LOCUS       sequence-182608-       17286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: ZmUbi:TaCas9 + 
            TaU6:gRNA, Plant Selection: PvUbi2:hpt II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17286)
  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 17286)
  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_25F
FEATURES             Location/Qualifiers
     source          1..17286
                     /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(4969..5763)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6188..6212
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    6290..6464
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(6505..7530)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     protein_bind    9470..9491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9506..9511,9512..9529,9530..9536)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9544..9560
                     /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     9568..9584
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             11641..15744
                     /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             11641..15744
                     /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             15745..15792
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      15803..16051
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     CDS             complement(16429..16734)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGTCL"
     primer_bind     16599..16615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     16625..16644
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        16625..16643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(16691..16711)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(16691..16709)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(16730..16746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    16754..16770
                     /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(16778..16784,16785..16802,16803..16808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    16823..16844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        16883..16958
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(16975..16991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    17194..17218
                     /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 gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact aaaacaattc
     4981 atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa
     5041 aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc
     5101 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt
     5161 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc
     5221 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc
     5281 ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc
     5341 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa
     5401 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata
     5461 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg
     5521 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc
     5581 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata
     5641 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc
     5701 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc
     5761 catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat
     5821 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca
     5881 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg
     5941 aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc
     6001 cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg
     6061 gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg
     6121 atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc
     6181 tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca
     6241 cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg atctggattt
     6301 tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat tttacaaata
     6361 caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct ataggaaccc
     6421 taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag cttgtcgatc
     6481 gacagatccc ggtcggcatc tactctattt ctttgccctc ggacgagtgc tggggcgtcg
     6541 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg
     6601 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc
     6661 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag
     6721 accaatgcgg agcatatacg cccggagtcg tggcgatcct gcaagctccg gatgcctccg
     6781 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt
     6841 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat
     6901 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac
     6961 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact
     7021 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat
     7081 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct
     7141 cgtctggcta agatcggccg cagcgatcgc atccatagcc tccgcgaccg gttgtagaac
     7201 agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgaac ggcgggagat
     7261 gcaataggtc aggctctcgc taaactcccc aatgtcaagc acttccggaa tcgggagcgc
     7321 ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt
     7381 tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc
     7441 ctccgagagc tgcatcaggt cggacacgct gtcgaacttt tcgatcagaa acttctcgac
     7501 agacgtcgcg gtgagttcag gctttttcat aagcttctgc aaaagagaac cagacaacag
     7561 ggtaagtgcc tagcagtaaa caaacagaac tcatcacaag caaacagcaa catcatattc
     7621 ataccaacag gtcatgtgtg ttcatcacat cattagtact aagcatgcca tcatccaagt
     7681 atatcaaagt aagggcaaag agcatgcatg atcatcaggt gcacaaaaga atcatcaaat
     7741 tgtagcagta catatcttca tctatcatgc atatctatcc ataacaggac gatgcatgtt
     7801 gaccaggtaa aagctacagg atcctatagg aacagcagcg tatatatctt caccaatctt
     7861 agcatcttaa tcatgtggca catgcagttt caatttaagc acatgagcta gttgattatg
     7921 aggtaccaga gaatcatcaa actgatgtag cagcatatat cctcatctat catgcaatct
     7981 aatctaatct aactaaacag gaaaggtgtg ctattcagtt aaaagctacc gcatcatata
     8041 caaacggcag cataagaaaa agcataatca tcttaatcat gcaacaaacg cagattcata
     8101 ataagcccaa gagctagctt gtgatgatct tattctactc tgatctacag caatcagata
     8161 acgacctaac cttgcacatg gcaacaaaac aatcgatcgg acgaatcagt tgtttgttcc
     8221 tagctagcac catcgaacca gataatagat gcacgtacag atcccgaaaa cgaacccaaa
     8281 aacagggcag acctagctga acctaggcag cgacccagca gatcgtgaga acgatctcat
     8341 ctacgaacag cctagaagca accccacgat tcccggacaa acgacctaaa atccccacaa
     8401 atcacatgag catgacagga taaacagcgg aaccgatcag atctacacga aaaccccacc
     8461 tcccagccac ccacgatcag gaaacacgcg gatctagcat gatttcgtca acgcctcagc
     8521 ctagttccta gccacagacc aagcagaacc accaaaccac gccgagcgag gagatggggc
     8581 aagaggacgg gggagacgat cgccgtacct tgaagcgggg gaaggatcgc cgagggtcgc
     8641 gaggagagca atttggattt ggaacccggg ggttgtgcgc tccgaacgat gagacgatgt
     8701 gagattgtgg gaagaggcgc ggagggccct gtatttatgg gctgcgacgg ggggaggaga
     8761 ggtggggagg gttggggaag gaatccccca cccgtgccgt gacggttccg ggccgtgtga
     8821 gaggagcccg ctcgtctccg ccacgcaatt tccgcgatcg gagcggagct ttcgagaggc
     8881 ggctggatgg ttggtggccg ttagatttgt agacgccgtt aacgcctcgc ctccaccggg
     8941 aagagttttg agcagccgct tatgacaatg gcttaacgac gttagacgga gcgttagtgg
     9001 caggccatga tagaatagac gtattgcaat gggattataa ttaaataaat aagaatataa
     9061 taagatatgg caagtcggca ctcatgacat ggtcttcgaa atgatagtgc tcactttctt
     9121 agccgagaaa gttgacgcga ctgatttaga agttaagata ttatttctct cttcttttct
     9181 ttcctcgtca tataaggatg aaataaactt tagagattgc cggtagtgat tttggatttc
     9241 ggcgatcagg cttggtttgc cggtttcgga cggtgtgcct taggccaccc gcagtgtatc
     9301 ttgtaatgtt caaccgataa gcaagggtgg ggctcaagca agtagtaaac aactatgtca
     9361 aatgtcacca tggttatggt cttgtttagt tggcttcttg gccgattcat taatgcagct
     9421 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     9481 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     9541 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaattcga
     9601 gctcggtacc cggggatctg caggcgcgcc tgcagtgcag cgtgacccgg tcgtgcccct
     9661 ctctagagat aatgagcatt gcatgtctaa gttataaaaa attaccacat attttttttg
     9721 tcacacttgt ttgaagtgca gtttatctat ctttatacat atatttaaac tttactctac
     9781 gaataatata atctatagta ctacaataat atcagtgttt tagagaatca tataaatgaa
     9841 cagttagaca tggtctaaag gacaattgag tattttgaca acaggactct acagttttat
     9901 ctttttagtg tgcatgtgtt ctcctttttt tttgcaaata gcttcaccta tataatactt
     9961 catccatttt attagtacat ccatttaggg tttagggtta atggttttta tagactaatt
    10021 tttttagtac atctatttta ttctatttta gcctctaaat taagaaaact aaaactctat
    10081 tttagttttt ttatttaata atttagatat aaaatagaat aaaataaagt gactaaaaat
    10141 taaacaaata ccctttaaga aattaaaaaa actaaggaaa catttttctt gtttcgagta
    10201 gataatgcca gcctgttaaa cgccgtcgac gagtctaacg gacaccaacc agcgaaccag
    10261 cagcgtcgcg tcgggccaag cgaagcagac ggcacggcat ctctgtcgct gcctctggac
    10321 ccctctcgag agttccgctc caccgttgga cttgctccgc tgtcggcatc cagaaattgc
    10381 gtggcggagc ggcagacgtg agccggcacg gcaggcggcc tcctcctcct ctcacggcac
    10441 cggcagctac gggggattcc tttcccaccg ctccttcgct ttcccttcct cgcccgccgt
    10501 aataaataga caccccctcc acaccctctt tccccaacct cgtgttgttc ggagcgcaca
    10561 cacacacaac cagatctccc ccaaatccac ccgtcggcac ctccgcttca aggtacgccg
    10621 ctcgtcctcc cccccccccc tctctacctt ctctagatcg gcgttccggt ccatggttag
    10681 ggcccggtag ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt
    10741 gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt
    10801 gccagtgttt ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga
    10861 tttcatgatt ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa
    10921 tatatgccgt gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg
    10981 atgatgtggt ctggttgggc ggtcgttcta gatcggagta gaattaattc tgtttcaaac
    11041 tacctggtgg atttattaat tttggatctg tatgtgtgtg ccatacatat tcatagttac
    11101 gaattgaaga tgatggatgg aaatatcgat ctaggatagg tatacatgtt gatgcgggtt
    11161 ttactgatgc atatacagag atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt
    11221 gggcggtcgt tcattcgttc tagatcggag tagaatactg tttcaaacta cctggtgtat
    11281 ttattaattt tggaactgta tgtgtgtgtc atacatcttc atagttacga gtttaagatg
    11341 gatggaaata tcgatctagg ataggtatac atgttgatgt gggttttact gatgcatata
    11401 catgatggca tatgcagcat ctattcatat gctctaacct tgagtaccta tctattataa
    11461 taaacaagta tgttttataa ttattttgat cttgatatac ttggatgatg gcatatgcag
    11521 cagctatatg tggatttttt tagccctgcc ttcatacgct atttatttgc ttggtactgt
    11581 ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc ctgcagggcg atctattcga
    11641 atggacaaga agtactcgat cggcctcgac atcgggacga actcagttgg ctgggccgtg
    11701 atcaccgacg agtacaaggt gccctctaag aagttcaagg tcctggggaa caccgaccgc
    11761 cattccatca agaagaacct catcggcgct ctcctgttcg acagcgggga gaccgctgag
    11821 gctacgaggc tcaagagaac cgctaggcgc cggtacacga gaaggaagaa caggatctgc
    11881 tacctccaag agattttctc caacgagatg gccaaggttg acgattcatt cttccaccgc
    11941 ctggaggagt ctttcctcgt ggaggaggat aagaagcacg agcggcatcc catcttcggc
    12001 aacatcgtgg acgaggttgc ctaccacgag aagtacccta cgatctacca tctgcggaag
    12061 aagctcgtgg actccaccga taaggcggac ctcagactga tctacctcgc tctggcccac
    12121 atgatcaagt tccgcggcca tttcctgatc gagggggatc tcaacccaga caacagcgat
    12181 gttgacaagc tgttcatcca actcgtgcag acctacaacc aactcttcga ggagaacccg
    12241 atcaacgcct ctggcgtgga cgcgaaggct atcctgtccg cgaggctctc gaagtccagg
    12301 aggctggaga acctgatcgc tcagctccca ggcgagaaga agaacggcct gttcgggaac
    12361 ctcatcgctc tcagcctggg gctcaccccg aacttcaagt cgaacttcga tctcgctgag
    12421 gacgccaagc tgcaactctc caaggacacc tacgacgatg acctcgataa cctcctggcc
    12481 cagatcggcg atcaatacgc ggacctgttc ctcgctgcca agaacctgtc ggacgccatc
    12541 ctcctgtcag atatcctccg cgtgaacacc gagatcacga aggctccact ctctgcctcc
    12601 atgatcaagc gctacgacga gcaccatcag gatctgaccc tcctgaaggc gctggtccgc
    12661 caacagctcc cggagaagta caaggagatt ttcttcgatc agtcgaagaa cggctacgct
    12721 gggtacatcg acggcggggc ctcacaagag gagttctaca agttcatcaa gccaatcctg
    12781 gagaagatgg acggcacgga ggagctcctg gtgaagctca acagggagga cctcctgcgg
    12841 aagcagagaa ccttcgataa cggcagcatc ccccaccaaa tccatctcgg ggagctgcac
    12901 gccatcctga gaaggcaaga ggacttctac cctttcctca aggataaccg ggagaagatc
    12961 gagaagatcc tgaccttcag aatcccatac tacgtcggcc ctctcgcgcg ggggaactca
    13021 agattcgctt ggatgacccg caagtctgag gagaccatca cgccgtggaa cttcgaggag
    13081 gtggtggaca agggcgctag cgctcagtcg ttcatcgaga ggatgaccaa cttcgacaag
    13141 aacctgccca acgagaaggt gctccctaag cactcgctcc tgtacgagta cttcaccgtc
    13201 tacaacgagc tcacgaaggt gaagtacgtc accgagggca tgcgcaagcc agcgttcctg
    13261 tccggggagc agaagaaggc tatcgtggac ctcctgttca agaccaaccg gaaggtcacg
    13321 gttaagcaac tcaaggagga ctacttcaag aagatcgagt gcttcgattc ggtcgagatc
    13381 agcggcgttg aggaccgctt caacgccagc ctcgggacct accacgatct cctgaagatc
    13441 atcaaggata aggacttcct ggacaacgag gagaacgagg atatcctgga ggacatcgtg
    13501 ctgaccctca cgctgttcga ggacagggag atgatcgagg agcgcctgaa gacgtacgcc
    13561 catctcttcg atgacaaggt catgaagcaa ctcaagcgcc ggagatacac cggctggggg
    13621 aggctgtccc gcaagctcat caacggcatc cgggacaagc agtccgggaa gaccatcctc
    13681 gacttcctga agagcgatgg cttcgccaac aggaacttca tgcaactgat ccacgatgac
    13741 agcctcacct tcaaggagga tatccaaaag gctcaagtga gcggccaggg ggactcgctg
    13801 cacgagcata tcgcgaacct cgctggctcc cccgcgatca agaagggcat cctccagacc
    13861 gtgaaggttg tggacgagct cgtgaaggtc atgggccggc acaagcctga gaacatcgtc
    13921 atcgagatgg ccagagagaa ccaaaccacg cagaaggggc aaaagaactc tagggagcgc
    13981 atgaagcgca tcgaggaggg catcaaggag ctggggtccc aaatcctcaa ggagcaccca
    14041 gtggagaaca cccaactgca gaacgagaag ctctacctgt actacctcca gaacggcagg
    14101 gatatgtacg tggaccaaga gctggatatc aaccgcctca gcgattacga cgtcgatcat
    14161 atcgttcccc agtctttcct gaaggatgac tccatcgaca acaaggtcct caccaggtcg
    14221 gacaagaacc gcggcaagtc agataacgtt ccatctgagg aggtcgttaa gaagatgaag
    14281 aactactgga ggcagctcct gaacgccaag ctgatcacgc aaaggaagtt cgacaacctc
    14341 accaaggctg agagaggcgg gctctcagag ctggacaagg ccggcttcat caagcggcag
    14401 ctggtcgaga ccagacaaat cacgaagcac gttgcgcaaa tcctcgactc tcggatgaac
    14461 acgaagtacg atgagaacga caagctgatc agggaggtta aggtgatcac cctgaagtct
    14521 aagctcgtct ccgacttcag gaaggatttc cagttctaca aggttcgcga gatcaacaac
    14581 taccaccatg cccatgacgc ttacctcaac gctgtggtcg gcaccgctct gatcaagaag
    14641 tacccaaagc tggagtccga gttcgtgtac ggggactaca aggtttacga tgtgcgcaag
    14701 atgatcgcca agtcggagca agagatcggc aaggctaccg ccaagtactt cttctactca
    14761 aacatcatga acttcttcaa gaccgagatc acgctggcca acggcgagat ccggaagaga
    14821 ccgctcatcg agaccaacgg cgagacgggg gagatcgtgt gggacaaggg cagggatttc
    14881 gcgaccgtcc gcaaggttct ctccatgccc caggtgaaca tcgtcaagaa gaccgaggtc
    14941 caaacgggcg ggttctcaaa ggagtctatc ctgcctaagc ggaacagcga caagctcatc
    15001 gccagaaaga aggactggga cccaaagaag tacggcgggt tcgacagccc taccgtggcc
    15061 tactcggtcc tggttgtggc gaaggttgag aagggcaagt ccaagaagct caagagcgtg
    15121 aaggagctcc tggggatcac catcatggag aggtccagct tcgagaagaa cccaatcgac
    15181 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctcccgaag
    15241 tactctctct tcgagctgga gaacggcagg aagagaatgc tggcttccgc tggcgagctc
    15301 cagaagggga acgagctcgc gctgccaagc aagtacgtga acttcctcta cctggcttcc
    15361 cactacgaga agctcaaggg cagcccggag gacaacgagc aaaagcagct gttcgtcgag
    15421 cagcacaagc attacctcga cgagatcatc gagcaaatct ccgagttcag caagcgcgtg
    15481 atcctcgccg acgcgaacct ggataaggtc ctctccgcct acaacaagca ccgggacaag
    15541 cccatcagag agcaagcgga gaacatcatc catctcttca ccctgacgaa cctcggcgct
    15601 cctgctgctt tcaagtactt cgacaccacg atcgatcgga agagatacac ctccacgaag
    15661 gaggtcctgg acgcgaccct catccaccag tcgatcaccg gcctgtacga gacgaggatc
    15721 gacctctcac aactcggcgg ggataagaga cccgcagcaa ccaagaaggc agggcaagca
    15781 aagaagaaga agtgactcga gatatgaaga tgaagatgaa atatttggtg tgtcaaataa
    15841 aaagcttgtg tgcttaagtt tgtgtttttt tcttggcttg ttgtgttatg aatttgtggc
    15901 tttttctaat attaaatgaa tgtaagatca cattataatg aataaacaaa tgtttctata
    15961 atccattgtg aatgttttgt tggatctctt ctgcagcata taactactgt atgtgctatg
    16021 gtatggacta tggaatatga ttaaagataa ggagctccgg tgacggacga ccaagcccgt
    16081 tattctgaca gttctggtgc tcaacacatt tatatttatc aaggagcaca ttgttactca
    16141 ctgctaggag ggaatcgaac taggaatatt gatcagagga actacgagag agctgaagat
    16201 aactgccctc tagctctcac tgatctgggt cgcatagtga gatgcagccc acgtgagttc
    16261 agcaacggtc tagcgctggg cttttaggcc cgcatgatcg ggcttttgtc gggtggtcga
    16321 cgtgttcacg attggggaga gcaacgcagc agttcctctt agtttagtcc cacctcgcct
    16381 gtccagcaga gttctgaccg gtttataaac tcgcttgctg catcagactt agaggcaggt
    16441 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
    16501 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
    16561 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
    16621 cgcgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca
    16681 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
    16741 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
    16801 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
    16861 tgcccgcttt ccacctgcgg tcgttttaga gctagaaata gcaagttaaa ataaggctag
    16921 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttcccgg tggcactggc
    16981 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
    17041 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
    17101 ccaacagttg cgcagcctga atggcgaatg ctagagcagc ttgagcttgg atcagattgt
    17161 cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggc gggtaaacct
    17221 aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaa aaggtttatc
    17281 cgttcg
//
