LOCUS       sequence-379952-       10296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid encodes S. pyogenes Cas9 and eGFP separated by a T2A 
            peptide under an Aedes aegypti polyubiquitin promoter. It also 
            expresses a sgRNA scaffold under a Culex quinquefasciatus U6 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10296)
  AUTHORS   Torres TZB, Prince BC, Robison A, Ruckert C
  TITLE     Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile 
            Virus Mosquito Vector, Culex quinquefasciatus.
  JOURNAL   Insects. 2022 Sep 19;13(9):856. doi: 10.3390/insects13090856.
  PUBMED    36135557
REFERENCE   2  (bases 1 to 10296)
  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: "Insects."; 
            date: "2022-09-19"; volume: "13(9):856. doi"; pages: " 
            10.3390/insects13090856"
COMMENT     Sequence Label: pUb-Cas9-EGFP_cU6:6-sgRNA
FEATURES             Location/Qualifiers
     source          1..10296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(727..1518,1519..1587))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1350..1369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1588..1692)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      3268..3277
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3280..3300
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3325..7425
                     /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             3325..7425
                     /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             7426..7473
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7489..7542
                     /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"
     regulatory      7567..7576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7573..7575,7576..7578,7579..8289)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     8226..8247
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             8293..8346
                     /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             8353..9156
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(8416..8435)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9026..9045
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    9164..9298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9188..9207)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     9242..9261
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9299..9315
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9299..9315
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        9979..10054
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(join(10264..10296,1..556))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
       61 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
      121 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
      181 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
      241 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
      301 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
      361 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
      421 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
      481 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
      541 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
      601 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
      661 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
      721 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      781 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      841 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
      901 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      961 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1021 gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     1081 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1141 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     1201 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     1261 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     1321 gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa
     1381 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     1441 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     1501 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     1561 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     1621 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1681 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     1741 catgacatta acctataaaa ataggcgtat cacgaggcgg ctatctttac atgtagcttg
     1801 tgcattgaat ccaattataa tttgccttgg caccagctga gccagacaag aaagaaagct
     1861 tcccagaagt atatcgattt agaagggttg acgtcacttg ctgactgcac taatacagca
     1921 aatgatgcaa ttagaatgat tcaagtgaaa ttcccaaatt actgattttt ctctggattt
     1981 ggttatcaga ttacattcga agctaagatt agctaccgaa attgtcgatc aaatcaggaa
     2041 atcctttctc tatcgaaaaa ggcattcgca catcttcctc tctatgccat atacacgaag
     2101 ggtaggtaca ttgacgtctt tgccagaagt tgaactgcat cgttcaaggt acagaatgaa
     2161 cgactaacag acacaagcac gttttgctgt ccattcagac acagggatgg tacccatatt
     2221 cgatcgatat agagccatcc aaccgaacag aggtatatgt atgaatgtat tgctgaaatt
     2281 ttctagaagt acaaccacca ctacgacagt gtctataaaa cgcccctgca aaggcgaaac
     2341 cagctcaatc gaatacgttt cctagtggag tgaacattac gcggcccaag taagcagtgc
     2401 cagtgcaagt gaagtgaagt ctctagtgaa aaagagtgat ccaattagcc agaggagaaa
     2461 atttcagagt gaacaaagct ttattcaaag gacaattact attaaattgg tgaaagtgca
     2521 tttcggtgaa gggaatcttc tagtgaaggt aggtaaatta attgatgaaa ttatagctat
     2581 gagcgaaaac tagtttggtg aatgattcct ttgtctttga atgagcaaac tattttccaa
     2641 gatggcgact attgagcttt gagtgattag tgaaaatttg caacgcagtt tcatcatcat
     2701 tgataaaacc caattgtgat tcacagcgat aatcatattt cgttgaatca tcgctactaa
     2761 ttgaattaaa tttctagaat aataagaata acgtatttgc tccgtcacat atctaaaata
     2821 aatattttga tggtaattac ccattaaggt aatattaaca catatcgaga aaaaccttga
     2881 ggaaatcgtg aaaacttgaa gatacgcaat ttcaaaacta cgtagttcaa agtcgaaaac
     2941 aagttaattt ttcacttaaa agtagggcgt tgttgtgacg tcatcacctt caagtgtata
     3001 tttttcactt ggcctgcgac tgcaaacgca gacaaagcaa aacaagttta aaacctgtcg
     3061 tgtcgtgctc gaagccaaag gcaatgaatc aatatcaaat gagagtttgc atttcacaac
     3121 caattactca agcgtttcct cgtttctttt tctgctcaac agagatttca acaccaagct
     3181 cgataagctt gttcgaatct cgagtgcgcg cttccggagg tatacaccta ggcggtacca
     3241 ctgcagtgaa ttcggagctc taccggtgcc accatggccc caaagaagaa gcggaaggtc
     3301 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     3361 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     3421 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     3481 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     3541 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     3601 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     3661 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     3721 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     3781 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     3841 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     3901 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     3961 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     4021 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     4081 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     4141 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     4201 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     4261 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     4321 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     4381 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     4441 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     4501 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     4561 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     4621 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     4681 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     4741 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     4801 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     4861 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     4921 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     4981 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     5041 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     5101 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     5161 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     5221 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     5281 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     5341 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     5401 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     5461 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     5521 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     5581 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     5641 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     5701 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     5761 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     5821 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     5881 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     5941 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     6001 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     6061 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     6121 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     6181 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     6241 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     6301 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     6361 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     6421 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     6481 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     6541 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     6601 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     6661 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     6721 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     6781 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     6841 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     6901 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     6961 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     7021 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     7081 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     7141 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     7201 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     7261 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     7321 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     7381 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc
     7441 acgaaaaagg ccggccaggc aaaaaagaaa aagggatcag gcaagcttga gggcagagga
     7501 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctgctagcgg tagcggcagc
     7561 ggtagcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     7621 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     7681 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     7741 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     7801 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     7861 cgcaccatct tcttcaaggg cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     7921 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     7981 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     8041 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     8101 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     8161 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     8221 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     8281 ctgtacaagc ttgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc
     8341 ggccctgcta gcatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc
     8401 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     8461 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     8521 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     8581 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     8641 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     8701 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     8761 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     8821 gatcaggatg atctggacga agaacatcag gggctcgcgc cagccgaact gttcgccagg
     8881 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     8941 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     9001 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agaacttggc
     9061 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     9121 atcgccttct atcgccttct tgacgagttc ttctgactcg agggatccag acatgataag
     9181 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     9241 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttca
     9301 ggaaacagct atgaccatga ttacgccaag cttggtaccg agctcggatc cactagtaac
     9361 ggccgccagt gtgctggaat tcgcccttca aaatctacga agataataag gtctactatt
     9421 acatttaata aatattctta tttaatttga gctgtgtaaa attagtgcgt ttatccgggg
     9481 aatgtttata ttttatccga ggatttcccg aaattgatta aaaaaatcaa atctcttaaa
     9541 aaattagaaa caacctgtta aaaataagct tatagaaaaa atgtaaagtg ttgctacaag
     9601 gacaatcaat taatcatcat aacgaaaact tttaggatct tgaaaagcac gtcccgagat
     9661 gggaaagtgt gtttcatttc caggcgatcc ttttcgcttc acgaaatttt tgattgctac
     9721 ctcgtttaga gccctaagac aaaatacttc ttcaaaaaaa agcgaactcg aacccaaact
     9781 ttgtactact atgctaaaat gtctgcatgc atgggatgtc gcgcaaacga gtccaataag
     9841 aaataaaact gctcgaattc aacgtgacgc tacccgcaat gctctaccct taactttata
     9901 catcgctaga accgcgttcg cattcgctaa ctatataagc actttcccgg ccctacattc
     9961 ggaagagcga gctcttccgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
    10021 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt agatctggaa aaatgatgtg
    10081 acagtggaaa tgagaagtag gtgcatctgc aaagatttga ttcagagttg atgccattca
    10141 tgattttttt ttagaagtat catttgtgtt tttagaatat agaattaatt cttgaagacg
    10201 aaagggcctc gtgatacgcc tatttttata ggttaaccgt aaaaaggccg cgttgctggc
    10261 gtttttccat aggctccgcc cccctgacga gcatca
//
