LOCUS       sequence-469000-       17021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty Cas9 vector for multiplex gene editing in fungi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17021)
  AUTHORS   Yuan G, Deng S, Czajka JJ, Dai Z, Hofstad BA, Kim J, Pomraning KR
  TITLE     CRISPR-Cas9/Cas12a systems for efficient genome editing and large 
            genomic fragment deletions in Aspergillus niger.
  JOURNAL   Front Bioeng Biotechnol. 2024 Oct 16;12:1452496. doi: 
            10.3389/fbioe.2024.1452496. eCollection 2024.
  PUBMED    39479294
REFERENCE   2  (bases 1 to 17021)
  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: "Front 
            Bioeng Biotechnol."; date: "2024-10-16"; volume: "12:1452496. doi"; 
            pages: " 10.3389/fbioe.2024.1452496. eCollection 2024"
COMMENT     Sequence Label: pGY18
FEATURES             Location/Qualifiers
     source          1..17021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      104..5351
                     /label=AMA1
                     /note="replication origin from Aspergillus nidulans 
                     (Aleksenko and Clutterbuck, 1996)"
     misc_RNA        6012..6087
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             7214..11317
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7214..11317
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             11321..11341
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12810..13604
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(12864..12883)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     13474..13493
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(14039..14072)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(14542..14558)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14657..14674)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14828..15416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14908..14927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15538..16329,16330..16398))
                     /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     16161..16180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16399..16490)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     16532..16551
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     16557..16576
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    16783..16816
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 ccaactgacc agaggcaggg tagcggattg acggctccat ctccttcact tacaaggtct
       61 attgaaagcc ctttagcatc accaagcgga gaatagattg ttaagcttat tttttgtata
      121 ctgttttgtg atagcacgaa gtttttccac ggtatcttgt taaaaatata tatttgtggc
      181 gggcttacct acatcaaatt aataagagac taattataaa ctaaacacac aagcaagcta
      241 ctttagggta aaagtttata aatgcttttg acgtataaac gttgcttgta tttattatta
      301 caattaaagg tggatagaaa acctagagac tagttagaaa ctaatctcag gtttgcgtta
      361 aactaaatca gagcccgaga ggttaacaga acctagaagg ggactagata tccgggtagg
      421 gaaacaaaaa aaaaaaacaa gacagccaca tattagggag actagttaga agctagttcc
      481 aggactagga aaataaaaga caatgatacc acagtctagt tgacaactag atagattcta
      541 gattgaggcc aaagtctctg agatccaggt tagttgcaac taatactagt tagtatctag
      601 tctcctataa ctctgaagct agaataactt actactatta tcctcaccac tgttcagctg
      661 cgcaaacgga gtgattgcaa ggtgttcaga gactagttat tgactagtca gtgactagca
      721 ataactaaca aggtattaac ctaccatgtc tgccatcacc ctgcacttcc tcgggctcag
      781 cagccttttc ctcctcattt tcatgctcat tttccttgtt taagactgtg actagtcaaa
      841 gactagtcca gaaccacaaa ggagaaatgt cttaccactt tcttcattgc ttgtctcttt
      901 tgcattatcc atgtctgcaa ctagttagag tctagttagt gactagtccg acgaggactt
      961 gcttgtctcc ggattgttgg aggaactctc cagggcctca agatccacaa cagagccttc
     1021 tagaagactg gtcaataact agttggtctt tgtctgagtc tgacttacga ggttgcatac
     1081 tcgctccctt tgcctcgtca atcgatgaga aaaagcgcca aaactcgcaa tatggctttg
     1141 aaccacacgg tgctgagact agttagaatc tagtcccaaa ctagcttgga tagcttacct
     1201 ttgccctttg cgttgcgaca ggtcttgcag ggtatggttc ctttctcacc agctgattta
     1261 gctgccttgc taccctcacg gcggatctgc ataaagagtg gctagaggtt ataaattagc
     1321 actgatccta ggtacggggc tgaatgtaac ttgcccttcc tttctcatcg cgcggcaaga
     1381 caggcttgct caaattccta ccagtcacag gggtatgcac ggcgtacgga ccacttgaac
     1441 tagtcacaga ttagttagca actagtctgc attgaatggc tgtacttacg ggccctcgcc
     1501 attgtcctga tcatttccag cttcaccctc gttgctgcaa agtagttagt gactagtcaa
     1561 ggactagttg aaatgggaga agaaactcac gaattctcga cacccttagt attgtggtcc
     1621 ttggacttgg tgctgctata tattagctaa tacactagtt agactcacag aaacttacgc
     1681 agctcgcttg cgcttcttgg taggagtcgg ggttgggaga acagtgcctt caaacaagcc
     1741 ttcataccat gctacttgac tagtcaggga ctagtcacca agtaatctag ataggacttg
     1801 cctttggcct ccatcagttc cttcatagtg ggaggtccat tgtgcaatgt aaactccatg
     1861 ccgtgggagt tcttgtcctt caagtgcttg accaatatgt ttctgttggc agagggaacc
     1921 tgtcaactag ttaataacta gtcagaaact agtatagcag tagactcact gtacgcttga
     1981 ggcatccctt cactcggcag tagacttcat atggatggat atcaggcacg ccattgtcgt
     2041 cctgtggact agtcagtaac taggcttaaa gctagtcggg tcggcttact atcttgaaat
     2101 ccggcagcgt aagctccccg tccttaactg cctcgagata gtgacagtac tctggggact
     2161 ttcggagatc gttatcgcga atgctcggca tactaatcgt tgactagtct tggactagtc
     2221 ccgagcaaaa aggattggag gaggaggagg aaggtgagag tgagacaaag agcgaaataa
     2281 gagcttcaaa ggctatctct aagcagtatg aaggttaagt atctagttct tgactagatt
     2341 taaaagagat ttcgactagt tatgtacctg gagtttggat ataggaatgt gttgtggtaa
     2401 cgaaatgtaa gggggaggaa agaaaaagtc ggtcaagagg taactctaag tcggccattc
     2461 ctttttggga ggcgctaacc ataaacggca tggtcgactt agagttagct cagggaattt
     2521 agggagttat ctgcgaccac tggactctca ccgtttccat tggggtacta acatagccat
     2581 caaatgccgg cccgtgttta atctcctcga gatggctggg ctcacatgcg ttctactcaa
     2641 aaactcttat acctaggccc tctcccaatc gtgtatgttt ggaccttcct ctctcacact
     2701 ttggataaca tcaagcatac tgcagatttt gacctagtta acaaggcaca tcatgctatc
     2761 acttccaaca ctcttgctga gcgcgttctg ccttgccttt ctggttctac gaagtatcca
     2821 gtcccgcctg caacgccgca gaaacgccaa aaggtggggt tgtcaaccgc cagctagagc
     2881 tccatcggga ttctttggca ttccgccgtt cctcggtggt cgcagataac tccctaaatt
     2941 ccctgagcta actctaagtc gaccatgccg tttatggtta gcgcctccca aaaaggaatg
     3001 gccgacttag agttacctct tgaccgactt tttctttcct cccccttaca tttcgttacc
     3061 acaacacatt cctatatcca aactccaggt acataactag tcgaaatctc ttttaaatct
     3121 agtcaagaac tagatactta accttcatac tgcttagaga tagcctttga agctcttatt
     3181 tcgctctttg tctcactctc accttcctcc tcctcctcca atcctttttg ctcgggacta
     3241 gtccaagact agtcaacgat tagtatgccg agcattcgcg ataacgatct ccgaaagtcc
     3301 ccagagtact gtcactatct cgaggcagtt aaggacgggg agcttacgct gccggatttc
     3361 aagatagtaa gccgacccga ctagctttaa gcctagttac tgactagtcc acaggacgac
     3421 aatggcgtgc ctgatatcca tccatatgaa gtctactgcc gagtgaaggg atgcctcaag
     3481 cgtacagtga gtctactgct atactagttt ctgactagtt attaactagt tgacaggttc
     3541 cctctgccaa cagaaacata ttggtcaagc acttgaagga taagaactcc cacggcatgg
     3601 agtttacatt gcacaatgga cctcccacta tgaaggaact gatggaggcc aaaggcaagt
     3661 cctatctaga ttacttggtg actagtccct gactagtcaa gtagcatggt atgaaggctt
     3721 gtttgaaggc actgttctcc caaccccgac tcctaccaag aagcgcaagc gagctgcgta
     3781 agtttctgtg agtctaacta gtgtattagc taatatatag cagcaccaag tccaaggacc
     3841 acaatactaa gggtgtcgag aattcgtgag tttcttctcc catttcaact agtccttgac
     3901 tagtcactaa ctactttgca gcaacgaggg tgaagctgga aatgatcagg acaatggcga
     3961 gggcccgtaa gtacagccat tcaatgcaga ctagttgcta actaatctgt gactagttca
     4021 agtggtccgt acgccgtgca tacccctgtg actggtagga atttgagcaa gcctgtcttg
     4081 ccgcgcgatg agaaaggaaa ggcaagttac attcagcccc gtacctagga tcagtgctaa
     4141 tttataacct ctagccactc tttatgcaga tccgccgtga gggtagcaag gcagctaaat
     4201 cagctggtga gaaaggaacc ataccctgca agacctgtcg caacgcaaag ggcaaaggta
     4261 agctatccaa gctagtttgg gactagattc taactagtct cagcaccgtg tggttcaaag
     4321 ccatattgcg agttttggcg ctttttctca tcgattgacg aggcaaaggg agcgagtatg
     4381 caacctcgta agtcagactc agacaaagac caactagtta ttgaccagtc ttctagaagg
     4441 ctctgttgtg gatcttgagg ccctggagag ttcctccaac aatccggaga caagcaagtc
     4501 ctcgtcggac tagtcactaa ctagactcta actagttgca gacatggata atgcaaaaga
     4561 gacaagcaat gaagaaagtg gtaagacatt tctcctttgt ggttctggac tagtctttga
     4621 ctagtcacag tcttaaacaa ggaaaatgag catgaaaatg aggaggaaaa ggctgctgag
     4681 cccgaggaag tgcagggtga tggcagacat ggtaggttaa taccttgtta gttattgcta
     4741 gtcactgact agtcaataac tagtctctga acaccttgca atcactccgt ttgcgcagct
     4801 gaacagtggt gaggataata gtagtaagtt attctagctt cagagttata ggagactaga
     4861 tactaactag tattagttgc aactaacctg gatctcagag actttggcct caatctagaa
     4921 tctatctagt tgtcaactag actgtggtat cattgtcttt tattttccta gtcctggaac
     4981 tagcttctaa ctagtctccc taatatgtgg ctgtcttgtt tttttttttg tttccctacc
     5041 cggatatcta gtccccttct aggttctgtt aacctctcgg gctctgattt agtttaacgc
     5101 aaacctgaga ttagtttcta actagtctct aggttttcta tccaccttta attgtaataa
     5161 taaatacaag caacgtttat acgtcaaaag catttataaa cttttaccct aaagtagctt
     5221 gcttgtgtgt ttagtttata attagtctct tattaatttg atgtaggtaa gcccgccaca
     5281 aatatatatt tttaacaaga taccgtggaa aaacttcgtg ctatcacaaa acagtataca
     5341 aaaaataagc ttaacaatct attctccgct tggtgatgct aaagggcttt caatagacct
     5401 tgtaagtgaa ggagatggag ccgtcaatcc gctaccctgc ctctggtcag ttgggatcac
     5461 atagatgctc ggttgacagg accacctata gcaagtactt tgtaggaggt tctcctaacg
     5521 cttggcttca aatccaatca ggatatcagt gagttactgt ttgtcatcgc atcctcatga
     5581 agtcgctcaa cagccactga gaagcaaata tttgggacac catccctgat gttgaaattt
     5641 tacgctggag cccattcacc ggtgagctcg gggacagtct ggccagtgga gcggaaaatc
     5701 ttcaactaac tctgattggt accacaggta gccccacaga acaaaccgag caaacatgaa
     5761 aattttcgct tgaggttagc gcactcgcta gcgcctgccc gcaaatataa aaggccccga
     5821 aatcccatgc attttggaaa tcaagcgact ctacgtatgt catccccctc cctacctttg
     5881 ggaaaacctc tcaactgcaa gtcagaacat tttgctaaca gcaggggctg tggtttagtg
     5941 gtataatatt cccttagcat gggagaggtc cggggttcga ttccccgcag ctccaggaga
     6001 ccatggtctc cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     6061 cttgaaaaag tggcaccgag tcggtgctgt caatttttac acttgatgtt ggtgtaatca
     6121 ggcgatcaga gtttgctcaa attctctctt gttttagatt ctgaagaata taatttttac
     6181 atgactagtt cattttactt ccaagtagac tatttgtaga ttgagattgc cgtcaacttt
     6241 attcatagaa tcacattaaa ttgctcattg acctgcatgt ctatgcacac attcacatct
     6301 atcttctcag ggtattaaga cctcagccga gacagcagaa tcaccgccca agttaagcct
     6361 ttgtgctgat catgctctcg aacgggccaa gttcgggaaa agcaaaggag cgtttagtga
     6421 ggggcaattt gactcacctc ccaggcaaca gatgaggggg gcaaaaagaa agaaattttc
     6481 gtgagtcaat atggattccg agcatcattt tcttgcggtc tatcttgcta cgtatgttga
     6541 tcttgacgct gtggatcaag caacgccact cgctcgctcc atcgcaggct ggtcgcagac
     6601 aaattaaaag gcggcaaact cgtacagccg cggggttgtc cgctgcaaag tacagagtga
     6661 taaaagccgc catgcgacca tcaacgcgtt gatgcccagc tttttcgatc cgagaatcca
     6721 ccgtagaggc gatagcaagt aaagaaaagc taaacaaaaa aaaatttctg cccctaagcc
     6781 atgaaaacga gatggggtgg agcagaacca aggaaagagt cgcgctgggc tgccgttccg
     6841 gaaggtgttg taaaggctcg acgcccaagg tgggagtcta ggagaagaat ttgcatcggg
     6901 agtggggcgg gttacccctc catatccaat gacagatatc taccagccaa gggtttgagc
     6961 ccgcccgctt agtcgtcgtc ctcgcttgcc cctccataaa aggatttccc ctccccctcc
     7021 cacaaaattt tctttccctt cctctccttg tccgcttcag tacgtatatc ttcccttccc
     7081 tcgcttctct cctccatcct tctttcatcc atctcctgct aacttctctg ctcagcacct
     7141 ctacgcatta ctagccgtag tatctgagca cttctccctt ttatattcca caaaacataa
     7201 cacaaccttc accatggaca agaagtatag catcgggctg gacattggaa cgaactcggt
     7261 tggttgggct gtgattacgg acgaatacaa ggtgccatcc aagaagttta aggtcctggg
     7321 aaacaccgac cgtcactcaa tcaagaagaa tctcattgga gccctgctct tcgatagtgg
     7381 ggaaactgcc gaagctactc gactgaagcg aacggctcgc cggcgttata cacgacgcaa
     7441 gaatcgcatc tgctacctcc aggagatttt cagcaacgaa atggctaagg ttgatgactc
     7501 attctttcat cgactcgaag aaagtttctt ggtcgaggag gataagaagc acgagcgcca
     7561 tccgatcttt ggtaacattg tggatgaggt tgcctatcac gaaaagtacc caactatcta
     7621 tcatcttcgt aagaagctgg tcgatagcac ggacaaggct gatttgcgac ttatctacct
     7681 ggcactcgcg cacatgatta agttccgcgg ccattttctt atcgagggtg acctgaaccc
     7741 cgataattct gacgttgata agctcttcat ccagttggtc caaacctaca atcagctgtt
     7801 tgaggaaaac cctattaatg catctggcgt ggacgccaag gctatccttt cggcgcgcct
     7861 gtctaagtcg cggcgtttgg agaaccttat cgcacaactc cccggcgaaa agaagaacgg
     7921 cctcttcggt aatttgattg cgttgtcact tggtctgact cctaacttca agagtaattt
     7981 tgacctggca gaggatgcga agctccagtt gtctaaggat acgtatgatg acgatctcga
     8041 caacttgctt gcccaaatcg gtgaccagta cgctgatctt ttcctggccg ctaagaatct
     8101 ctcagatgca atcctgctca gtgacatttt gcgggtcaac accgagatta ctaaggcccc
     8161 cctgtcagct agtatgatca agcggtatga tgagcaccat caggacctca ccttgcttaa
     8221 ggccctcgtg cgtcagcaat tgcctgagaa gtataaggaa atcttctttg accaatccaa
     8281 gaacggatac gcagggtata ttgatggcgg tgcgagccag gaggaattct acaagtttat
     8341 caagccgatt ttggagaaga tggacggcac tgaggaactg ctcgtcaagc tgaatcgcga
     8401 agatttgctt cgtaagcaac gaacgttcga caacggctcc atcccgcacc agattcatct
     8461 gggcgagctc cacgccatcc ttcgacgcca ggaagatttc tacccatttc tgaaggacaa
     8521 ccgtgagaag atcgaaaaga ttcttacatt ccgaatcccc tactatgtgg gacctttggc
     8581 ccgtgggaat tcccgatttg cttggatgac ccgaaagagc gaggaaacca tcactccgtg
     8641 gaacttcgag gaagtcgtgg acaagggtgc atccgcgcag agcttcattg agcggatgac
     8701 caattttgat aagaaccttc cgaatgaaaa ggtcctgcca aagcattcgc tgctctacga
     8761 gtatttcacc gtgtataacg aactgactaa ggtcaagtac gtgacggagg gaatgcggaa
     8821 gccagccttc ctctcagggg aacaaaagaa ggctatcgtc gatttgcttt ttaagaccaa
     8881 tcgtaaagtg actgttaagc agctgaagga ggattatttc aagaagattg aatgtttcga
     8941 ctccgtcgag atcagcggcg tggaagatcg ctttaacgct tccctcggta cctaccacga
     9001 cctgctcaag atcattaagg acaaggattt cctcgataac gaggaaaatg aggacatctt
     9061 ggaagatatt gtcctcacgt tgacactttt tgaggaccgc gaaatgatcg aggaacggct
     9121 caagacatat gcccatttgt tcgacgataa ggtgatgaag cagctgaagc ggcgtcgata
     9181 caccggatgg ggtcgcctta gccggaagct gatcaacggc attcgagata agcaatctgg
     9241 taagactatc ttggatttcc ttaagtcgga cggcttcgcc aaccgcaatt ttatgcagct
     9301 tattcacgac gattccctga cgttcaagga ggacatccag aaggcacaag tctcaggaca
     9361 aggggattcc ctgcacgagc atatcgccaa cctggctgga tccccggcga tcaagaaggg
     9421 gattcttcag accgtcaagg ttgtcgacga gctggtcaag gtgatgggcc gtcataagcc
     9481 agaaaacatc gtgattgaga tggcccgaga aaatcagacc actcaaaagg gtcagaagaa
     9541 cagccgcgag cggatgaagc ggatcgagga aggcattaag gaacttggtt ctcagatcct
     9601 gaaggagcac cctgttgaaa acacacagct ccaaaatgag aagctgtatc tctactattt
     9661 gcaaaatgga cgcgacatgt acgtcgatca ggagctcgac attaaccggt tgtcggacta
     9721 cgatgttgac catatcgtcc cgcaatcctt ccttaaggac gatagcattg ataacaaggt
     9781 gctgactcgc tcagataaga accggggcaa gtccgacaat gttccaagcg aggaagtggt
     9841 taagaagatg aagaactact ggcgccaatt gcttaatgcc aagctcatca cacagcgcaa
     9901 gtttgacaac ttgaccaagg ccgagcgggg agggctgagt gaactcgata aggctggctt
     9961 catcaagcgt caactcgtgg agacgcgaca gatcacaaag cacgttgctc agattctgga
    10021 ctcccggatg aacacaaagt acgacgagaa tgataagctc atccgtgaag ttaaggtcat
    10081 taccctcaag tctaagttgg tgtcggattt ccgcaaggac ttccaatttt ataaggttcg
    10141 ggagatcaac aattatcacc atgcacatga tgcgtacctc aacgcagtcg tgggaactgc
    10201 gctcatcaag aagtatccca agttggagtc cgaattcgtc tacggggatt ataaggttta
    10261 cgacgtccgc aagatgatcg ccaagagtga gcaggaaatt ggcaaggcca cggctaagta
    10321 tttcttttac tccaacatca tgaatttctt taagacggag atcacactcg ccaatggaga
    10381 aatccgtaag cgacctttga ttgaaactaa cggcgagact ggtgaaatcg tttgggataa
    10441 ggggcgcgac ttcgctaccg tgcggaaggt tctgagcatg ccgcaagtca atatcgtcaa
    10501 gaaaaccgag gtgcagacag gcggtttctc taaggaatcg attcttccaa agcgtaactc
    10561 tgacaagctg atcgctcgaa agaaggattg ggaccccaag aagtatggag ggttcgattc
    10621 tcctacagtg gcatactcgg ttctcgttgt cgcgaaggtt gagaagggaa agtctaagaa
    10681 gctgaagtcg gtcaaggaac tgctcgggat caccattatg gagcgctcca gcttcgaaaa
    10741 gaatcccatc gactttctcg aggccaaggg ctataaggaa gtcaagaagg atcttatcat
    10801 taagctgcct aagtactctt tgttcgagct tgaaaacggt cgaaagcgaa tgctcgcatc
    10861 ggcaggagag ttgcagaagg ggaatgaatt ggcacttccc tcaaagtacg tgaacttcct
    10921 gtatctcgcg tcccactacg agaagctgaa gggtagccct gaggacaacg aacagaagca
    10981 actttttgtt gagcaacaca agcattatct ggatgagatc attgaacaga tttcagagtt
    11041 cagtaagcgc gtcatcctcg ccgatgctaa tctcgacaag gtgttgtcgg cctacaacaa
    11101 gcaccgtgac aagccgatcc gagagcaggc tgaaaatatc attcatctgt tcaccctcac
    11161 taacttggga gcaccagcag cgttcaagta ttttgatacg acaatcgacc gtaagcgata
    11221 cacgtccaca aaggaggtgc ttgatgcgac cctgattcat caatccatca ctgggctcta
    11281 tgaaacccgt atcgacctta gtcaactggg gggcgaccct cccaagaaga agcgcaaggt
    11341 ctgagcggac attcgattta tgccgttatg acttccttaa aaaagccttt acgaatgaaa
    11401 gaaatggaat tagacttgtt atgtagttga ttctacaatg gattatgatt cctgaacttc
    11461 aaatccgctg ttcattatta atctcagctc ttcccgtaaa gccaatgttg aaactattcg
    11521 taaatgtacc tcgttttgcg tgtaccttgc ttatcacgtg atattacatg acctggacag
    11581 agttctgcgc gaaagtcata acgtaaatcc cgggcggtag gtgcgtcccg ggcggaaggt
    11641 agttttctcg tccaccccaa cgcgtttatc aacctcaact ttcaacaacc atcatgccac
    11701 caaaagcgcg taaaacaaag cgagatttga ttgagcaaga gggcaggatc caatgcgcga
    11761 ttcaagacat taaaaatgga aaatttcaaa aaattgcgcc cgcagcgcgt gcatacaaaa
    11821 ttcatcccaa tacaagtgta atgcgaacga ctccagaagt gactaagcaa ccgtaaaccg
    11881 gaagcaacaa tccggctctc atggcaaaga tggcatgtcc ggattgcgga ggaaagattt
    11941 ggcgtgtgcc tgacggtttt gttccacaag gaccctgtaa acttggtgat ggtaatgagg
    12001 tgctctggcc ttgattgagc aaaagtcagc tcgcggggac tacgtgagtt gatggtggag
    12061 gatcatgtac taaatagagc atacctaggg gatggtgaac ggacgtccag ggttccgtag
    12121 aattgtaaca actctactcg acttgctgcc cactggggta tgtaaataca tgccttttca
    12181 aagttgccga gtgtactaaa tggagtgtat gtttggccaa tgtcatttgg gaggggccgg
    12241 gaaccctttg ggacacccgc ggatgggctg gatctcggtc actggagaca tagcctagtg
    12301 gtctagtagt ctagcagcag gacagcttca tggttaaccc cacatgtgat cctccgtccg
    12361 gtttccgtag tatataatgg acctgaagct tctccctctc tcgatgcggt tgaaacataa
    12421 atttgctcat aacgtatgtc catctaaccc tttcattgtt tcttgaccac tgtctccttt
    12481 ctggaaaaaa aaagactctg gtaagtatgg gcctaggatc gagctactga tcccccaatc
    12541 cttcctaatc cctctgccac ttgccccgac ctgttcaccg tgaccgtacc accaccaccc
    12601 cgccccctcc tcaactaaac ccctcccttc ccctcccccc tgatgttttc tcttttcttt
    12661 aatctgactg tcacctccct ctctacttct tttccctcaa ttttcttctc ctcttcttac
    12721 tctctgtcat ctgtttcgat acccatacta atctcgcgtg ctttttcttc accttcgtgt
    12781 atagatttgt cacaatccca aatttcacca tgattgaaca agatggattg cacgcaggtt
    12841 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
    12901 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
    12961 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
    13021 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
    13081 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
    13141 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
    13201 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
    13261 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
    13321 tcgccaggct caaggcgcgc atgcccgacg gcgatgatct cgtcgtgacc catggcgatg
    13381 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
    13441 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
    13501 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
    13561 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
    13621 cgaaatgacc gaccaagcga cgatccactt aacgttactg aaatcatcaa acagcttgac
    13681 gaatctggat ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt
    13741 caggatctcg ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt
    13801 aatagctcca tgtcaacaag aataaaacgc gtttcgggtt tacctcttcc agatacagct
    13861 catctgcaat gcattaatgc attggacctc gcaaccctag tacgcccttc aggctccggc
    13921 gaagcagaag aatagcttag cagagtccat gcggatttcg gctccaaagt agatgccgac
    13981 cgatccccac taccgcatta agacctcagc gcggccgcaa atttaaataa aatgaagtga
    14041 agttcctata ctttctagag aataggaact tctatagtga gtcgaataag ggcgacacaa
    14101 aatttattct aaatgcataa taaatactga taacatctta tagtttgtat tatattttgt
    14161 attatcgttg acatgtataa ttttgatatc aaaaactgat tttcccttta ttattttcga
    14221 gatttatttt cttaattctc tttaacaaac tagaaatatt gtatatacaa aaaatcataa
    14281 ataatagatg aatagtttaa ttataggtgt tcatcaatcg aaaaagcaac gtatcttatt
    14341 taaagtgcgt tgcttttttc tcatttataa ggttaaataa ttctcatata tcaagcaaag
    14401 tgacaggcgc ccttaaatat tctgacaaat gctctttccc taaactcccc ccataaaaaa
    14461 acccgccgaa gcgggttttt acgttatttg cggattaacg attactcgtt atcagaaccg
    14521 cccagggggc ccgagcttaa gactggccgt cgttttacaa cacagaaaga gtttgtagaa
    14581 acgcaaaaag gccatccgtc aggggccttc tgcttagttt gatgcctggc agttccctac
    14641 tctcgccttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    14701 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    14761 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    14821 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
    14881 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
    14941 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
    15001 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
    15061 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
    15121 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
    15181 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
    15241 ggtgggctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
    15301 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
    15361 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
    15421 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgacgcgcgc gtaactcacg
    15481 ttaagggatt ttggtcatga gcttgcgccg tcccgtcaag tcagcgtaat gctctgctta
    15541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
    15601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
    15661 cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag caataaacca
    15721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
    15781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
    15841 tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
    15901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
    15961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
    16021 ggttatggca gcgctacata attctcttac tgtcatgcca tccgtaagat gcttttctgt
    16081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
    16141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
    16201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
    16261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
    16321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
    16381 gaaatgttga atactcatat tcttcctttt tcaatattat tgaagcattt atcagggtta
    16441 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggtcag
    16501 tgttacaacc aattaaccaa ttctgaacat tatcgcgagc ccatttatac ctgaatatgg
    16561 ctcataacac cccttgtttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
    16621 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggactccc catgcgagag
    16681 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgc
    16741 ccgggctaat tatggggtgt cgcccttatt cgactctata gtgaagttcc tattctctag
    16801 aaagtatagg aacttctgaa gtggggattt aaatgcggcc gcgctgaggg tttaatcgac
    16861 gaagcagctg acggccagtg ccaagcttaa cgcgtaccgg gcccagtata tgttccgcag
    16921 atgactggag ctctgccata cgtgccctct caagcaccat ttgttccatc tacagagact
    16981 agtcaccaac tagtctatca agactcacag ggtacattgc t
//
