LOCUS       sequence-444271-       17674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for Aspergillus CRISPR-Cas9 genetic engineering with Golden 
            Gate cloning drop-out cassette for protospacer insertion and pyrG 
            selectable marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17674)
  AUTHORS   Modaffari D, Finlayson A, Miao Y, Wallace EWJ, Sawin KE
  TITLE     Improved gene editing and fluorescent-protein tagging in Aspergillus
            nidulans using a Golden Gate-based CRISPR-Cas9 plasmid system.
  JOURNAL   Wellcome Open Res. 2024 Oct 17;9:602. doi: 
            10.12688/wellcomeopenres.23086.1. eCollection 2024.
  PUBMED    39640368
REFERENCE   2  (bases 1 to 17674)
  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: "Wellcome 
            Open Res."; date: "2024-10-17"; volume: "9:602. doi"; pages: " 
            10.12688/wellcomeopenres.23086.1. eCollection 2024"
COMMENT     Sequence Label: pDM026
FEATURES             Location/Qualifiers
     source          1..17674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     622..641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(860..951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     993..1012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1018..1037
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    1244..1277
                     /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)"
     rep_origin      1590..6837
                     /label=AMA1
                     /note="replication origin from Aspergillus nidulans 
                     (Aleksenko and Clutterbuck, 1996)"
     ncRNA           7546..7613
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     RBS             7773..7784
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             7791..8504
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      8516..8587
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8603..8630
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        8648..8723
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             9929..14032
                     /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             9929..14032
                     /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             14036..14056
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(16172..16205)
                     /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(16675..16691)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(16790..16807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(16961..17549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(17041..17060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(17673..17674,1..790,791..859))
                     /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"
ORIGIN
        1 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
       61 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
      121 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc
      181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
      241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
      301 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
      361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
      421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
      481 tggttatggc agcgctacat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
      541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
      601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
      661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      841 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
      901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
      961 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg
     1021 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     1081 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga
     1141 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1201 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta
     1261 gaaagtatag gaacttctga agtggggatt taaatgcggc cgcgctgagg gtttaatcga
     1321 cgaagcagct gacggccagt gccaagctta acgcgtaccg ggcccagtat atgttccgca
     1381 gatgactgga gctctgccat acgtgccctc tcaagcacca tttgttccat ctacagagac
     1441 tagtcaccaa ctagtctatc aagactcaca gggtacattg ctgagaccaa ctgaccagag
     1501 gcagggtagc ggattgacgg ctccatctcc ttcacttaca aggtctattg aaagcccttt
     1561 agcatcacca agcggagaat agattgttaa gcttattttt tgtatactgt tttgtgatag
     1621 cacgaagttt ttccacggta tcttgttaaa aatatatatt tgtggcgggc ttacctacat
     1681 caaattaata agagactaat tataaactaa acacacaagc aagctacttt agggtaaaag
     1741 tttataaatg cttttgacgt ataaacgttg cttgtattta ttattacaat taaaggtgga
     1801 tagaaaacct agagactagt tagaaactaa tctcaggttt gcgttaaact aaatcagagc
     1861 ccgagaggtt aacagaacct agaaggggac tagatatccg ggtagggaaa caaaaaaaaa
     1921 aacaagacag ccacatatta gggagactag ttagaagcta gttccaggac taggaaaata
     1981 aaagacaatg ataccacagt ctagttgaca actagataga ttctagattg aggccaaagt
     2041 ctctgagatc caggttagtt gcaactaata ctagttagta tctagtctcc tataactctg
     2101 aagctagaat aacttactac tattatcctc accactgttc agctgcgcaa acggagtgat
     2161 tgcaaggtgt tcagagacta gttattgact agtcagtgac tagcaataac taacaaggta
     2221 ttaacctacc atgtctgcca tcaccctgca cttcctcggg ctcagcagcc ttttcctcct
     2281 cattttcatg ctcattttcc ttgtttaaga ctgtgactag tcaaagacta gtccagaacc
     2341 acaaaggaga aatgtcttac cactttcttc attgcttgtc tcttttgcat tatccatgtc
     2401 tgcaactagt tagagtctag ttagtgacta gtccgacgag gacttgcttg tctccggatt
     2461 gttggaggaa ctctccaggg cctcaagatc cacaacagag ccttctagaa gactggtcaa
     2521 taactagttg gtctttgtct gagtctgact tacgaggttg catactcgct ccctttgcct
     2581 cgtcaatcga tgagaaaaag cgccaaaact cgcaatatgg ctttgaacca cacggtgctg
     2641 agactagtta gaatctagtc ccaaactagc ttggatagct tacctttgcc ctttgcgttg
     2701 cgacaggtct tgcagggtat ggttcctttc tcaccagctg atttagctgc cttgctaccc
     2761 tcacggcgga tctgcataaa gagtggctag aggttataaa ttagcactga tcctaggtac
     2821 ggggctgaat gtaacttgcc tttcctttct catcgcgcgg caagacaggc ttgctcaaat
     2881 tcctaccagt cacaggggta tgcacggcgt acggaccact tgaactagtc acagattagt
     2941 tagcaactag tctgcattga atggctgtac ttacgggccc tcgccattgt cctgatcatt
     3001 tccagcttca ccctcgttgc tgcaaagtag ttagtgacta gtcaaggact agttgaaatg
     3061 ggagaagaaa ctcacgaatt ctcgacaccc ttagtattgt ggtccttgga cttggtgctg
     3121 ctatatatta gctaatacac tagttagact cacagaaact tacgcagctc gcttgcgctt
     3181 cttggtagga gtcggggttg ggagaacagt gccttcaaac aagccttcat accatgctac
     3241 ttgactagtc agggactagt caccaagtaa tctagatagg acttgccttt ggcctccatc
     3301 agttccttca tagtgggagg tccattgtgc aatgtaaact ccatgccgtg ggagttcttg
     3361 tccttcaagt gcttgaccaa tatgtttctg ttggcagagg gaacctgtca actagttaat
     3421 aactagtcag aaactagtat agcagtagac tcactgtacg cttgaggcat cccttcactc
     3481 ggcagtagac ttcatatgga tggatatcag gcacgccatt gtcgtcctgt ggactagtca
     3541 gtaactaggc ttaaagctag tcgggtcggc ttactatctt gaaatccggc agcgtaagct
     3601 ccccgtcctt aactgcctcg agatagtgac agtactctgg ggactttcgg agatcgttat
     3661 cgcgaatgct cggcatacta atcgttgact agtcttggac tagtcccgag caaaaaggat
     3721 tggaggagga ggaggaaggt gagagtgaga caaagagcga aataagagct tcaaaggcta
     3781 tctctaagca gtatgaaggt taagtatcta gttcttgact agatttaaaa gagatttcga
     3841 ctagttatgt acctggagtt tggatatagg aatgtgttgt ggtaacgaaa tgtaaggggg
     3901 aggaaagaaa aagtcggtca agaggtaact ctaagtcggc cattcctttt tgggaggcgc
     3961 taaccataaa cggcatggtc gacttagagt tagctcaggg aatttaggga gttatctgcg
     4021 accaccgagg aacggcggaa tgccaaagaa tcccgatgga gctctagctg gcggttgaca
     4081 accccacctt ttggcgtttc tgcggcgttg caggcgggac tggatacttc gtagaaccag
     4141 aaaggcaagg cagaacgcgc tcagcaagag tgttggaagt gatagcatga tgtgccttgt
     4201 taactaggtc aaaatctgca gtatgcttga tgttatccaa agtgtgagag aggaaggtcc
     4261 aaacatacac gattgggaga gggcctaggt ataagagttt ttgagtagaa cgcatgtgag
     4321 cccagccatc tcgaggagat taaacacggg ccggcatttg atggctatgt tagtacccca
     4381 atggaaacgg tgagagtcca gtggtcgcag ataactccct aaattccctg agctaactct
     4441 aagtcgacca tgccgtttat ggttagcgcc tcccaaaaag gaatggccga cttagagtta
     4501 cctcttgacc gactttttct ttcctccccc ttacatttcg ttaccacaac acattcctat
     4561 atccaaactc caggtacata actagtcgaa atctctttta aatctagtca agaactagat
     4621 acttaacctt catactgctt agagatagcc tttgaagctc ttatttcgct ctttgtctca
     4681 ctctcacctt cctcctcctc ctccaatcct ttttgctcgg gactagtcca agactagtca
     4741 acgattagta tgccgagcat tcgcgataac gatctccgaa agtccccaga gtactgtcac
     4801 tatctcgagg cagttaagga cggggagctt acgctgccgg atttcaagat agtaagccga
     4861 cccgactagc tttaagccta gttactgact agtccacagg acgacaatgg cgtgcctgat
     4921 atccatccat atgaagtcta ctgccgagtg aagggatgcc tcaagcgtac agtgagtcta
     4981 ctgctatact agtttctgac tagttattaa ctagttgaca ggttccctct gccaacagaa
     5041 acatattggt caagcacttg aaggacaaga actcccacgg catggagttt acattgcaca
     5101 atggacctcc cactatgaag gaactgatgg aggccaaagg caagtcctat ctagattact
     5161 tggtgactag tccctgacta gtcaagtagc atggtatgaa ggcttgtttg aaggcactgt
     5221 tctcccaacc ccgactccta ccaagaagcg caagcgagct gcgtaagttt ccgtgagtct
     5281 aactagtgta ttagctaata tatagcagca ccaagtccaa ggaccacaat actaagggtg
     5341 tcgagaattc gtgagtttct tctcccattt caactagtcc ttgactagtc actaactact
     5401 ttgcagcaac gagggtgaag ctggaaatga tcaggacaat ggcgagggcc cgtaagtaca
     5461 gccattcaat gcagactagt tgctaactaa tctgtgacta gttcaagtgg tccgtacgcc
     5521 gtgcataccc ctgtgactgg taggaatttg agcaagcctg tcttgccgcg cgatgagaaa
     5581 ggaagggcaa gttacattca gccccgtacc taggatcagt gctaatttat aacctctagc
     5641 cactctttat gcagatccgc cgtgagggta gcaaggcagc taaatcagct ggtgagaaag
     5701 gaaccatacc ctgcaagacc tgtcgcaacg caaagggcaa aggtaagcta tccaagctag
     5761 tttgggacta gattctaact agtctcagca ccgtgtggtt caaagccata ttgcgagttt
     5821 tggcgctttt tctcatcgat tgacgaggca aagggagcga gtatgcaacc tcgtaagtca
     5881 gactcagaca aagaccaact agttattgac cagtcttcta gaaggctctg ttgtggatct
     5941 tgaggccctg gagagttcct ccaacaatcc ggagacaagc aagtcctcgt cggactagtc
     6001 actaactaga ctctaactag ttgcagacat ggataatgca aaagagacaa gcaatgaaga
     6061 aagtggtaag acatttctcc tttgtggttc tggactagtc ttcgactagt cacagtctta
     6121 aacaaggaaa atgagcatga aaatgaggag gaaaaggctg ctgagcccga ggaagtgcag
     6181 ggtgatggca gacatggtag gttaatacct tgttagttat tgctagtcac tgactagtca
     6241 ataactagtc tctgaacacc ttgcaatcac tccgtttgcg cagctgaaca gtggtgagga
     6301 taatagtagt aagttattct agcttcagag ttataggaga ctagatacta actagtatta
     6361 gttgcaacta acctggatct cagagacttt ggcctcaatc tagaatctat ctagttgtca
     6421 actagactgt ggtatcattg tcttttattt tcctagtcct ggaactagct tctaactagt
     6481 ctccctaata tgtggctgtc ttgttttttt tttttgtttc cctacccgga tatctagtcc
     6541 ccttctaggt tctgttaacc tctcgggctc tgatttagtt taacgcaaac ctgagattag
     6601 tttctaacta gtctctaggt tttctatcca cctttaattg taataataaa tacaagcaac
     6661 gtttatacgt caaaagcatt tataaacttt taccctaaag tagcttgctt gtgtgtttag
     6721 tttataatta gtctcttatt aatttgatgt aggtaagccc gccacaaata tatattttta
     6781 acaagatacc gtggaaaaac ttcgtgctat cacaaaacag tatacaaaaa ataagcttaa
     6841 caatctattc tccgcttggt gatgctaaag ggctttcaat agaccttgta agtgaaggag
     6901 atggagccgt caatccgcta ccctgcctct ggtcagttgg tctcagcaat gtaccctgtg
     6961 agtcttgata gactagttgg tgactagtct ctgtagatgg aacaaatggt gcttgagagg
     7021 gcacgtatgg cagagctcca gtcatctgcg gaacatatac tgggcccggg aagatctgat
     7081 cacatagatg ctcggttgac aggaccacct atagcaagta ctttgtagga ggttctccta
     7141 acgcttggct tcaaatccaa tcaggatatc agtgagttac tgtttgtcat cgcatcctca
     7201 tgaagtcgct caacagccac tgagaagcaa atatttggga gaccatccct gatgttgaaa
     7261 ttttacgctg gagcccattc accggtgagc tcggggacag tctggccagt ggagcggaaa
     7321 atcttcaact aactctgatt ggtaccacag gtagccccac agaacaaacc gagcaaacat
     7381 gaaaattttc gcttgaggtt agcgcactcg ctagcgcctg cccgcaaata taaaaggccc
     7441 cgaaatccca tgcattttgg aaatcaagcg actctacgta tgtcatcccc ctccctacct
     7501 ttgggaaaac ctctcaactg caagtcagaa cattttgcta acagcggccg gcatggtccc
     7561 agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca tggcgaatgg gaccgatgca
     7621 ggtggaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat cttatttaat
     7681 aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc ggcaactcaa
     7741 gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag atgcgtaaag
     7801 gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg
     7861 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
     7921 tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc
     7981 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
     8041 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
     8101 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
     8161 agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca
     8221 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
     8281 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
     8341 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
     8401 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
     8461 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat
     8521 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     8581 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatacacc
     8641 tgcagtcgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     8701 aaaaagtggc accgagtcgg tgcttttgca tcattggtct agtggtagaa ttcatcgttg
     8761 ccatcgatga ggcccgtgtt cgattcacgg atgatgcatg tcaattttta cacttgatgt
     8821 tggtgtaatc aggcgatcag agtttgctca aattctctct tgttttagat tctgaagaat
     8881 ataattttta catgactagt tcattttact tccaagtaga ctatttgtag attgagattg
     8941 ccgtcaactt tattcataga atcacattaa attgctcatt gacctgcatg tctatgcaca
     9001 cattcacatc tatcttctca gggtattaat taagacctca gccgagacag cagaatcacc
     9061 gcccaagtta agcctttgtg ctgatcatgc tctcgaacgg gccaagttcg ggaaaagcaa
     9121 aggagcgttt agtgaggggc aatttgactc acctcccagg caacagatga ggggggcaaa
     9181 aagaaagaaa ttttcgtgag tcaatatgga ttccgagcat cattttcttg cggtctatct
     9241 tgctacgtat gttgatcttg acgctgtgga tcaagcaacg ccactcgctc gctccatcgc
     9301 aggctggtcg cagacaaatt aaaaggcggc aaactcgtac agccgcgggg ttgtccgctg
     9361 caaagtacag agtgataaaa gccgccatgc gaccatcaac gcgttgatgc ccagcttttt
     9421 cgatccgaga atccaccgta gaggcgatag caagtaaaga aaagctaaac aaaaaaaaat
     9481 ttctgcccct aagccatgaa aacgagatgg ggtggagcag aaccaaggaa agagtcgcgc
     9541 tgggctgccg ttccggaagg tgttgtaaag gctcgacgcc caaggtggga gtctaggaga
     9601 agaatttgca tcgggagtgg ggcgggttac ccctccatat ccaatgacag atatctacca
     9661 gccaagggtt tgagcccgcc cgcttagtcg tcgtcctcgc ttgcccctcc ataaaaggat
     9721 ttcccctccc cctcccacaa aattttcttt cccttcctct ccttgtccgc ttcagtacgt
     9781 atatcttccc ttccctcgct tctctcctcc atccttcttt catccatctc ctgctaactt
     9841 ctctgctcag cacctctacg cattactagc cgtagtatct gagcacttct cccttttata
     9901 ttccacaaaa cataacacaa ccttcaccat ggacaagaag tatagcatcg ggctggacat
     9961 tggaacgaac tcggttggtt gggctgtgat tacggacgaa tacaaggtgc catccaagaa
    10021 gtttaaggtc ctgggaaaca ccgaccgtca ctcaatcaag aagaatctca ttggagccct
    10081 gctcttcgat agtggggaga ccgccgaagc tactcgactg aagcgaacgg ctcgccggcg
    10141 ttatacacga cgcaagaatc gcatctgcta cctccaggag attttcagca acgaaatggc
    10201 taaggttgat gactcattct ttcatcgact cgaagaaagt ttcttggtcg aggaggataa
    10261 gaagcacgag cgccatccga tctttggtaa cattgtggat gaggttgcct atcacgaaaa
    10321 gtacccaact atctatcatc ttcgtaagaa gctggtcgat agcacggaca aggctgattt
    10381 gcgacttatc tacctggcac tcgcgcacat gattaagttc cgcggccatt ttcttatcga
    10441 gggtgacctg aaccccgata attctgacgt tgataagctc ttcatccagt tggtccaaac
    10501 ctacaatcag ctgtttgagg aaaaccctat taatgcatct ggcgtggacg ccaaggctat
    10561 cctttcggcg cgcctgtcta agtcgcggcg tttggagaac cttatcgcac aactccccgg
    10621 cgaaaagaag aacggcctct tcggtaattt gattgcgttg tcacttggtc tgactcctaa
    10681 cttcaagagt aattttgacc tggcagagga tgcgaagctc cagttgtcta aggatacgta
    10741 tgatgacgat ctcgacaact tgcttgccca aatcggtgac cagtacgctg atcttttcct
    10801 ggccgctaag aatctctcag atgcaatcct gctcagtgac attttgcggg tcaacaccga
    10861 gattactaag gcccccctgt cagctagtat gatcaagcgg tatgatgagc accatcagga
    10921 cctcaccttg cttaaggccc tcgtgcgtca gcaattgcct gagaagtata aggaaatctt
    10981 ctttgaccaa tccaagaacg gatacgcagg gtatattgat ggcggtgcga gccaggagga
    11041 attctacaag tttatcaagc cgattttgga gaagatggac ggcactgagg aactgctcgt
    11101 caagctgaat cgcgaagatt tgcttcgtaa gcaacgaacg ttcgacaacg gctccatccc
    11161 gcaccagatt catctgggcg agctccacgc catccttcga cgccaggaag atttctaccc
    11221 atttctgaag gacaaccgtg agaagatcga aaagattctt acattccgaa tcccctacta
    11281 tgtgggacct ttggcccgtg ggaattcccg atttgcttgg atgacccgaa agagcgagga
    11341 aaccatcact ccgtggaact tcgaggaagt cgtggacaag ggtgcatccg cgcagagctt
    11401 cattgagcgg atgaccaatt ttgataagaa ccttccgaat gaaaaggtcc tgccaaagca
    11461 ttcgctgctc tacgagtatt tcaccgtgta taacgaactg actaaggtca agtacgtgac
    11521 ggagggaatg cggaagccag ccttcctctc aggggaacaa aagaaggcta tcgtcgattt
    11581 gctttttaag accaatcgta aagtgactgt taagcagctg aaggaggatt atttcaagaa
    11641 gattgaatgt ttcgactccg tcgagatcag cggcgtggaa gatcgcttta acgcttccct
    11701 cggtacctac cacgacctgc tcaagatcat taaggacaag gatttcctcg ataacgagga
    11761 aaatgaggac atcttggaag atattgtcct cacgttgaca ctttttgagg accgcgaaat
    11821 gatcgaggaa cggctcaaga catatgccca tttgttcgac gataaggtga tgaagcagct
    11881 gaagcggcgt cgatacaccg gatggggtcg ccttagccgg aagctgatca acggcattcg
    11941 agataagcaa tctggtaaga ctatcttgga tttccttaag tcggacggct tcgccaaccg
    12001 caattttatg cagcttattc acgacgattc cctgacgttc aaggaggaca tccagaaggc
    12061 acaagtctca ggacaagggg attccctgca cgagcatatc gccaacctgg ctggatcccc
    12121 ggcgatcaag aaggggattc ttcagaccgt caaggttgtc gacgagctgg tcaaggtgat
    12181 gggccgtcat aagccagaaa acatcgtgat tgagatggcc cgagaaaatc agaccactca
    12241 aaagggtcag aagaacagcc gcgagcggat gaagcggatc gaggaaggca ttaaggaact
    12301 tggttctcag atcctgaagg agcaccctgt tgaaaacaca cagctccaaa atgagaagct
    12361 gtatctctac tatttgcaaa atggacgcga catgtacgtc gatcaggagc tcgacattaa
    12421 ccggttgtcg gactacgatg ttgaccatat cgtcccgcaa tccttcctta aggacgatag
    12481 cattgataac aaggtgctga ctcgctcaga taagaaccgg ggcaagtccg acaatgttcc
    12541 aagcgaggaa gtggttaaga agatgaagaa ctactggcgc caattgctta atgccaagct
    12601 catcacacag cgcaagtttg acaacttgac caaggccgag cggggagggc tgagtgaact
    12661 cgataaggct ggcttcatca agcgtcaact cgtggagacg cgacagatca caaagcacgt
    12721 tgctcagatt ctggactccc ggatgaacac aaagtacgac gagaatgata agctcatccg
    12781 tgaagttaag gtcattaccc tcaagtctaa gttggtgtcg gatttccgca aggacttcca
    12841 attttataag gttcgggaga tcaacaatta tcaccatgca catgatgcgt acctcaacgc
    12901 agtcgtggga actgcgctca tcaagaagta tcccaagttg gagtccgaat tcgtctacgg
    12961 ggattataag gtttacgacg tccgcaagat gatcgccaag agtgagcagg aaattggcaa
    13021 ggccacggct aagtatttct tttactccaa catcatgaat ttctttaaga cggagatcac
    13081 actcgccaat ggagaaatcc gtaagcgacc tttgattgag accaacggcg agactggtga
    13141 aatcgtttgg gataaggggc gcgacttcgc taccgtgcgg aaggttctga gcatgccgca
    13201 agtcaatatc gtcaagaaaa ccgaggtgca gacaggcggt ttctctaagg aatcgattct
    13261 tccaaagcgt aactctgaca agctgatcgc tcgaaagaag gattgggacc ccaagaagta
    13321 tggagggttc gattctccta cagtggcata ctcggttctc gttgtcgcga aggttgagaa
    13381 gggaaagtct aagaagctga agtcggtcaa ggaactgctc gggatcacca ttatggagcg
    13441 ctccagcttc gaaaagaatc ccatcgactt tctcgaggcc aagggctata aggaagtcaa
    13501 gaaggatctt atcattaagc tgcctaagta ctctttgttc gagcttgaaa acggtcgaaa
    13561 gcgaatgctc gcatcggcag gagagttgca gaaggggaat gaattggcac ttccctcaaa
    13621 gtacgtgaac ttcctgtatc tcgcgtccca ctacgagaag ctgaagggta gccctgagga
    13681 caacgaacag aagcaacttt ttgttgagca acacaagcat tatctggatg agatcattga
    13741 acagatttca gagttcagta agcgcgtcat cctcgccgat gctaatctcg acaaggtgtt
    13801 gtcggcctac aacaagcacc gtgacaagcc gatccgagag caggctgaaa atatcattca
    13861 tctgttcacc ctcactaact tgggagcacc agcagcgttc aagtattttg atacgacaat
    13921 cgaccgtaag cgatacacgt ccacaaagga ggtgcttgat gcgaccctga ttcatcaatc
    13981 catcactggg ctctatgaaa cccgtatcga ccttagtcaa ctggggggcg accctcccaa
    14041 gaagaagcgc aaggtctgag cggacattcg atttatgccg ttatgacttc cttaaaaaag
    14101 cctttacgaa tgaaagaaat ggaattagac ttgttatgta gttgattcta caatggatta
    14161 tgattcctga acttcaaatc cgctgttcat tattaatctc agctcttccc gtaaagccaa
    14221 tgttgaaact attcgtaaat gtacctcgtt ttgcgtgtac cttgcttatc acgtgatatt
    14281 acatgacctg gacagagttc tgcgcgaaag tcataacgta aatcccgggc ggtaggtgcg
    14341 tcccgggcgg aaggtagttt tctcgtccac cccaacgcgt ttatcaacct caactttcaa
    14401 caaccatcat gccaccaaaa gcgcgtaaaa caaagcgaga tttgattgag caagagggca
    14461 ggatccaatg cgcgattcaa gacattaaaa atggaaaatt tcaaaaaatt gcgcccgcag
    14521 cgcgtgcata caaaattcat cccaatacaa gtgtaattcc tcgtgtactg tgtaagcgcc
    14581 cactaggtaa tatgacatga ttacgaattc gagctcggta cccggccgga actccacgtc
    14641 tagaggatcc accagtgatt gaccaatgtt ttatcttcta cagttctgcc tgtctacccc
    14701 attctagctg tacctgacta cagagtagtt taattgtggt tgaccccaca gtcggaggcg
    14761 gaggaataca gcaccgatgt ggcctgtctc catccagatt ggcacgcaat ttttacacgc
    14821 ggaaaagatc gagatagagt acgactttaa atttagtccc cggcggcttc tattttagaa
    14881 tatttgagat ttgattctca agcaattgat ttggttgggt caccctcaat tggataatat
    14941 acctcattgc tcggctactt caactcatca atcaccgtca taccccgcat ataaccctcc
    15001 attcccacga tgtcgtccaa gtcgcaattg acttacggtg ctcgagccag caagcacccc
    15061 aatcctctgg caaagagact ttttgagatt gccgaagcaa agaagacaaa cgttaccgtc
    15121 tctgctgatg tgacgacaac ccgagaactc ctggacctcg ctgaccgtac ggaagctgtt
    15181 ggatccaata catatgccgt ctagcaatgg actaatcaac ttttgatgat acaggtctcg
    15241 gtccctacat cgccgtcatc aagacacaca tcgacatcct caccgatttc agcgtcgaca
    15301 ctatcaatgg cctgaatgtg ctggctcaaa agtacaactt tttgatcttc gaggaccgca
    15361 aattcatcga catcggcaat accgtccaga agcaatacca cggcggtgct ctgaggatct
    15421 ccgaatgggc ccacattatc aactgcagcg ttctccctgg cgagggcatc gtcgaggctc
    15481 tggcccagac cgcatctgcg caagacttcc cctatggtcc tgagagagga ctgttggtcc
    15541 tggcagagat gacccccaaa ggatcgctgg ctacgggcga gtataccaag gcatcggttg
    15601 actacgctcg caaatacaag aacttcgtta tgggtttcgt gtcgacgcgg gccctgacgg
    15661 aagtgcagtc ggatgtgtct tcagcctcgg aggatgaaga tttcgtggtc ttcacgacgg
    15721 gtgtgaacct ctcttccaaa ggagataagc ttggacagca ataccagact cctgcatcgg
    15781 ctattggacg cggtgccgac tttatcatcg ccggtcgagg catctacgct gctcccgacc
    15841 cggttgaagc tgcacagcgg taccagaaag aaggctggga agcttatatg gccagagtat
    15901 gcggcaagtc atgatttcct cttggagcaa aagtgtagtg ccagtacgag tgttgtggag
    15961 gaaggctgca tacattgtgc ctgtcattaa acgatgagct cgtccgtatt ggcccctgta
    16021 atgccatgtt ttccgccccc aatcgtcaag gttttccctt tgttagattc ctaccagtca
    16081 tctagcaagg cggccgcagc tagcacaatt gaggcatccc cactaccgca ttaagacctc
    16141 agcgcggccg caaatttaaa taaaatgaag tgaagttcct atactttcta gagaatagga
    16201 acttctatag tgagtcgaat aagggcgaca caaaatttat tctaaatgca taataaatac
    16261 tgataacatc ttatagtttg tattatattt tgtattatcg ttgacatgta taattttgat
    16321 atcaaaaact gattttccct ttattatttt cgagatttat tttcttaatt ctctttaaca
    16381 aactagaaat attgtatata caaaaaatca taaataatag atgaatagtt taattatagg
    16441 tgttcatcaa tcgaaaaagc aacgtatctt atttaaagtg cgttgctttt ttctcattta
    16501 taaggttaaa taattctcat atatcaagca aagtgacagg cgcccttaaa tattctgaca
    16561 aatgctcttt ccctaaactc cccccataaa aaaacccgcc gaagcgggtt tttacgttat
    16621 ttgcggatta acgattactc gttatcagaa ccgcccaggg ggcccgagct taagactggc
    16681 cgtcgtttta caacacagaa agagtttgta gaaacgcaaa aaggccatcc gtcaggggcc
    16741 ttctgcttag tttgatgcct ggcagttccc tactctcgcc ttccgcttcc tcgctcactg
    16801 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
    16861 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
    16921 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
    16981 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
    17041 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
    17101 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
    17161 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
    17221 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
    17281 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
    17341 ggtatgtagg cggtgctaca gagttcttga agtggtgggc taactacggc tacactagaa
    17401 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
    17461 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
    17521 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
    17581 acgctcagtg gaacgacgcg cgcgtaactc acgttaaggg attttggtca tgagcttgcg
    17641 ccgtcccgtc aagtcagcgt aatgctctgc tttt
//
