LOCUS       sequence-352460-       17112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Egg cell-specific promoter-controlled expression of 
            3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target
            sequence (U6-26 promoter), Hyg resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17112)
  AUTHORS   Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ
  TITLE     Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently 
            generates homozygous mutants for multiple target genes in 
            Arabidopsis in a single generation.
  JOURNAL   Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0.
  PUBMED    26193878
REFERENCE   2  (bases 1 to 17112)
  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: "2015-07-21"; volume: "16:144. doi"; pages: " 
            10.1186/s13059-015-0715-0"
COMMENT     Sequence Label: pHEE401E
FEATURES             Location/Qualifiers
     source          1..17112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..780
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     717..737
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             847..1872
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     polyA_signal    1913..2087
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    2165..2189
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             2614..3408
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3495..4083
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3984..4003
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4237..4254
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4269..4409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4335..4354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4495..4517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      4753..4947
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(5013..6086)
                     /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(6515..7144)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    8445..8469
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     8657..8679
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8671..8688
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8672..8688
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             9401..10192
                     /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        10344..10419
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             11989..12054
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             12061..12081
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12106..16206
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             12106..16206
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             16119..16127
                     /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             16207..16254
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(16911..16927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(16911..16927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(16924..16946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    16935..16951
                     /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(16959..16965,16966..16983,16984..16989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    17004..17025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggtttgcgta ttggctagag cagcttgcca acatggtgga gcacgacact ctcgtctact
       61 ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa
      121 gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa
      181 ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta
      241 tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca
      301 tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgaacatg
      361 gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc agaagaccaa
      421 agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc
      481 ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc
      541 catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag tggtcccaaa
      601 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca
      661 aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat
      721 ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gacacgctga
      781 aatcaccagt ctctctctac aaatctatct ctctcgagct ttcgcagatc cggggggcaa
      841 tgagatatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
      901 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
      961 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     1021 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     1081 gacattgggg agtttagcga gagcctgacc tattgcatct cccgccgttc acagggtgtc
     1141 acgttgcaag acctgcctga aaccgaactg cccgctgttc tacaaccggt cgcggaggct
     1201 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     1261 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     1321 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     1381 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     1441 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     1501 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     1561 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     1621 tcgccacgac tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     1681 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     1741 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     1801 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     1861 gcaaagaaat agagtagatg ccgaccggga tctgtcgatc gacaagctcg agtttctcca
     1921 taataatgtg tgagtagttc ccagataagg gaattagggt tcctataggg tttcgctcat
     1981 gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac ttctatcaat
     2041 aaaatttcta attcctaaaa ccaaaatcca gtactaaaat ccagatcccc cgaattaatt
     2101 cggcgttaat tcagtacatt aaaaacgtcc gcaatgtgtt attaagttgt ctaagcgtca
     2161 atttgtttac accacaatat atcctgccac cagccagcca acagctcccc gaccggcagc
     2221 tcggcacaaa atcaccactc gatacaggca gcccatcagt ccgggacggc gtcagcggga
     2281 gagccgttgt aaggcggcag actttgctca tgttaccgat gctattcgga agaacggcaa
     2341 ctaagctgcc gggtttgaaa cacggatgat ctcgcggagg gtagcatgtt gattgtaacg
     2401 atgacagagc gttgctgcct gtgatcaccg cggtttcaaa atcggctccg tcgatactat
     2461 gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt taaggtttta
     2521 gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt ggggtatctt
     2581 taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat atcaccggaa
     2641 ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat gtctcctgct
     2701 aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac ggacagccgg
     2761 tataaaggga ccacctatga tgtggaacgg gaaaaggaca tgatgctatg gctggaagga
     2821 aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag caatctgctc
     2881 atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca aagccctgaa
     2941 aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga catatcggat
     3001 tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt actgaataac
     3061 gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa agatccgcgc
     3121 gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt ttcccacggc
     3181 gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt tattgatctt
     3241 gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg gtcgatcagg
     3301 gaggatatcg gggaagaaca gtatgtcgag ctattttttg acttactggg gatcaagcct
     3361 gattgggaga aaataaaata ttatatttta ctggatgaat tgttttagta cctagaatgc
     3421 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3481 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3541 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3601 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3661 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3721 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3781 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3841 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3901 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3961 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4021 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4081 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4141 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4201 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4261 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4321 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4381 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4441 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4501 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ggtgccttga
     4561 tgtgggcgcc ggcggtcgag tggcgacggc gcggcttgtc cgcgccctgg tagattgcct
     4621 ggccgtaggc cagccatttt tgagcggcca gcggccgcga taggccgacg cgaagcggcg
     4681 gggcgtaggg agcgcagcga ccgaagggta ggcgcttttt gcagctcttc ggctgtgcgc
     4741 tggccagaca gttatgcaca ggccaggcgg gttttaagag ttttaataag ttttaaagag
     4801 ttttaggcgg aaaaatcgcc ttttttctct tttatatcag tcacttacat gtgtgaccgg
     4861 ttcccaatgt acggctttgg gttcccaatg tacgggttcc ggttcccaat gtacggcttt
     4921 gggttcccaa tgtacgtgct atccacagga aacagacctt ttcgaccttt ttcccctgct
     4981 agggcaattt gccctagcat ctgctccgta cattaggaac cggcggatgc ttcgccctcg
     5041 atcaggttgc ggtagcgcat gactaggatc gggccagcct gccccgcctc ctccttcaaa
     5101 tcgtactccg gcaggtcatt tgacccgatc agcttgcgca cggtgaaaca gaacttcttg
     5161 aactctccgg cgctgccact gcgttcgtag atcgtcttga acaaccatct ggcttctgcc
     5221 ttgcctgcgg cgcggcgtgc caggcggtag agaaaacggc cgatgccggg atcgatcaaa
     5281 aagtaatcgg ggtgaaccgt cagcacgtcc gggttcttgc cttctgtgat ctcgcggtac
     5341 atccaatcag ctagctcgat ctcgatgtac tccggccgcc cggtttcgct ctttacgatc
     5401 ttgtagcggc taatcaaggc ttcaccctcg gataccgtca ccaggcggcc gttcttggcc
     5461 ttcttcgtac gctgcatggc aacgtgcgtg gtgtttaacc gaatgcaggt ttctaccagg
     5521 tcgtctttct gctttccgcc atcggctcgc cggcagaact tgagtacgtc cgcaacgtgt
     5581 ggacggaaca cgcggccggg cttgtctccc ttcccttccc ggtatcggtt catggattcg
     5641 gttagatggg aaaccgccat cagtaccagg tcgtaatccc acacactggc catgccggcc
     5701 ggccctgcgg aaacctctac gtgcccgtct ggaagctcgt agcggatcac ctcgccagct
     5761 cgtcggtcac gcttcgacag acggaaaacg gccacgtcca tgatgctgcg actatcgcgg
     5821 gtgcccacgt catagagcat cggaacgaaa aaatctggtt gctcgtcgcc cttgggcggc
     5881 ttcctaatcg acggcgcacc ggctgccggc ggttgccggg attctttgcg gattcgatca
     5941 gcggccgctt gccacgattc accggggcgt gcttctgcct cgatgcgttg ccgctgggcg
     6001 gcctgcgcgg ccttcaactt ctccaccagg tcatcaccca gcgccgcgcc gatttgtacc
     6061 gggccggatg gtttgcgacc gctcacgccg attcctcggg cttgggggtt ccagtgccat
     6121 tgcagggccg gcaggcaacc cagccgctta cgcctggcca accgcccgtt cctccacaca
     6181 tggggcattc cacggcgtcg gtgcctggtt gttcttgatt ttccatgccg cctcctttag
     6241 ccgctaaaat tcatctactc atttattcat ttgctcattt actctggtag ctgcgcgatg
     6301 tattcagata gcagctcggt aatggtcttg ccttggcgta ccgcgtacat cttcagcttg
     6361 gtgtgatcct ccgccggcaa ctgaaagttg acccgcttca tggctggcgt gtctgccagg
     6421 ctggccaacg ttgcagcctt gctgctgcgt gcgctcggac ggccggcact tagcgtgttt
     6481 gtgcttttgc tcattttctc tttacctcat taactcaaat gagttttgat ttaatttcag
     6541 cggccagcgc ctggacctcg cgggcagcgt cgccctcggg ttctgattca agaacggttg
     6601 tgccggcggc ggcagtgcct gggtagctca cgcgctgcgt gatacgggac tcaagaatgg
     6661 gcagctcgta cccggccagc gcctcggcaa cctcaccgcc gatgcgcgtg cctttgatcg
     6721 cccgcgacac gacaaaggcc gcttgtagcc ttccatccgt gacctcaatg cgctgcttaa
     6781 ccagctccac caggtcggcg gtggcccata tgtcgtaagg gcttggctgc accggaatca
     6841 gcacgaagtc ggctgccttg atcgcggaca cagccaagtc cgccgcctgg ggcgctccgt
     6901 cgatcactac gaagtcgcgc cggccgatgg ccttcacgtc gcggtcaatc gtcgggcggt
     6961 cgatgccgac aacggttagc ggttgatctt cccgcacggc cgcccaatcg cgggcactgc
     7021 cctggggatc ggaatcgact aacagaacat cggccccggc gagttgcagg gcgcgggcta
     7081 gatgggttgc gatggtcgtc ttgcctgacc cgcctttctg gttaagtaca gcgataacct
     7141 tcatgcgttc cccttgcgta tttgtttatt tactcatcgc atcatatacg cagcgaccgc
     7201 atgacgcaag ctgttttact caaatacaca tcaccttttt agacggcggc gctcggtttc
     7261 ttcagcggcc aagctggccg gccaggccgc cagcttggca tcagacaaac cggccaggat
     7321 ttcatgcagc cgcacggttg agacgtgcgc gggcggctcg aacacgtacc cggccgcgat
     7381 catctccgcc tcgatctctt cggtaatgaa aaacggttcg tcctggccgt cctggtgcgg
     7441 tttcatgctt gttcctcttg gcgttcattc tcggcggccg ccagggcgtc ggcctcggtc
     7501 aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg tgggcgtcac ttcctcgctg
     7561 cgctcaagtg cgcggtacag ggtcgagcga tgcacgccaa gcagtgcagc cgcctctttc
     7621 acggtgcggc cttcctggtc gatcagctcg cgggcgtgcg cgatctgtgc cggggtgagg
     7681 gtagggcggg ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc gctccgggtg
     7741 cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt caacaccatg
     7801 cggccggccg gcgtggtggt gtcggcccac ggctctgcca ggctacgcag gcccgcgccg
     7861 gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg tgctgcgggc caggcggtct
     7921 agcctggtca ctgtcacaac gtcgccaggg cgtaggtggt caagcatcct ggccagctcc
     7981 gggcggtcgc gcctggtgcc ggtgatcttc tcggaaaaca gcttggtgca gccggccgcg
     8041 tgcagttcgg cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg ggcatagccc
     8101 agcaggccag cggcggcgct cttgttcatg gcgtaatgtc tccggttcta gtcgcaagta
     8161 ttctacttta tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg cagggcggca
     8221 gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt cagaagacgg ctgcactgaa
     8281 cgtcagaagc cgactgcact atagcagcgg aggggttgga tcaaagtact ttgatcccga
     8341 ggggaaccct gtggttggca tgcacataca aatggacgaa cggataaacc ttttcacgcc
     8401 cttttaaata tccgattatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat
     8461 atcctgtcaa acactgatag tttaaactga aggcgggaaa cgacaatctg atccaagctc
     8521 aagctgctct agcattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     8581 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     8641 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc caagcttcga
     8701 cttgccttcc gcacaataca tcatttcttc ttagcttttt ttcttcttct tcgttcatac
     8761 agtttttttt tgtttatcag cttacatttt cttgaaccgt agctttcgtt ttcttctttt
     8821 taactttcca ttcggagttt ttgtatcttg tttcatagtt tgtcccagga ttagaatgat
     8881 taggcatcga accttcaaga atttgattga ataaaacatc ttcattctta agatatgaag
     8941 ataatcttca aaaggcccct gggaatctga aagaagagaa gcaggcccat ttatatggga
     9001 aagaacaata gtatttctta tataggccca tttaagttga aaacaatctt caaaagtccc
     9061 acatcgctta gataagaaaa cgaagctgag tttatataca gctagagtcg aagtagtgat
     9121 tgggagacca acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg
     9181 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     9241 ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc
     9301 caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt
     9361 tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc atgggggaag cggtgatcgc
     9421 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac
     9481 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga
     9541 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat
     9601 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga
     9661 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact
     9721 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat
     9781 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg
     9841 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa
     9901 tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt
     9961 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt
    10021 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc
    10081 tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga
    10141 agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat aatgtctagc
    10201 tagaaattcg ttcaagccga cgccgcttcg cggcgcggct taactcaagc gttagatgca
    10261 ctaagcacat aattgctcac agccaaacta tcaggtcaag tctgctttta ttatttttaa
    10321 gcgtgcataa taagccggtc tcggttttag agctagaaat agcaagttaa aataaggcta
    10381 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttttgca aaattttcca
    10441 gatcgatttc ttcttcctct gttcttcggc gttcaatttc tggggttttc tcttcgtttt
    10501 ctgtaactga aacctaaaat ttgacctaaa aaaaatctca aataatatga ttcagtggtt
    10561 ttgtactttt cagttagttg agttttgcag ttccgatgag ataaaccaat accatggtta
    10621 tactagtgaa taaaagcatt tgcgtttggt ttatcattgc gtttatacaa ggacagagat
    10681 ccactgagct ggaatagctt aaaaccatta tcagaacaaa ataaaccatt ttttgttaag
    10741 aatcagagca tagtaaacaa cagaaacaac ctaagagagg taacttgtcc aagaagatag
    10801 ctaattatat ctattttata aaagttatca tagtttgtaa gtcacaaaag atgcaaataa
    10861 cagagaaact aggagacttg agaatataca ttcttgtata tttgtattcg agattgtgaa
    10921 aatttgacca taagtttaaa ttcttaaaaa gatatatctg atctaggtga tggttataga
    10981 ctgtaatttt accacatgtt taatgatgga tagtgacaca catgacacat cgacaacact
    11041 atagcatctt atttagatta caacatgaaa tttttctgta atacatgtct ttgtacataa
    11101 tttaaaagta attcctaaga aatatattta tacaaggagt ttaaagaaaa catagcataa
    11161 agttcaatga gtagtaaaaa ccatatacag tatatagcat aaagttcaat gagtttatta
    11221 caaaagcatt ggttcacttt ctgtaacacg acgttaaacc ttcgtctcca ataggagcgc
    11281 tactgattca acatgccaat atatactaaa tacgtttcta cagtcaaatg ctttaacgtt
    11341 tcatgattaa gtgactattt accgtcaatc ctttcccatt cctcccacta atccaacttt
    11401 ttaattactc ttaaatcacc actaagctag taacgcctat catgaattag ctctactaaa
    11461 tctagcaacc tttcaaattt gcagtattgc aggtgtctct gtgtctttaa aatagttgcc
    11521 ttatgatttc ttcggtttca agatgatcaa atagttatag atttcatgct cacacatgct
    11581 cattagatgt gtacatactt tacttaccca aatctatttt ctcgcaaaga ttttgatggt
    11641 aaagctgatt tggttctatt gaactaaatc aaacgagttt cagactgagt gattctaatc
    11701 cggcccatta gcccctaaac agacccacta attacgcagc ttttaataga gtaattacac
    11761 ctagtttacc cactaaacca ctaagcacta attatctcac aatctaatga gcttccctcg
    11821 taattacttg ggctttcact ctaccattta tttgtaacag tcaagtctct actgtctcta
    11881 tataaactct ctaaagttaa cacacaattc tcatcacaaa caaatcaacc aaagcaactt
    11941 ctactctttc ttctttcgac cttatcaatc tgttgagaaa tctagatgga ttacaaggac
    12001 cacgacgggg attacaagga ccacgacatt gattacaagg atgatgatga caagatggct
    12061 ccgaagaaga agaggaaggt tggcatccac ggggtgccag ctgctgacaa gaagtactcg
    12121 atcggcctcg atattgggac taactctgtt ggctgggccg tgatcaccga cgagtacaag
    12181 gtgccctcaa agaagttcaa ggtcctgggc aacaccgatc ggcattccat caagaagaat
    12241 ctcattggcg ctctcctgtt cgacagcggc gagacggctg aggctacgcg gctcaagcgc
    12301 accgcccgca ggcggtacac gcgcaggaag aatcgcatct gctacctgca ggagattttc
    12361 tccaacgaga tggcgaaggt tgacgattct ttcttccaca ggctggagga gtcattcctc
    12421 gtggaggagg ataagaagca cgagcggcat ccaatcttcg gcaacattgt cgacgaggtt
    12481 gcctaccacg agaagtaccc tacgatctac catctgcgga agaagctcgt ggactccaca
    12541 gataaggcgg acctccgcct gatctacctc gctctggccc acatgattaa gttcaggggc
    12601 catttcctga tcgaggggga tctcaacccg gacaatagcg atgttgacaa gctgttcatc
    12661 cagctcgtgc agacgtacaa ccagctcttc gaggagaacc ccattaatgc gtcaggcgtc
    12721 gacgcgaagg ctatcctgtc cgctaggctc tcgaagtctc ggcgcctcga gaacctgatc
    12781 gcccagctgc cgggcgagaa gaagaacggc ctgttcggga atctcattgc gctcagcctg
    12841 gggctcacgc ccaacttcaa gtcgaatttc gatctcgctg aggacgccaa gctgcagctc
    12901 tccaaggaca catacgacga tgacctggat aacctcctgg cccagatcgg cgatcagtac
    12961 gcggacctgt tcctcgctgc caagaatctg tcggacgcca tcctcctgtc tgatattctc
    13021 agggtgaaca ccgagattac gaaggctccg ctctcagcct ccatgatcaa gcgctacgac
    13081 gagcaccatc aggatctgac cctcctgaag gcgctggtca ggcagcagct ccccgagaag
    13141 tacaaggaga tcttcttcga tcagtcgaag aacggctacg ctgggtacat tgacggcggg
    13201 gcctctcagg aggagttcta caagttcatc aagccgattc tggagaagat ggacggcacg
    13261 gaggagctgc tggtgaagct caatcgcgag gacctcctga ggaagcagcg gacattcgat
    13321 aacggcagca tcccacacca gattcatctc ggggagctgc acgctatcct gaggaggcag
    13381 gaggacttct accctttcct caaggataac cgcgagaaga tcgagaagat tctgactttc
    13441 aggatcccgt actacgtcgg cccactcgct aggggcaact cccgcttcgc ttggatgacc
    13501 cgcaagtcag aggagacgat cacgccgtgg aacttcgagg aggtggtcga caagggcgct
    13561 agcgctcagt cgttcatcga gaggatgacg aatttcgaca agaacctgcc aaatgagaag
    13621 gtgctcccta agcactcgct cctgtacgag tacttcacag tctacaacga gctgactaag
    13681 gtgaagtatg tgaccgaggg catgaggaag ccggctttcc tgtctgggga gcagaagaag
    13741 gccatcgtgg acctcctgtt caagaccaac cggaaggtca cggttaagca gctcaaggag
    13801 gactacttca agaagattga gtgcttcgat tcggtcgaga tctctggcgt tgaggaccgc
    13861 ttcaacgcct ccctggggac ctaccacgat ctcctgaaga tcattaagga taaggacttc
    13921 ctggacaacg aggagaatga ggatatcctc gaggacattg tgctgacact cactctgttc
    13981 gaggaccggg agatgatcga ggagcgcctg aagacttacg cccatctctt cgatgacaag
    14041 gtcatgaagc agctcaagag gaggaggtac accggctggg ggaggctgag caggaagctc
    14101 atcaacggca ttcgggacaa gcagtccggg aagacgatcc tcgacttcct gaagagcgat
    14161 ggcttcgcga accgcaattt catgcagctg attcacgatg acagcctcac attcaaggag
    14221 gatatccaga aggctcaggt gagcggccag ggggactcgc tgcacgagca tatcgcgaac
    14281 ctcgctggct cgccagctat caagaagggg attctgcaga ccgtgaaggt tgtggacgag
    14341 ctggtgaagg tcatgggcag gcacaagcct gagaacatcg tcattgagat ggcccgggag
    14401 aatcagacca cgcagaaggg ccagaagaac tcacgcgaga ggatgaagag gatcgaggag
    14461 ggcattaagg agctggggtc ccagatcctc aaggagcacc cggtggagaa cacgcagctg
    14521 cagaatgaga agctctacct gtactacctc cagaatggcc gcgatatgta tgtggaccag
    14581 gagctggata ttaacaggct cagcgattac gacgtcgatc atatcgttcc acagtcattc
    14641 ctgaaggatg actccattga caacaaggtc ctcaccaggt cggacaagaa ccggggcaag
    14701 tctgataatg ttccttcaga ggaggtcgtt aagaagatga agaactactg gcgccagctc
    14761 ctgaatgcca agctgatcac gcagcggaag ttcgataacc tcacaaaggc tgagaggggc
    14821 gggctctctg agctggacaa ggcgggcttc atcaagaggc agctggtcga gacacggcag
    14881 atcactaagc acgttgcgca gattctcgac tcacggatga acactaagta cgatgagaat
    14941 gacaagctga tccgcgaggt gaaggtcatc accctgaagt caaagctcgt ctccgacttc
    15001 aggaaggatt tccagttcta caaggttcgg gagatcaaca attaccacca tgcccatgac
    15061 gcgtacctga acgcggtggt cggcacagct ctgatcaaga agtacccaaa gctcgagagc
    15121 gagttcgtgt acggggacta caaggtttac gatgtgagga agatgatcgc caagtcggag
    15181 caggagattg gcaaggctac cgccaagtac ttcttctact ctaacattat gaatttcttc
    15241 aagacagaga tcactctggc caatggcgag atccggaagc gccccctcat cgagacgaac
    15301 ggcgagacgg gggagatcgt gtgggacaag ggcagggatt tcgcgaccgt caggaaggtt
    15361 ctctccatgc cacaagtgaa tatcgtcaag aagacagagg tccagactgg cgggttctct
    15421 aaggagtcaa ttctgcctaa gcggaacagc gacaagctca tcgcccgcaa gaaggactgg
    15481 gatccgaaga agtacggcgg gttcgacagc cccactgtgg cctactcggt cctggttgtg
    15541 gcgaaggttg agaagggcaa gtccaagaag ctcaagagcg tgaaggagct gctggggatc
    15601 acgattatgg agcgctccag cttcgagaag aacccgatcg atttcctgga ggcgaagggc
    15661 tacaaggagg tgaagaagga cctgatcatt aagctcccca agtactcact cttcgagctg
    15721 gagaacggca ggaagcggat gctggcttcc gctggcgagc tgcagaaggg gaacgagctg
    15781 gctctgccgt ccaagtatgt gaacttcctc tacctggcct cccactacga gaagctcaag
    15841 ggcagccccg aggacaacga gcagaagcag ctgttcgtcg agcagcacaa gcattacctc
    15901 gacgagatca ttgagcagat ttccgagttc tccaagcgcg tgatcctggc cgacgcgaat
    15961 ctggataagg tcctctccgc gtacaacaag caccgcgaca agccaatcag ggagcaggct
    16021 gagaatatca ttcatctctt caccctgacg aacctcggcg cccctgctgc tttcaagtac
    16081 ttcgacacaa ctatcgatcg caagaggtac acaagcacta aggaggtcct ggacgcgacc
    16141 ctcatccacc agtcgattac cggcctctac gagacgcgca tcgacctgtc tcagctcggg
    16201 ggcgacaagc ggccagcggc gacgaagaag gcggggcagg cgaagaagaa gaagtgagct
    16261 cagagctttc gttcgtatca tcggtttcga caacgttcgt caagttcaat gcatcagttt
    16321 cattgcgcac acaccagaat cctactgagt ttgagtatta tggcattggg aaaactgttt
    16381 ttcttgtacc atttgttgtg cttgtaattt actgtgtttt ttattcggtt ttcgctatcg
    16441 aactgtgaaa tggaaatgga tggagaagag ttaatgaatg atatggtcct tttgttcatt
    16501 ctcaaattaa tattatttgt tttttctctt atttgttgtg tgttgaattt gaaattataa
    16561 gagatatgca aacattttgt tttgagtaaa aatgtgtcaa atcgtggcct ctaatgaccg
    16621 aagttaatat gaggagtaaa acacttgtag ttgtaccatt atgcttattc actaggcaac
    16681 aaatatattt tcagacctag aaaagctgca aatgttactg aatacaagta tgtcctcttg
    16741 tgttttagac atttatgaac tttcctttat gtaattttcc agaatccttg tcagattcta
    16801 atcattgctt tataattata gttatactca tggatttgta gttgagtatg aaaatatttt
    16861 ttaatgcatt ttatgacttg ccaattgatt gacaacgaat tcgtaatcat gtcatagctg
    16921 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
    16981 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
    17041 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
    17101 gcggggagag gc
//
