LOCUS       sequence-182272-       18623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: ZmUbi:TaCas9_dead 
            + TaU6:gRNA , Plant Selection: PvUbi2:hpt II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 18623)
  AUTHORS   Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, 
            Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF
  TITLE     A multi-purpose toolkit to enable advanced genome engineering in 
            plants.
  JOURNAL   Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 
            10.1105/tpc.16.00922.
  PUBMED    28522548
REFERENCE   2  (bases 1 to 18623)
  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: "Plant Cell.
            2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922."
COMMENT     Sequence Label: pDIRECT_25G
FEATURES             Location/Qualifiers
     source          1..18623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1233..1862
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2291..3364
                     /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"
     rep_origin      3430..3624
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3860..3882)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3968..4108
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4023..4042
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4123..4140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4294..4882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4374..4393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6307..7101)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    7526..7550
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    7628..7802
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(7843..8868)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     protein_bind    10808..10829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10844..10849,10850..10867,10868..10874)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10882..10898
                     /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)."
     primer_bind     10906..10922
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(12978..13004,13005..13007,13008..15494,15495..15497,
                     15498..17081)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             17082..17129
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      17140..17388
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     CDS             complement(17766..18071)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGTCL"
     primer_bind     17936..17952
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     17962..17981
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        17962..17980
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(18028..18048)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(18028..18046)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(18067..18083)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    18091..18107
                     /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(18115..18121,18122..18139,18140..18145))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    18160..18181
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        18220..18295
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(18312..18328)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    18531..18555
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta ctttgatcca
       61 acccctccgc tgctatagtg cagtcggctt ctgacgttca gtgcagccgt cttctgaaaa
      121 cgacatgtcg cacaagtcct aagttacgcg acaggctgcc gccctgccct tttcctggcg
      181 ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact tgcgactaga accggagaca
      241 ttacgccatg aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga
      301 cgaccaggac ttgaccaacc aacgggccga actgcacgcg gccggctgca ccaagctgtt
      361 ttccgagaag atcaccggca ccaggcgcga ccgcccggag ctggccagga tgcttgacca
      421 cctacgccct ggcgacgttg tgacagtgac caggctagac cgcctggccc gcagcacccg
      481 cgacctactg gacattgccg agcgcatcca ggaggccggc gcgggcctgc gtagcctggc
      541 agagccgtgg gccgacacca ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg
      601 cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc
      661 caaggcccga ggcgtgaagt ttggcccccg ccctaccctc accccggcac agatcgcgca
      721 cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg cactgcttgg
      781 cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc gaggaagtga cgcccaccga
      841 ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc gaggccgacg ccctggcggc
      901 cgccgagaat gaacgccaag aggaacaagc atgaaaccgc accaggacgg ccaggacgaa
      961 ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg cggccgggta cgtgttcgag
     1021 ccgcccgcgc acggctcaac cgtgcggctg catgaaatcc tggccggttt gtctgatgcc
     1081 aagctggcgg cctggccggc cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa
     1141 aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga
     1201 tgagtaaata aacaaatacg caaggggaac gcatgaaggt tatcgctgta cttaaccaga
     1261 aaggcgggtc aggcaagacg accatcgcaa cccatctagc ccgcgccctg caactcgccg
     1321 gggccgatgt tctgttagtc gattccgatc cccagggcag tgcccgcgat tgggcggccg
     1381 tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg
     1441 tgaaggccat cggccggcgc gacttcgtag tgatcgacgg agcgccccag gcggcggact
     1501 tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat tccggtgcag ccaagccctt
     1561 acgacatatg ggccaccgcc gacctggtgg agctggttaa gcagcgcatt gaggtcacgg
     1621 atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg
     1681 gtgaggttgc cgaggcgctg gccgggtacg agctgcccat tcttgagtcc cgtatcacgc
     1741 agcgcgtgag ctacccaggc actgccgccg ccggcacaac cgttcttgaa tcagaacccg
     1801 agggcgacgc tgcccgcgag gtccaggcgc tggccgctga aattaaatca aaactcattt
     1861 gagttaatga ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc
     1921 gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa
     1981 gcgggtcaac tttcagttgc cggcggagga tcacaccaag ctgaagatgt acgcggtacg
     2041 ccaaggcaag accattaccg agctgctatc tgaatacatc gcgcagctac cagagtaaat
     2101 gagcaaatga ataaatgagt agatgaattt tagcggctaa aggaggcggc atggaaaatc
     2161 aagaacaacc aggcaccgac gccgtggaat gccccatgtg tggaggaacg ggcggttggc
     2221 caggcgtaag cggctgggtt gcctgccggc cctgcaatgg cactggaacc cccaagcccg
     2281 aggaatcggc gtgagcggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg
     2341 tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc
     2401 agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg
     2461 gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc
     2521 agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga
     2581 cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga
     2641 gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga
     2701 ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga
     2761 agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt
     2821 ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt
     2881 aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac
     2941 ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg
     3001 gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa
     3061 gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg
     3121 ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa
     3181 gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg
     3241 caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc
     3301 tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc
     3361 ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg
     3421 cctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa
     3481 cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga
     3541 tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct
     3601 taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagcccaag agctgcaaaa
     3661 agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc
     3721 ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca
     3781 agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt
     3841 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggaaacggtc acagcttgtc
     3901 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3961 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     4021 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     4081 atgcgtaagg agaaaatacc gcatcaggcc ctcttccgct tcctcgctca ctgactcgct
     4141 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4201 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4261 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4321 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4381 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4441 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4501 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4561 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4621 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4681 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4741 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4801 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4861 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4921 gtgctgatga atcccctaat gatttttatc aaaatcatta agttaaggta gatacacatc
     4981 ttgtcatatg atcaaatggt ttcgccaaaa atcaataatc agacaacaaa atgtgcgaac
     5041 tcgatatttt acacgactct ctttaccaat tctgccccga attacactta aaacgactca
     5101 acagcttaac gttggcttgc cacgccttac ttgactgtaa aactctcact cttaccgaac
     5161 ttggccgtaa cctgccaacc aaagcgagaa caaaacataa catcaaacga atcgaccgat
     5221 tgttaggtaa tcgtcacctc cacaaagagc gactcgctgt ataccgttgg catgctagct
     5281 ttatctgttc gggcaatacg atgcccattg tacttgttga ctggtctgat atccgtgagc
     5341 aaaaacggct tatggtattg cgagcttcag tcgcactaca cggtcgttct gttactcttt
     5401 atgagaaagc gttcccgctt tcagagcaat gttcaaagaa agctcatgac caatttctag
     5461 ccgaccttgc gagcattcta ccgagtaaca ccacaccgct cattgtcagt gatgctggct
     5521 ttaaagtgcc atggtataaa tccgttgaga agctgggttg gtactggtta agtcgagtaa
     5581 gaggaaaagt acaatatgca gacctaggag cggaaaactg gaaacctatc agcaacttac
     5641 atgatatgtc atctagtcac tcaaagactt taggctataa gaggctgact aaaagcaatc
     5701 caatctcatg ccaaattcta ttgtataaat ctcgctctaa aggccgaaaa aatcagcgct
     5761 cgacacggac tcattgtcac cacccgtcac ctaaaatcta ctcagcgtcg gcaaaggagc
     5821 catggattct agcaactaac ttacctgttg aaattcgaac acccaaacaa cttgttaata
     5881 tctattcgaa gcgaatgcag attgaagaaa ccttccgaga cttgaaaagt cctgcctacg
     5941 gactaggcct acgccatagc cgaacgagca gctcagagcg ttttgatatc atgctgctaa
     6001 tcgccctgat gcttcaacta acatgttggc ttgcgggcgt tcatgctcag aaacaaggtt
     6061 gggacaagca cttccaggct aacacagtca gaaatcgaaa cgtactctca acagttcgct
     6121 taggcatgga agttttgcgg cattctggct acacaataac aagggaagac ttactcgtgg
     6181 ctgcaaccct actagctcaa aatttattca cacatggtta cgctttgggg aaattatgag
     6241 gggatctctc agcgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc
     6301 taggtactaa aacaattcat ccagtaaaat ataatatttt attttctccc aatcaggctt
     6361 gatccccagt aagtcaaaaa atagctcgac atactgttct tccccgatat cctccctgat
     6421 cgaccggacg cagaaggcaa tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc
     6481 aataaagcca cttactttgc catctttcac aaagatgttg ctgtctccca ggtcgccgtg
     6541 ggaaaagaca agttcctctt cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg
     6601 atctttaaat ggagtgtctt cttcccagtt ttcgcaatcc acatcggcca gatcgttatt
     6661 cagtaagtaa tccaattcgg ctaagcggct gtctaagcta ttcgtatagg gacaatccga
     6721 tatgtcgatg gagtgaaaga gcctgatgca ctccgcatac agctcgataa tcttttcagg
     6781 gctttgttca tcttcatact cttccgagca aaggacgcca tcggcctcac tcatgagcag
     6841 attgctccag ccatcatgcc gttcaaagtg caggaccttt ggaacaggca gctttccttc
     6901 cagccatagc atcatgtcct tttcccgttc cacatcatag gtggtccctt tataccggct
     6961 gtccgtcatt tttaaatata ggttttcatt ttctcccacc agcttatata ccttagcagg
     7021 agacattcct tccgtatctt ttacgcagcg gtatttttcg atcagttttt tcaattccgg
     7081 tgatattctc attttagcca tttattattt ccttcctctt ttctacagta tttaaagata
     7141 ccccaagaag ctaattataa caagacgaac tccaattcac tgttccttgc attctaaaac
     7201 cttaaatacc agaaaacagc tttttcaaag ttgttttcaa agttggcgta taacatagta
     7261 tcgacggagc cgattttgaa accgcggtga tcacaggcag caacgctctg tcatcgttac
     7321 aatcaacatg ctaccctccg cgagatcatc cgtgtttcaa acccggcagc ttagttgccg
     7381 ttcttccgaa tagcatcggt aacatgagca aagtctgccg ccttacaacg gctctcccgc
     7441 tgacgccgtc ccggactgat gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc
     7501 ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac aaattgacgc
     7561 ttagacaact taataacaca ttgcggacgt ttttaatgta ctgaattaac gccgaattaa
     7621 ttcgggggat ctggatttta gtactggatt ttggttttag gaattagaaa ttttattgat
     7681 agaagtattt tacaaataca aatacatact aagggtttct tatatgctca acacatgagc
     7741 gaaaccctat aggaacccta attcccttat ctgggaacta ctcacacatt attatggaga
     7801 aactcgagct tgtcgatcga cagatcccgg tcggcatcta ctctatttct ttgccctcgg
     7861 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
     7921 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
     7981 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
     8041 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg gcgatcctgc
     8101 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
     8161 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
     8221 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
     8281 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
     8341 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
     8401 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
     8461 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatagcctc
     8521 cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
     8581 ctgtgaacgg cgggagatgc aataggtcag gctctcgcta aactccccaa tgtcaagcac
     8641 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
     8701 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
     8761 agcacgagat tcttcgccct ccgagagctg catcaggtcg gacacgctgt cgaacttttc
     8821 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcataa gcttctgcaa
     8881 aagagaacca gacaacaggg taagtgccta gcagtaaaca aacagaactc atcacaagca
     8941 aacagcaaca tcatattcat accaacaggt catgtgtgtt catcacatca ttagtactaa
     9001 gcatgccatc atccaagtat atcaaagtaa gggcaaagag catgcatgat catcaggtgc
     9061 acaaaagaat catcaaattg tagcagtaca tatcttcatc tatcatgcat atctatccat
     9121 aacaggacga tgcatgttga ccaggtaaaa gctacaggat cctataggaa cagcagcgta
     9181 tatatcttca ccaatcttag catcttaatc atgtggcaca tgcagtttca atttaagcac
     9241 atgagctagt tgattatgag gtaccagaga atcatcaaac tgatgtagca gcatatatcc
     9301 tcatctatca tgcaatctaa tctaatctaa ctaaacagga aaggtgtgct attcagttaa
     9361 aagctaccgc atcatataca aacggcagca taagaaaaag cataatcatc ttaatcatgc
     9421 aacaaacgca gattcataat aagcccaaga gctagcttgt gatgatctta ttctactctg
     9481 atctacagca atcagataac gacctaacct tgcacatggc aacaaaacaa tcgatcggac
     9541 gaatcagttg tttgttccta gctagcacca tcgaaccaga taatagatgc acgtacagat
     9601 cccgaaaacg aacccaaaaa cagggcagac ctagctgaac ctaggcagcg acccagcaga
     9661 tcgtgagaac gatctcatct acgaacagcc tagaagcaac cccacgattc ccggacaaac
     9721 gacctaaaat ccccacaaat cacatgagca tgacaggata aacagcggaa ccgatcagat
     9781 ctacacgaaa accccacctc ccagccaccc acgatcagga aacacgcgga tctagcatga
     9841 tttcgtcaac gcctcagcct agttcctagc cacagaccaa gcagaaccac caaaccacgc
     9901 cgagcgagga gatggggcaa gaggacgggg gagacgatcg ccgtaccttg aagcggggga
     9961 aggatcgccg agggtcgcga ggagagcaat ttggatttgg aacccggggg ttgtgcgctc
    10021 cgaacgatga gacgatgtga gattgtggga agaggcgcgg agggccctgt atttatgggc
    10081 tgcgacgggg ggaggagagg tggggagggt tggggaagga atcccccacc cgtgccgtga
    10141 cggttccggg ccgtgtgaga ggagcccgct cgtctccgcc acgcaatttc cgcgatcgga
    10201 gcggagcttt cgagaggcgg ctggatggtt ggtggccgtt agatttgtag acgccgttaa
    10261 cgcctcgcct ccaccgggaa gagttttgag cagccgctta tgacaatggc ttaacgacgt
    10321 tagacggagc gttagtggca ggccatgata gaatagacgt attgcaatgg gattataatt
    10381 aaataaataa gaatataata agatatggca agtcggcact catgacatgg tcttcgaaat
    10441 gatagtgctc actttcttag ccgagaaagt tgacgcgact gatttagaag ttaagatatt
    10501 atttctctct tcttttcttt cctcgtcata taaggatgaa ataaacttta gagattgccg
    10561 gtagtgattt tggatttcgg cgatcaggct tggtttgccg gtttcggacg gtgtgcctta
    10621 ggccacccgc agtgtatctt gtaatgttca accgataagc aagggtgggg ctcaagcaag
    10681 tagtaaacaa ctatgtcaaa tgtcaccatg gttatggtct tgtttagttg gcttcttggc
    10741 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
    10801 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
    10861 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
    10921 acatgattac gaattcgagc tcggtacccg gggatctgca ggcgcgcctg cagtgcagcg
    10981 tgacccggtc gtgcccctct ctagagataa tgagcattgc atgtctaagt tataaaaaat
    11041 taccacatat tttttttgtc acacttgttt gaagtgcagt ttatctatct ttatacatat
    11101 atttaaactt tactctacga ataatataat ctatagtact acaataatat cagtgtttta
    11161 gagaatcata taaatgaaca gttagacatg gtctaaagga caattgagta ttttgacaac
    11221 aggactctac agttttatct ttttagtgtg catgtgttct cctttttttt tgcaaatagc
    11281 ttcacctata taatacttca tccattttat tagtacatcc atttagggtt tagggttaat
    11341 ggtttttata gactaatttt tttagtacat ctattttatt ctattttagc ctctaaatta
    11401 agaaaactaa aactctattt tagttttttt atttaataat ttagatataa aatagaataa
    11461 aataaagtga ctaaaaatta aacaaatacc ctttaagaaa ttaaaaaaac taaggaaaca
    11521 tttttcttgt ttcgagtaga taatgccagc ctgttaaacg ccgtcgacga gtctaacgga
    11581 caccaaccag cgaaccagca gcgtcgcgtc gggccaagcg aagcagacgg cacggcatct
    11641 ctgtcgctgc ctctggaccc ctctcgagag ttccgctcca ccgttggact tgctccgctg
    11701 tcggcatcca gaaattgcgt ggcggagcgg cagacgtgag ccggcacggc aggcggcctc
    11761 ctcctcctct cacggcaccg gcagctacgg gggattcctt tcccaccgct ccttcgcttt
    11821 cccttcctcg cccgccgtaa taaatagaca ccccctccac accctctttc cccaacctcg
    11881 tgttgttcgg agcgcacaca cacacaacca gatctccccc aaatccaccc gtcggcacct
    11941 ccgcttcaag gtacgccgct cgtcctcccc ccccccctct ctaccttctc tagatcggcg
    12001 ttccggtcca tggttagggc ccggtagttc tacttctgtt catgtttgtg ttagatccgt
    12061 gtttgtgtta gatccgtgct gctagcgttc gtacacggat gcgacctgta cgtcagacac
    12121 gttctgattg ctaacttgcc agtgtttctc tttggggaat cctgggatgg ctctagccgt
    12181 tccgcagacg ggatcgattt catgattttt tttgtttcgt tgcatagggt ttggtttgcc
    12241 cttttccttt atttcaatat atgccgtgca cttgtttgtc gggtcatctt ttcatgcttt
    12301 tttttgtctt ggttgtgatg atgtggtctg gttgggcggt cgttctagat cggagtagaa
    12361 ttaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat gtgtgtgcca
    12421 tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta ggataggtat
    12481 acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc gcttggttgt
    12541 gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag aatactgttt
    12601 caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata catcttcata
    12661 gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg ttgatgtggg
    12721 ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct ctaaccttga
    12781 gtacctatct attataataa acaagtatgt tttataatta ttttgatctt gatatacttg
    12841 gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc atacgctatt
    12901 tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg ttacttcctg
    12961 cagggcgatc tattcgaatg gacaagaagt actcgatcgg cctcgccatc gggacgaact
    13021 cagttggctg ggccgtgatc accgacgagt acaaggtgcc ctctaagaag ttcaaggtcc
    13081 tggggaacac cgaccgccat tccatcaaga agaacctcat cggcgctctc ctgttcgaca
    13141 gcggggagac cgctgaggct acgaggctca agagaaccgc taggcgccgg tacacgagaa
    13201 ggaagaacag gatctgctac ctccaagaga ttttctccaa cgagatggcc aaggttgacg
    13261 attcattctt ccaccgcctg gaggagtctt tcctcgtgga ggaggataag aagcacgagc
    13321 ggcatcccat cttcggcaac atcgtggacg aggttgccta ccacgagaag taccctacga
    13381 tctaccatct gcggaagaag ctcgtggact ccaccgataa ggcggacctc agactgatct
    13441 acctcgctct ggcccacatg atcaagttcc gcggccattt cctgatcgag ggggatctca
    13501 acccagacaa cagcgatgtt gacaagctgt tcatccaact cgtgcagacc tacaaccaac
    13561 tcttcgagga gaacccgatc aacgcctctg gcgtggacgc gaaggctatc ctgtccgcga
    13621 ggctctcgaa gtccaggagg ctggagaacc tgatcgctca gctcccaggc gagaagaaga
    13681 acggcctgtt cgggaacctc atcgctctca gcctggggct caccccgaac ttcaagtcga
    13741 acttcgatct cgctgaggac gccaagctgc aactctccaa ggacacctac gacgatgacc
    13801 tcgataacct cctggcccag atcggcgatc aatacgcgga cctgttcctc gctgccaaga
    13861 acctgtcgga cgccatcctc ctgtcagata tcctccgcgt gaacaccgag atcacgaagg
    13921 ctccactctc tgcctccatg atcaagcgct acgacgagca ccatcaggat ctgaccctcc
    13981 tgaaggcgct ggtccgccaa cagctcccgg agaagtacaa ggagattttc ttcgatcagt
    14041 cgaagaacgg ctacgctggg tacatcgacg gcggggcctc acaagaggag ttctacaagt
    14101 tcatcaagcc aatcctggag aagatggacg gcacggagga gctcctggtg aagctcaaca
    14161 gggaggacct cctgcggaag cagagaacct tcgataacgg cagcatcccc caccaaatcc
    14221 atctcgggga gctgcacgcc atcctgagaa ggcaagagga cttctaccct ttcctcaagg
    14281 ataaccggga gaagatcgag aagatcctga ccttcagaat cccatactac gtcggccctc
    14341 tcgcgcgggg gaactcaaga ttcgcttgga tgacccgcaa gtctgaggag accatcacgc
    14401 cgtggaactt cgaggaggtg gtggacaagg gcgctagcgc tcagtcgttc atcgagagga
    14461 tgaccaactt cgacaagaac ctgcccaacg agaaggtgct ccctaagcac tcgctcctgt
    14521 acgagtactt caccgtctac aacgagctca cgaaggtgaa gtacgtcacc gagggcatgc
    14581 gcaagccagc gttcctgtcc ggggagcaga agaaggctat cgtggacctc ctgttcaaga
    14641 ccaaccggaa ggtcacggtt aagcaactca aggaggacta cttcaagaag atcgagtgct
    14701 tcgattcggt cgagatcagc ggcgttgagg accgcttcaa cgccagcctc gggacctacc
    14761 acgatctcct gaagatcatc aaggataagg acttcctgga caacgaggag aacgaggata
    14821 tcctggagga catcgtgctg accctcacgc tgttcgagga cagggagatg atcgaggagc
    14881 gcctgaagac gtacgcccat ctcttcgatg acaaggtcat gaagcaactc aagcgccgga
    14941 gatacaccgg ctgggggagg ctgtcccgca agctcatcaa cggcatccgg gacaagcagt
    15001 ccgggaagac catcctcgac ttcctgaaga gcgatggctt cgccaacagg aacttcatgc
    15061 aactgatcca cgatgacagc ctcaccttca aggaggatat ccaaaaggct caagtgagcg
    15121 gccaggggga ctcgctgcac gagcatatcg cgaacctcgc tggctccccc gcgatcaaga
    15181 agggcatcct ccagaccgtg aaggttgtgg acgagctcgt gaaggtcatg ggccggcaca
    15241 agcctgagaa catcgtcatc gagatggcca gagagaacca aaccacgcag aaggggcaaa
    15301 agaactctag ggagcgcatg aagcgcatcg aggagggcat caaggagctg gggtcccaaa
    15361 tcctcaagga gcacccagtg gagaacaccc aactgcagaa cgagaagctc tacctgtact
    15421 acctccagaa cggcagggat atgtacgtgg accaagagct ggatatcaac cgcctcagcg
    15481 attacgacgt cgatgctatc gttccccagt ctttcctgaa ggatgactcc atcgacaaca
    15541 aggtcctcac caggtcggac aagaaccgcg gcaagtcaga taacgttcca tctgaggagg
    15601 tcgttaagaa gatgaagaac tactggaggc agctcctgaa cgccaagctg atcacgcaaa
    15661 ggaagttcga caacctcacc aaggctgaga gaggcgggct ctcagagctg gacaaggccg
    15721 gcttcatcaa gcggcagctg gtcgagacca gacaaatcac gaagcacgtt gcgcaaatcc
    15781 tcgactctcg gatgaacacg aagtacgatg agaacgacaa gctgatcagg gaggttaagg
    15841 tgatcaccct gaagtctaag ctcgtctccg acttcaggaa ggatttccag ttctacaagg
    15901 ttcgcgagat caacaactac caccatgccc atgacgctta cctcaacgct gtggtcggca
    15961 ccgctctgat caagaagtac ccaaagctgg agtccgagtt cgtgtacggg gactacaagg
    16021 tttacgatgt gcgcaagatg atcgccaagt cggagcaaga gatcggcaag gctaccgcca
    16081 agtacttctt ctactcaaac atcatgaact tcttcaagac cgagatcacg ctggccaacg
    16141 gcgagatccg gaagagaccg ctcatcgaga ccaacggcga gacgggggag atcgtgtggg
    16201 acaagggcag ggatttcgcg accgtccgca aggttctctc catgccccag gtgaacatcg
    16261 tcaagaagac cgaggtccaa acgggcgggt tctcaaagga gtctatcctg cctaagcgga
    16321 acagcgacaa gctcatcgcc agaaagaagg actgggaccc aaagaagtac ggcgggttcg
    16381 acagccctac cgtggcctac tcggtcctgg ttgtggcgaa ggttgagaag ggcaagtcca
    16441 agaagctcaa gagcgtgaag gagctcctgg ggatcaccat catggagagg tccagcttcg
    16501 agaagaaccc aatcgacttc ctggaggcca agggctacaa ggaggtgaag aaggacctga
    16561 tcatcaagct cccgaagtac tctctcttcg agctggagaa cggcaggaag agaatgctgg
    16621 cttccgctgg cgagctccag aaggggaacg agctcgcgct gccaagcaag tacgtgaact
    16681 tcctctacct ggcttcccac tacgagaagc tcaagggcag cccggaggac aacgagcaaa
    16741 agcagctgtt cgtcgagcag cacaagcatt acctcgacga gatcatcgag caaatctccg
    16801 agttcagcaa gcgcgtgatc ctcgccgacg cgaacctgga taaggtcctc tccgcctaca
    16861 acaagcaccg ggacaagccc atcagagagc aagcggagaa catcatccat ctcttcaccc
    16921 tgacgaacct cggcgctcct gctgctttca agtacttcga caccacgatc gatcggaaga
    16981 gatacacctc cacgaaggag gtcctggacg cgaccctcat ccaccagtcg atcaccggcc
    17041 tgtacgagac gaggatcgac ctctcacaac tcggcgggga taagagaccc gcagcaacca
    17101 agaaggcagg gcaagcaaag aagaagaagt gactcgagat atgaagatga agatgaaata
    17161 tttggtgtgt caaataaaaa gcttgtgtgc ttaagtttgt gtttttttct tggcttgttg
    17221 tgttatgaat ttgtggcttt ttctaatatt aaatgaatgt aagatcacat tataatgaat
    17281 aaacaaatgt ttctataatc cattgtgaat gttttgttgg atctcttctg cagcatataa
    17341 ctactgtatg tgctatggta tggactatgg aatatgatta aagataagga gctccggtga
    17401 cggacgacca agcccgttat tctgacagtt ctggtgctca acacatttat atttatcaag
    17461 gagcacattg ttactcactg ctaggaggga atcgaactag gaatattgat cagaggaact
    17521 acgagagagc tgaagataac tgccctctag ctctcactga tctgggtcgc atagtgagat
    17581 gcagcccacg tgagttcagc aacggtctag cgctgggctt ttaggcccgc atgatcgggc
    17641 ttttgtcggg tggtcgacgt gttcacgatt ggggagagca acgcagcagt tcctcttagt
    17701 ttagtcccac ctcgcctgtc cagcagagtt ctgaccggtt tataaactcg cttgctgcat
    17761 cagacttaga ggcaggtgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
    17821 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
    17881 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
    17941 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
    18001 cccctcgagg tcctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
    18061 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
    18121 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
    18181 aattgcgttg cgctcactgc ccgctttcca cctgcggtcg ttttagagct agaaatagca
    18241 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
    18301 ttcccggtgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
    18361 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
    18421 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcta gagcagcttg
    18481 agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgacaggata
    18541 tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg
    18601 gcgtgaaaag gtttatccgt tcg
//
