LOCUS       sequence-459824-       17949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nCas9 mediated-base editing with NGG PAM recognition motif in 
            Arabidopsis. A3A-nCas9-UGI includes D10A in Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17949)
  AUTHORS   Wang X, Pan W, Sun C, Yang H, Cheng Z, Yan F, Ma G, Shang Y, Zhang 
            R, Gao C, Liu L, Zhang H
  TITLE     Creating large-scale genetic diversity in Arabidopsis via base 
            editing-mediated deep artificial evolution.
  JOURNAL   Genome Biol. 2024 Aug 9;25(1):215. doi: 10.1186/s13059-024-03358-9.
  PUBMED    39123212
REFERENCE   2  (bases 1 to 17949)
  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: "Genome 
            Biol."; date: "2024-08-9"; volume: "25(1):215. doi"; pages: " 
            10.1186/s13059-024-03358-9"
COMMENT     Sequence Label: pHEE901-A3A
FEATURES             Location/Qualifiers
     source          1..17949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..25
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     213..235
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     227..244
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     228..244
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             957..1748
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        1900..1975
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(4194..4217,4218..4220,4221..8294)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             8207..8215
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             8295..8342
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8364..8612
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8625..8645
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(9304..9320)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9304..9320)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9317..9339)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9328..9344
                     /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(9352..9358,9359..9376,9377..9382))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9397..9418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9609..10285
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     10222..10242
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             10352..11377
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     polyA_signal    11418..11592
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11670..11694
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             12119..12913
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      13000..13588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13489..13508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13742..13759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13774..13914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13840..13859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     14000..14022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      14258..14452
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(14518..15591)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(16020..16649)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac
       61 aatctgatcc aagctcaagc tgctctagca ttcgccattc aggctgcgca actgttggga
      121 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
      181 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
      241 cagtgccaag cttcgacttg ccttccgcac aatacatcat ttcttcttag ctttttttct
      301 tcttcttcgt tcatacagtt tttttttgtt tatcagctta cattttcttg aaccgtagct
      361 ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc atagtttgtc
      421 ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa aacatcttca
      481 ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga agagaagcag
      541 gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta agttgaaaac
      601 aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta tatacagcta
      661 gagtcgaagt agtgattggg agaccaaccc agtggacata agcctgttcg gttcgtaagc
      721 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
      781 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
      841 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
      901 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatgg
      961 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     1021 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     1081 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     1141 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     1201 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     1261 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     1321 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     1381 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     1441 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     1501 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     1561 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     1621 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     1681 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     1741 gcaaataatg tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac
     1801 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg
     1861 cttttattat ttttaagcgt gcataataag ccggtctcgg ttttagagct agaaatagca
     1921 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1981 tttgcaaaat tttccagatc gatttcttct tcctctgttc ttcggcgttc aatttctggg
     2041 gttttctctt cgttttctgt aactgaaacc taaaatttga cctaaaaaaa atctcaaata
     2101 atatgattca gtggttttgt acttttcagt tagttgagtt ttgcagttcc gatgagataa
     2161 accaatacca tggttatact agtgaataaa agcatttgcg tttggtttat cattgcgttt
     2221 atacaaggac agagatccac tgagctggaa tagcttaaaa ccattatcag aacaaaataa
     2281 accatttttt gttaagaatc agagcatagt aaacaacaga aacaacctaa gagaggtaac
     2341 ttgtccaaga agatagctaa ttatatctat tttataaaag ttatcatagt ttgtaagtca
     2401 caaaagatgc aaataacaga gaaactagga gacttgagaa tatacattct tgtatatttg
     2461 tattcgagat tgtgaaaatt tgaccataag tttaaattct taaaaagata tatctgatct
     2521 aggtgatggt tatagactgt aattttacca catgtttaat gatggatagt gacacacatg
     2581 acacatcgac aacactatag catcttattt agattacaac atgaaatttt tctgtaatac
     2641 atgtctttgt acataattta aaagtaattc ctaagaaata tatttataca aggagtttaa
     2701 agaaaacata gcataaagtt caatgagtag taaaaaccat atacagtata tagcataaag
     2761 ttcaatgagt ttattacaaa agcattggtt cactttctgt aacacgacgt taaaccttcg
     2821 tctccaatag gagcgctact gattcaacat gccaatatat actaaatacg tttctacagt
     2881 caaatgcttt aacgtttcat gattaagtga ctatttaccg tcaatccttt cccattcctc
     2941 ccactaatcc aactttttaa ttactcttaa atcaccacta agctagtaac gcctatcatg
     3001 aattagctct actaaatcta gcaacctttc aaatttgcag tattgcaggt gtctctgtgt
     3061 ctttaaaata gttgccttat gatttcttcg gtttcaagat gatcaaatag ttatagattt
     3121 catgctcaca catgctcatt agatgtgtac atactttact tacccaaatc tattttctcg
     3181 caaagatttt gatggtaaag ctgatttggt tctattgaac taaatcaaac gagtttcaga
     3241 ctgagtgatt ctaatccggc ccattagccc ctaaacagac ccactaatta cgcagctttt
     3301 aatagagtaa ttacacctag tttacccact aaaccactaa gcactaatta tctcacaatc
     3361 taatgagctt ccctcgtaat tacttgggct ttcactctac catttatttg taacagtcaa
     3421 gtctctactg tctctatata aactctctaa agttaacaca caattctcat cacaaacaaa
     3481 tcaaccaaag caacttctac tctttcttct ttcgacctta tcaatctgtt gagaaatcta
     3541 ggatggaggc cagcccggct agcggcccaa ggcatctcat ggacccgcac atcttcacca
     3601 gcaacttcaa caacggcatc ggcaggcaca agacctactt gtgctacgag gtggagaggc
     3661 tcgacaacgg aacctccgtg aagatggacc aacacagggg gttcctccac aaccaagcca
     3721 agaacctcct ctgcggcttc tacggcaggc acgccgagtt gaggttcctc gacttggtgc
     3781 catccctcca actcgatcca gcccaaatct accgcgtgac ctggttcatc tcctggtccc
     3841 catgcttctc ctggggttgc gccggcgagg ttcgggcttt cctccaagaa aacacccacg
     3901 tccgcctccg cattttcgcc gccaggatct atgattacga ccctctctac aaggaggccc
     3961 tccagatgct gcgggacgcc ggtgctcagg tgagtatcat gacctacgac gagttcaagc
     4021 actgctggga caccttcgtt gaccaccagg gctgcccatt ccaaccatgg gacggtctgg
     4081 atgaacacag ccaagccttg tccggcaggc tccgggccat cctccaaaac caggggaact
     4141 ccgggagcga gacgccaggc acctccgagt cggccacccc agaatctagg cctgacaaga
     4201 agtactcgat cggcctcgcc attgggacta actctgttgg ctgggccgtg atcaccgacg
     4261 agtacaaggt gccctcaaag aagttcaagg tcctgggcaa caccgatcgg cattccatca
     4321 agaagaatct cattggcgct ctcctgttcg acagcggcga gacggctgag gctacgcggc
     4381 tcaagcgcac cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg
     4441 agattttctc caacgagatg gcgaaggttg acgattcttt cttccacagg ctggaggagt
     4501 cattcctcgt ggaggaggat aagaagcacg agcggcatcc aatcttcggc aacattgtcg
     4561 acgaggttgc ctaccacgag aagtacccta cgatctacca tctgcggaag aagctcgtgg
     4621 actccacaga taaggcggac ctccgcctga tctacctcgc tctggcccac atgattaagt
     4681 tcaggggcca tttcctgatc gagggggatc tcaacccgga caatagcgat gttgacaagc
     4741 tgttcatcca gctcgtgcag acgtacaacc agctcttcga ggagaacccc attaatgcgt
     4801 caggcgtcga cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg cgcctcgaga
     4861 acctgatcgc ccagctgccg ggcgagaaga agaacggcct gttcgggaat ctcattgcgc
     4921 tcagcctggg gctcacgccc aacttcaagt cgaatttcga tctcgctgag gacgccaagc
     4981 tgcagctctc caaggacaca tacgacgatg acctggataa cctcctggcc cagatcggcg
     5041 atcagtacgc ggacctgttc ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg
     5101 atattctcag ggtgaacacc gagattacga aggctccgct ctcagcctcc atgatcaagc
     5161 gctacgacga gcaccatcag gatctgaccc tcctgaaggc gctggtcagg cagcagctcc
     5221 ccgagaagta caaggagatc ttcttcgatc agtcgaagaa cggctacgct gggtacattg
     5281 acggcggggc ctctcaggag gagttctaca agttcatcaa gccgattctg gagaagatgg
     5341 acggcacgga ggagctgctg gtgaagctca atcgcgagga cctcctgagg aagcagcgga
     5401 cattcgataa cggcagcatc ccacaccaga ttcatctcgg ggagctgcac gctatcctga
     5461 ggaggcagga ggacttctac cctttcctca aggataaccg cgagaagatc gagaagattc
     5521 tgactttcag gatcccgtac tacgtcggcc cactcgctag gggcaactcc cgcttcgctt
     5581 ggatgacccg caagtcagag gagacgatca cgccgtggaa cttcgaggag gtggtcgaca
     5641 agggcgctag cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa
     5701 atgagaaggt gctccctaag cactcgctcc tgtacgagta cttcacagtc tacaacgagc
     5761 tgactaaggt gaagtatgtg accgagggca tgaggaagcc ggctttcctg tctggggagc
     5821 agaagaaggc catcgtggac ctcctgttca agaccaaccg gaaggtcacg gttaagcagc
     5881 tcaaggagga ctacttcaag aagattgagt gcttcgattc ggtcgagatc tctggcgttg
     5941 aggaccgctt caacgcctcc ctggggacct accacgatct cctgaagatc attaaggata
     6001 aggacttcct ggacaacgag gagaatgagg atatcctcga ggacattgtg ctgacactca
     6061 ctctgttcga ggaccgggag atgatcgagg agcgcctgaa gacttacgcc catctcttcg
     6121 atgacaaggt catgaagcag ctcaagagga ggaggtacac cggctggggg aggctgagca
     6181 ggaagctcat caacggcatt cgggacaagc agtccgggaa gacgatcctc gacttcctga
     6241 agagcgatgg cttcgcgaac cgcaatttca tgcagctgat tcacgatgac agcctcacat
     6301 tcaaggagga tatccagaag gctcaggtga gcggccaggg ggactcgctg cacgagcata
     6361 tcgcgaacct cgctggctcg ccagctatca agaaggggat tctgcagacc gtgaaggttg
     6421 tggacgagct ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc attgagatgg
     6481 cccgggagaa tcagaccacg cagaagggcc agaagaactc acgcgagagg atgaagagga
     6541 tcgaggaggg cattaaggag ctggggtccc agatcctcaa ggagcacccg gtggagaaca
     6601 cgcagctgca gaatgagaag ctctacctgt actacctcca gaatggccgc gatatgtatg
     6661 tggaccagga gctggatatt aacaggctca gcgattacga cgtcgatcat atcgttccac
     6721 agtcattcct gaaggatgac tccattgaca acaaggtcct caccaggtcg gacaagaacc
     6781 ggggcaagtc tgataatgtt ccttcagagg aggtcgttaa gaagatgaag aactactggc
     6841 gccagctcct gaatgccaag ctgatcacgc agcggaagtt cgataacctc acaaaggctg
     6901 agaggggcgg gctctctgag ctggacaagg cgggcttcat caagaggcag ctggtcgaga
     6961 cacggcagat cactaagcac gttgcgcaga ttctcgactc acggatgaac actaagtacg
     7021 atgagaatga caagctgatc cgcgaggtga aggtcatcac cctgaagtca aagctcgtct
     7081 ccgacttcag gaaggatttc cagttctaca aggttcggga gatcaacaat taccaccatg
     7141 cccatgacgc gtacctgaac gcggtggtcg gcacagctct gatcaagaag tacccaaagc
     7201 tcgagagcga gttcgtgtac ggggactaca aggtttacga tgtgaggaag atgatcgcca
     7261 agtcggagca ggagattggc aaggctaccg ccaagtactt cttctactct aacattatga
     7321 atttcttcaa gacagagatc actctggcca atggcgagat ccggaagcgc cccctcatcg
     7381 agacgaacgg cgagacgggg gagatcgtgt gggacaaggg cagggatttc gcgaccgtca
     7441 ggaaggttct ctccatgcca caagtgaata tcgtcaagaa gacagaggtc cagactggcg
     7501 ggttctctaa ggagtcaatt ctgcctaagc ggaacagcga caagctcatc gcccgcaaga
     7561 aggactggga tccgaagaag tacggcgggt tcgacagccc cactgtggcc tactcggtcc
     7621 tggttgtggc gaaggttgag aagggcaagt ccaagaagct caagagcgtg aaggagctgc
     7681 tggggatcac gattatggag cgctccagct tcgagaagaa cccgatcgat ttcctggagg
     7741 cgaagggcta caaggaggtg aagaaggacc tgatcattaa gctccccaag tactcactct
     7801 tcgagctgga gaacggcagg aagcggatgc tggcttccgc tggcgagctg cagaagggga
     7861 acgagctggc tctgccgtcc aagtatgtga acttcctcta cctggcctcc cactacgaga
     7921 agctcaaggg cagccccgag gacaacgagc agaagcagct gttcgtcgag cagcacaagc
     7981 attacctcga cgagatcatt gagcagattt ccgagttctc caagcgcgtg atcctggccg
     8041 acgcgaatct ggataaggtc ctctccgcgt acaacaagca ccgcgacaag ccaatcaggg
     8101 agcaggctga gaatatcatt catctcttca ccctgacgaa cctcggcgcc cctgctgctt
     8161 tcaagtactt cgacacaact atcgatcgca agaggtacac aagcactaag gaggtcctgg
     8221 acgcgaccct catccaccag tcgattaccg gcctctacga gacgcgcatc gacctgtctc
     8281 agctcggggg cgacaagcgg ccagcggcga cgaagaaggc ggggcaggcg aagaagaaga
     8341 agacccgcga ctccggcggc agcacgaacc tctccgacat catcgagaag gagacgggca
     8401 agcagctcgt gatccaggag agcatcctca tgctgccgga ggaggtggag gaggtcatcg
     8461 gcaacaagcc cgagtccgac atcctcgtgc acaccgccta cgacgagtcc acggacgaga
     8521 acgtcatgct cctgacgagc gacgctccag agtacaagcc atgggctctc gtgatccagg
     8581 acagcaacgg cgagaacaag atcaagatgc tgtccggcgg ctccccaaag aagaagcgga
     8641 aggtgtagga gctcagagct ttcgttcgta tcatcggttt cgacaacgtt cgtcaagttc
     8701 aatgcatcag tttcattgcg cacacaccag aatcctactg agtttgagta ttatggcatt
     8761 gggaaaactg tttttcttgt accatttgtt gtgcttgtaa tttactgtgt tttttattcg
     8821 gttttcgcta tcgaactgtg aaatggaaat ggatggagaa gagttaatga atgatatggt
     8881 ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt gtgtgttgaa
     8941 tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt caaatcgtgg
     9001 cctctaatga ccgaagttaa tatgaggagt aaaacacttg tagttgtacc attatgctta
     9061 ttcactaggc aacaaatata ttttcagacc tagaaaagct gcaaatgtta ctgaatacaa
     9121 gtatgtcctc ttgtgtttta gacatttatg aactttcctt tatgtaattt tccagaatcc
     9181 ttgtcagatt ctaatcattg ctttataatt atagttatac tcatggattt gtagttgagt
     9241 atgaaaatat tttttaatgc attttatgac ttgccaattg attgacaacg aattcgtaat
     9301 catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     9361 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     9421 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     9481 aatcggccaa cgcgcgggga gaggcggttt gcgtattggc tagagcagct tgccaacatg
     9541 gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc agaagaccaa
     9601 agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc
     9661 ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc
     9721 catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag tggtcccaaa
     9781 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca
     9841 aagcaagtgg attgatgtga acatggtgga gcacgacact ctcgtctact ccaagaatat
     9901 caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc
     9961 gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga
    10021 aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga
    10081 tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa
    10141 agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct ccactgacgt
    10201 aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc
    10261 atttcatttg gagaggacac gctgaaatca ccagtctctc tctacaaatc tatctctctc
    10321 gagctttcgc agatccgggg ggcaatgaga tatgaaaaag cctgaactca ccgcgacgtc
    10381 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
    10441 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
    10501 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
    10561 cgcgctcccg attccggaag tgcttgacat tggggagttt agcgagagcc tgacctattg
    10621 catctcccgc cgttcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
    10681 tgttctacaa ccggtcgcgg aggctatgga tgcgatcgct gcggccgatc ttagccagac
    10741 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
    10801 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
    10861 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
    10921 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
    10981 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
    11041 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
    11101 gcggaggcat ccggagcttg caggatcgcc acgactccgg gcgtatatgc tccgcattgg
    11161 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
    11221 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
    11281 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
    11341 ccgacgcccc agcactcgtc cgagggcaaa gaaatagagt agatgccgac cgggatctgt
    11401 cgatcgacaa gctcgagttt ctccataata atgtgtgagt agttcccaga taagggaatt
    11461 agggttccta tagggtttcg ctcatgtgtt gagcatataa gaaaccctta gtatgtattt
    11521 gtatttgtaa aatacttcta tcaataaaat ttctaattcc taaaaccaaa atccagtact
    11581 aaaatccaga tcccccgaat taattcggcg ttaattcagt acattaaaaa cgtccgcaat
    11641 gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatcct gccaccagcc
    11701 agccaacagc tccccgaccg gcagctcggc acaaaatcac cactcgatac aggcagccca
    11761 tcagtccggg acggcgtcag cgggagagcc gttgtaaggc ggcagacttt gctcatgtta
    11821 ccgatgctat tcggaagaac ggcaactaag ctgccgggtt tgaaacacgg atgatctcgc
    11881 ggagggtagc atgttgattg taacgatgac agagcgttgc tgcctgtgat caccgcggtt
    11941 tcaaaatcgg ctccgtcgat actatgttat acgccaactt tgaaaacaac tttgaaaaag
    12001 ctgttttctg gtatttaagg ttttagaatg caaggaacag tgaattggag ttcgtcttgt
    12061 tataattagc ttcttggggt atctttaaat actgtagaaa agaggaagga aataataaat
    12121 ggctaaaatg agaatatcac cggaattgaa aaaactgatc gaaaaatacc gctgcgtaaa
    12181 agatacggaa ggaatgtctc ctgctaaggt atataagctg gtgggagaaa atgaaaacct
    12241 atatttaaaa atgacggaca gccggtataa agggaccacc tatgatgtgg aacgggaaaa
    12301 ggacatgatg ctatggctgg aaggaaagct gcctgttcca aaggtcctgc actttgaacg
    12361 gcatgatggc tggagcaatc tgctcatgag tgaggccgat ggcgtccttt gctcggaaga
    12421 gtatgaagat gaacaaagcc ctgaaaagat tatcgagctg tatgcggagt gcatcaggct
    12481 ctttcactcc atcgacatat cggattgtcc ctatacgaat agcttagaca gccgcttagc
    12541 cgaattggat tacttactga ataacgatct ggccgatgtg gattgcgaaa actgggaaga
    12601 agacactcca tttaaagatc cgcgcgagct gtatgatttt ttaaagacgg aaaagcccga
    12661 agaggaactt gtcttttccc acggcgacct gggagacagc aacatctttg tgaaagatgg
    12721 caaagtaagt ggctttattg atcttgggag aagcggcagg gcggacaagt ggtatgacat
    12781 tgccttctgc gtccggtcga tcagggagga tatcggggaa gaacagtatg tcgagctatt
    12841 ttttgactta ctggggatca agcctgattg ggagaaaata aaatattata ttttactgga
    12901 tgaattgttt tagtacctag aatgcatgac caaaatccct taacgtgagt tttcgttcca
    12961 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
    13021 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
    13081 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
    13141 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
    13201 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
    13261 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
    13321 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
    13381 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
    13441 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
    13501 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
    13561 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
    13621 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
    13681 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
    13741 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
    13801 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
    13861 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
    13921 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
    13981 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
    14041 aaacgcgcga ggcagggtgc cttgatgtgg gcgccggcgg tcgagtggcg acggcgcggc
    14101 ttgtccgcgc cctggtagat tgcctggccg taggccagcc atttttgagc ggccagcggc
    14161 cgcgataggc cgacgcgaag cggcggggcg tagggagcgc agcgaccgaa gggtaggcgc
    14221 tttttgcagc tcttcggctg tgcgctggcc agacagttat gcacaggcca ggcgggtttt
    14281 aagagtttta ataagtttta aagagtttta ggcggaaaaa tcgccttttt tctcttttat
    14341 atcagtcact tacatgtgtg accggttccc aatgtacggc tttgggttcc caatgtacgg
    14401 gttccggttc ccaatgtacg gctttgggtt cccaatgtac gtgctatcca caggaaacag
    14461 accttttcga cctttttccc ctgctagggc aatttgccct agcatctgct ccgtacatta
    14521 ggaaccggcg gatgcttcgc cctcgatcag gttgcggtag cgcatgacta ggatcgggcc
    14581 agcctgcccc gcctcctcct tcaaatcgta ctccggcagg tcatttgacc cgatcagctt
    14641 gcgcacggtg aaacagaact tcttgaactc tccggcgctg ccactgcgtt cgtagatcgt
    14701 cttgaacaac catctggctt ctgccttgcc tgcggcgcgg cgtgccaggc ggtagagaaa
    14761 acggccgatg ccgggatcga tcaaaaagta atcggggtga accgtcagca cgtccgggtt
    14821 cttgccttct gtgatctcgc ggtacatcca atcagctagc tcgatctcga tgtactccgg
    14881 ccgcccggtt tcgctcttta cgatcttgta gcggctaatc aaggcttcac cctcggatac
    14941 cgtcaccagg cggccgttct tggccttctt cgtacgctgc atggcaacgt gcgtggtgtt
    15001 taaccgaatg caggtttcta ccaggtcgtc tttctgcttt ccgccatcgg ctcgccggca
    15061 gaacttgagt acgtccgcaa cgtgtggacg gaacacgcgg ccgggcttgt ctcccttccc
    15121 ttcccggtat cggttcatgg attcggttag atgggaaacc gccatcagta ccaggtcgta
    15181 atcccacaca ctggccatgc cggccggccc tgcggaaacc tctacgtgcc cgtctggaag
    15241 ctcgtagcgg atcacctcgc cagctcgtcg gtcacgcttc gacagacgga aaacggccac
    15301 gtccatgatg ctgcgactat cgcgggtgcc cacgtcatag agcatcggaa cgaaaaaatc
    15361 tggttgctcg tcgcccttgg gcggcttcct aatcgacggc gcaccggctg ccggcggttg
    15421 ccgggattct ttgcggattc gatcagcggc cgcttgccac gattcaccgg ggcgtgcttc
    15481 tgcctcgatg cgttgccgct gggcggcctg cgcggccttc aacttctcca ccaggtcatc
    15541 acccagcgcc gcgccgattt gtaccgggcc ggatggtttg cgaccgctca cgccgattcc
    15601 tcgggcttgg gggttccagt gccattgcag ggccggcagg caacccagcc gcttacgcct
    15661 ggccaaccgc ccgttcctcc acacatgggg cattccacgg cgtcggtgcc tggttgttct
    15721 tgattttcca tgccgcctcc tttagccgct aaaattcatc tactcattta ttcatttgct
    15781 catttactct ggtagctgcg cgatgtattc agatagcagc tcggtaatgg tcttgccttg
    15841 gcgtaccgcg tacatcttca gcttggtgtg atcctccgcc ggcaactgaa agttgacccg
    15901 cttcatggct ggcgtgtctg ccaggctggc caacgttgca gccttgctgc tgcgtgcgct
    15961 cggacggccg gcacttagcg tgtttgtgct tttgctcatt ttctctttac ctcattaact
    16021 caaatgagtt ttgatttaat ttcagcggcc agcgcctgga cctcgcgggc agcgtcgccc
    16081 tcgggttctg attcaagaac ggttgtgccg gcggcggcag tgcctgggta gctcacgcgc
    16141 tgcgtgatac gggactcaag aatgggcagc tcgtacccgg ccagcgcctc ggcaacctca
    16201 ccgccgatgc gcgtgccttt gatcgcccgc gacacgacaa aggccgcttg tagccttcca
    16261 tccgtgacct caatgcgctg cttaaccagc tccaccaggt cggcggtggc ccatatgtcg
    16321 taagggcttg gctgcaccgg aatcagcacg aagtcggctg ccttgatcgc ggacacagcc
    16381 aagtccgccg cctggggcgc tccgtcgatc actacgaagt cgcgccggcc gatggccttc
    16441 acgtcgcggt caatcgtcgg gcggtcgatg ccgacaacgg ttagcggttg atcttcccgc
    16501 acggccgccc aatcgcgggc actgccctgg ggatcggaat cgactaacag aacatcggcc
    16561 ccggcgagtt gcagggcgcg ggctagatgg gttgcgatgg tcgtcttgcc tgacccgcct
    16621 ttctggttaa gtacagcgat aaccttcatg cgttcccctt gcgtatttgt ttatttactc
    16681 atcgcatcat atacgcagcg accgcatgac gcaagctgtt ttactcaaat acacatcacc
    16741 tttttagacg gcggcgctcg gtttcttcag cggccaagct ggccggccag gccgccagct
    16801 tggcatcaga caaaccggcc aggatttcat gcagccgcac ggttgagacg tgcgcgggcg
    16861 gctcgaacac gtacccggcc gcgatcatct ccgcctcgat ctcttcggta atgaaaaacg
    16921 gttcgtcctg gccgtcctgg tgcggtttca tgcttgttcc tcttggcgtt cattctcggc
    16981 ggccgccagg gcgtcggcct cggtcaatgc gtcctcacgg aaggcaccgc gccgcctggc
    17041 ctcggtgggc gtcacttcct cgctgcgctc aagtgcgcgg tacagggtcg agcgatgcac
    17101 gccaagcagt gcagccgcct ctttcacggt gcggccttcc tggtcgatca gctcgcgggc
    17161 gtgcgcgatc tgtgccgggg tgagggtagg gcgggggcca aacttcacgc ctcgggcctt
    17221 ggcggcctcg cgcccgctcc gggtgcggtc gatgattagg gaacgctcga actcggcaat
    17281 gccggcgaac acggtcaaca ccatgcggcc ggccggcgtg gtggtgtcgg cccacggctc
    17341 tgccaggcta cgcaggcccg cgccggcctc ctggatgcgc tcggcaatgt ccagtaggtc
    17401 gcgggtgctg cgggccaggc ggtctagcct ggtcactgtc acaacgtcgc cagggcgtag
    17461 gtggtcaagc atcctggcca gctccgggcg gtcgcgcctg gtgccggtga tcttctcgga
    17521 aaacagcttg gtgcagccgg ccgcgtgcag ttcggcccgt tggttggtca agtcctggtc
    17581 gtcggtgctg acgcgggcat agcccagcag gccagcggcg gcgctcttgt tcatggcgta
    17641 atgtctccgg ttctagtcgc aagtattcta ctttatgcga ctaaaacacg cgacaagaaa
    17701 acgccaggaa aagggcaggg cggcagcctg tcgcgtaact taggacttgt gcgacatgtc
    17761 gttttcagaa gacggctgca ctgaacgtca gaagccgact gcactatagc agcggagggg
    17821 ttggatcaaa gtactttgat cccgagggga accctgtggt tggcatgcac atacaaatgg
    17881 acgaacggat aaaccttttc acgccctttt aaatatccga ttattctaat aaacgctctt
    17941 ttctcttag
//
