccag cttcctgtgt tgctaaccgc agccggacgc aactccttat
     6061 cggaacagga cgcgcctcca tatcagccgc gcgttatctc atgcgcgtga ccggacacga
     6121 ggcgcccgtc ccgcttatcg cgcctataaa tacagcccgc aacgatctgg taaacacagt
     6181 tgaacagcat ctgttacagc gacacaacat gagccggtcc aacaacgcca acgcgcccac
     6241 gccatccaac cgccgccgca acctgtctct ggtgatggtg cgctcctcca agaacgtcat
     6301 caaggagttc atgcgcttca aggtgcgcat ggagggcacc gtgaacggcc acgagttcga
     6361 gatcgagggc gagggcgagg gccgccccta cgagggccac aacaccgtga agctgaaggt
     6421 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg
     6481 ctccaaggtg tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc
     6541 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     6601 ccaggactcc tccctgcagg acggctgctt catctacaag gtgaagttca tcggcgtgaa
     6661 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     6721 gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc cacaaggccc tgaagctgaa
     6781 ggacggcggc cactacctgg tggagttcaa gtccatctac atggccaaga agcccgtgca
     6841 gctgcccggc tactactacg tggactccaa gctggacatc acctcccaca acgaggacta
     6901 caccatcgtg gagcagtacg agcgcaccga gggccgccac cacctgttcc tgtaggactc
     6961 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     7021 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     7081 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     7141 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aagcttctag
     7201 gccggccgat ctcggatctg acaatgttca gtgcagagac tcggctacgc ctcgtggact
     7261 ttgaagttga ccaacaatgt ttattcttac ctctaatagt cctctgtggc aaggtcaaga
     7321 ttctgttaga agccaatgaa gaacctggtt gttcaataac attttgttcg tctaatattt
     7381 cactaccgct tgacgttggc tgcacttcat gtacctcatc tataaacgct tcttctgtat
     7441 cgctctggac gtcatcttca cttacgtgat ctgatatttc actgtcagaa tcctcaccaa
     7501 caagctcgtc atcgctttgc agaagagcag agaggatatg ctcatcgtct aaagaactac
     7561 ccattttatt atatattagt cacgatatct ataacaagaa aatatatata taataagtta
     7621 tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc
     7681 acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg
     7741 acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct
     7801 aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat
     7861 gcgtcaattt tacgcagact atctttctag ggttaaaaaa gatttgcgct ttactcgacc
     7921 taaactttaa acacgtcata gaatcttcgt ttgacaaaaa ccacattgtg gccaagctgt
     7981 gtgacgcgac gcgcgctaaa gaatggcaaa ccaagtcgcg cgagcgtcga ctctagagga
     8041 tccccgggta ccgagctcga attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt
     8101 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     8161 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     8221 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     8281 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     8341 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     8401 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     8461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     8521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     8581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     8641 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     8701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     8761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     8821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     8881 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     8941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     9001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     9061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     9121 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     9181 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     9241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     9301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     9361 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     9421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     9481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     9541 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     9601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     9661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     9721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     9781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     9841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     9901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     9961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    10021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    10081 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    10141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    10201 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
    10261 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
    10321 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
    10381 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
    10441 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
    10501 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
    10561 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
    10621 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
    10681 acggccagtg ccaagctttg tttaaaaata taacaaaatt gtgatcccac aaaatgaagt
    10741 ggggcaaaat caaataatta atagtgtccg taaacttgtt ggtcttcaac tttttgagga
    10801 acacgttgga cggcaaatcc gtgactataa cacaagttga tttaataatt ttagccaaca
    10861 cgtcgggctg cgtgtttttt gccgacgcgt ctgtgtacac gttgattaac tggtcgatta
    10921 aactgttgaa ataatttaat ttttggttct tctttaaatc tgtgatgaaa ttttttaaaa
    10981 taactttaaa ttcttcattg gtaaaaaatg ccacgttttg caacttgtga gggtctaata
    11041 tgaggtcaaa ctcagtagga gttttatcca aaaaagaaaa catgattacg tctgtacacg
    11101 aacgcgtatt aacgcagagt gcaaagtata agagggttaa aaaatatatt ttacgcacca
    11161 tatacgcatc gggttgatat cgttaatatg gatcaatttg aacagttgat taacgtgtct
    11221 ctgctcaagt ctttgatcaa aacgcaaatc gacgaaaatg tgtcggacaa tatcaagtcg
    11281 atgagcgaaa aactaaaaag gctagaatac gacaatctca cagacagcgt tgagatatac
    11341 ggtattcacg acagcaggct gaataataaa aaaattagaa actattattt aaccctagaa
    11401 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
    11461 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
    11521 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
    11581 aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta aacattctct
    11641 cttttacaaa aataaactta ttttgtactt taaaaacagt catgttgtat tataaaataa
    11701 gtaattagct taacttatac ataatagaaa caaattatac ttattagtca gtcagaaaca
    11761 actttggcac atatcaatat tatgctctcg acaaataact tttttgcatt ttttgcacga
    11821 tgcatttgcc tttcgcctta ttttagaggg gcagtaagta cagtaagtac gttttttcat
    11881 tactggctct tcagtactgt catctgatgt accaggcact tcatttggca aaatattaga
    11941 gatattatcg cgcaaatatc tcttcaaagt aggagcttct aaacgcttac gcataaacga
    12001 tgacgtcagg ctcatgtaaa ggtttctcat aaattttttg cgactttgaa ccttttctcc
    12061 cttgctactg acattatggc tgtatataat aaaagaattt atgcaggcaa tgtttatcat
    12121 tccgtacaat aatgccatag gccacctatt cgtcttccta ctgcaggtca tcacagaaca
    12181 catttggtct agcgtgtcca ctccgccttt agtttgatta taatacataa ccatttgcgg
    12241 tttaccggta ctttcgttga tagaagcatc ctcatcacaa gatgataata agtataccat
    12301 cttagctggc ttcggtttat atgagacgag agtaaggggt ccgtcaaaac aaaacatcga
    12361 tgttcccact ggcctggagc gactgttttt cagtacttcc ggtatctcgc gtttgtttga
    12421 tcgcacggtt cccacaatgg ttaattcgag ctcgcccggg gtcctaggtc gacgatgtag
    12481 gtcacggtct cgaagccgcg gtgcgggtgc cagggcgtgc ccttgggctc cccgggcgcg
    12541 tactccacct cacccatctg gtccatcatg atgaacgggt cgaggtggcg gtagttgatc
    12601 ccggcgaacg cgcggcgcac cgggaagccc tcgccctcga aaccgctggg cgcggtggtc
    12661 acggtgagca cgggacgtgc gacggcgtcg gcgggtgcgg atacgcgggg cagcgtcagc
    12721 gggttctcga cggtcacggc gggcatgtcg acgcggccgc tcgacccatc ccattataca
    12781 cccatataaa catttacaaa ggagtaaaat ccaggacatc catgtcaata tcaatcgtat
    12841 catctggtcg gtagccttgg aatcctctat tgcttccaag gcaccgccaa atccatccca
    12901 tctcgaattt tagccgtata ttcgtttatc tatgtaagta ctattaaagt ttgtgctcaa
    12961 aacggagaac tgagttttct gaaatcgggg tgtgtgaaat gtgtcgaagt cggaaatcgt
    13021 agtagcctat ttgtgaacat tcggtgtagt aatccaagcc aggttcagtt cacctcagta
    13081 tcagctagca cgtacacgac taaaatctaa acctgaaaaa ttatacgttt aaatattcag
    13141 tcttttgccg atttttgccc cactcagact gttttaaaag ctcgattttt ttttgtacca
    13201 ttttttcggt gtgaaaaagg ggccctaact ttactatcaa actcgagatg
//
