LOCUS       sequence-320624-       15039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construction of inPTG-Cas9 plasmids expressing gRNA within an 
            engineered intron for binary vector plant genome editing. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15039)
  AUTHORS   Ding D, Chen K, Chen Y, Li H, Xie K
  TITLE     Engineering Introns to Express RNA Guides for Cas9- and 
            Cpf1-Mediated Multiplex Genome Editing.
  JOURNAL   Mol Plant. 2018 Apr 2;11(4):542-552. doi: 
            10.1016/j.molp.2018.02.005. Epub 2018 Feb 17.
  PUBMED    29462720
REFERENCE   2  (bases 1 to 15039)
  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"; doi: 
            "10.1016/j.molp.2018.02.005"; journalName: "Mol Plant"; date: 
            "2018-04-2- 2"; volume: "11"; issue: "4"; pages: "542-552"
COMMENT     Sequence Label: pRGEB33
FEATURES             Location/Qualifiers
     source          1..15039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    201..225
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    303..477
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(518..1543)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(1616..1961)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(1653..1673)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    2152..2173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2188..2193,2194..2211,2212..2218)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2226..2242
                     /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     2231..2253
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2250..2266
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2250..2266
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    4033..4057
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4102..4167
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4174..4194
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4219..8319
                     /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             4219..8319
                     /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             8320..8367
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(8400..8424)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8435..8458
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      8480..8732
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(8741..8758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8741..8757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8750..8772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    8960..8984
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             10285..10914
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             11343..12416
                     /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      12482..12676
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(12912..12934)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    13020..13160
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     13075..13094
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(13175..13192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13346..13934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13426..13445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(14021..14815)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
        1 acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt
       61 ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg
      121 ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg
      181 gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga
      241 caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg
      301 gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag
      361 tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac
      421 cctataggaa ccctaattcc cttatctggg aactactcac acattattat ggagaaactc
      481 gagcttgtcg atcgacagat cccggtcggc atctactcta tttctttgcc ctcggacgag
      541 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
      601 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
      661 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
      721 agagttggtc aagaccaatg cggagcatat acgcccggag tcgtggcgat cctgcaagct
      781 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
      841 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
      901 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
      961 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
     1021 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
     1081 caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg
     1141 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccata gcctccgcga
     1201 ccggttgtag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
     1261 aacggcggga gatgcaatag gtcaggctct cgctaaactc cccaatgtca agcacttccg
     1321 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
     1381 cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac
     1441 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
     1501 gaaacttctc gacagacgtc gcggtgagtt caggcttttt catatctcat tgccccccgg
     1561 atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt cagcgtgtcc
     1621 tctccaaatg aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt
     1681 gtgcgtcatc ccttacgtca gtggagatat cacatcaatc cacttgcttt gaagacgtgg
     1741 ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac
     1801 tgtcggcaga ggcatcttga acgatagcct ttcctttatc gcaatgatgg catttgtagg
     1861 tgccaccttc cttttctact gtccttttga tgaagtgaca gatagctggg caatggaatc
     1921 cgaggaggtt tcccgatatt accctttgtt gaaaagtctc aatagccctt tggtcttctg
     1981 agactgtatc tttgatattc ttggagtaga cgagagtgtc gtgctccacc atgttggcaa
     2041 gctgctctag ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2101 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2161 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2221 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgccaag
     2281 cttacaaatt cgggtcaagg cggaagccag cgcgccaccc cacgtcagca aatacggagg
     2341 cgcggggttg acggcgtcac ccggtcctaa cggcgaccaa caaaccagcc agaagaaatt
     2401 acagtaaaaa aaaagtaaat tgcactttga tccacctttt attacctaag tctcaatttg
     2461 gatcaccctt aaacctatct tttcaatttg ggccgggttg tggtttggac taccatgaac
     2521 aacttttcgt catgtctaac ttccctttca gcaaacatat gaaccatata tagaggagat
     2581 cggccgtata ctagagctga tgtgtttaag gtcgttgatt gcacgagaaa aaaaaatcca
     2641 aatcgcaaca atagcaaatt tatctggttc aaagtgaaaa gatatgttta aaggtagtcc
     2701 aaagtaaaac ttatagataa taaaatgtgg tccaaagcgt aattcactca aaaaaaatca
     2761 acgagacgtg taccaaacgg agacaaacgg catcttctcg aaatttccca accgctcgct
     2821 cgcccgcctc gtcttcccgg aaaccgcggt ggtttcagcg tggcggattc tccaagcaga
     2881 cggagacgtc acggcacggg actcctccca ccacccaacc gccataaata ccagccccct
     2941 catctcctct cctcgcatca gctccacccc cgaaaaattt ctccccaatc tcgcgaggct
     3001 ctcgtcgtcg aatcgaatcc tctcgcgtcc tcaaggtacg ctgcttctcc tctcctcgct
     3061 tcgtttcgat tcgatttcgg acgggtgagg ttgttttgtt gctagatccg attggtggtt
     3121 agggttgtcg atgtgattat cgtgagatgt ttaggggttg tagatctgat ggttgtgatt
     3181 tgggcacggt tggttcgata ggtggaatcg tggttaggtt ttgggattgg atgttggttc
     3241 tgatgattgg ggggaatttt tacggttaga tgaattgttg gatgattcga ttggggaaat
     3301 cggtgtagat ctgttgggga attgtggaac tagtcatgcc tgagtgattg gtgcgatttg
     3361 tagcgtgttc catcttgtag gccttgttgc gagcatgttc agatctactg ttccgctctt
     3421 gattgagtta ttggtgccat gggttggtgc aaacacaggc tttaatatgt tatatctgtt
     3481 ttgtgtttga tgtagatctg tagggtagtt cttcttagac atggttcaat tatgtagctt
     3541 gtgcgtttcg atttgatttc atatgttcac agattagata atgatgaact cttttaatta
     3601 attgtcaatg gtaaatagga agtcttgtcg ctatatctgt cataatgatc tcatgttact
     3661 atctgccagt aatttatgct aagaactata ttagaatatc atgttacaat ctgtagtaat
     3721 atcatgttac aatctgtagt tcatctatat aatctattgt ggtaatttct ttttactatc
     3781 tgtgtgaaga ttattgccac tagttcattc tacttatttc tgaagttcag gatacgtgtg
     3841 ctgttactac ctatctgaat acatgtgtga tgtgcctgtt actatctttt tgaatacatg
     3901 tatgttctgt tggaatatgg caggagaccg aggtctcggt ttgctgtttg atccgttgtt
     3961 gtgtccttaa tcttgtgcta gttcttaccc tatctgtttg gtgattattt cttgcagatt
     4021 ctagagttat caacaagttt gtacaaaaaa gcaggcttcg aaggagatag aaccaattct
     4081 ctaaggaaat acttaaccat ggactataag gaccacgacg gagactacaa ggatcatgat
     4141 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
     4201 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct
     4261 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     4321 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     4381 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     4441 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     4501 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     4561 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     4621 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     4681 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     4741 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     4801 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     4861 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     4921 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     4981 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     5041 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     5101 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     5161 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     5221 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     5281 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     5341 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     5401 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     5461 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     5521 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     5581 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     5641 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     5701 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     5761 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     5821 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     5881 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     5941 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     6001 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     6061 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     6121 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     6181 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     6241 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     6301 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     6361 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     6421 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     6481 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     6541 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     6601 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     6661 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     6721 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     6781 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     6841 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     6901 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     6961 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     7021 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     7081 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     7141 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     7201 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     7261 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     7321 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     7381 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     7441 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     7501 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     7561 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     7621 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     7681 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     7741 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     7801 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     7861 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     7921 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     7981 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     8041 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     8101 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     8161 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     8221 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     8281 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa
     8341 aaggccggcc aggcaaaaaa gaaaaagtaa gaattcgcgg ccgcactcga gatatctaga
     8401 cccagctttc ttgtacaaag tggttgataa cagcgactac aaggatgacg atgacaaggc
     8461 ttagagctcg aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa
     8521 tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt
     8581 aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc
     8641 gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt
     8701 atcgcgcgcg gtgtcatcta tgttactaga tcgggaattc actggccgtc gttttacaac
     8761 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     8821 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     8881 gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt tcccgccttc
     8941 agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg
     9001 tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg
     9061 tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc
     9121 gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt
     9181 cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg
     9241 tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca
     9301 tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg
     9361 acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga
     9421 agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc
     9481 ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac
     9541 tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt
     9601 gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg
     9661 agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc
     9721 gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg
     9781 agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc
     9841 gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc
     9901 ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga
     9961 atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt
    10021 cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc
    10081 gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc
    10141 ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg
    10201 tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa
    10261 taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg
    10321 tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat
    10381 gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa
    10441 gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc
    10501 atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg
    10561 tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata
    10621 tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg
    10681 ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt
    10741 gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg
    10801 agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac
    10861 gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat
    10921 gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac
    10981 gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca
    11041 actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca
    11101 agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat
    11161 gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa
    11221 ccaggcaccg acgccgtgga atgccccatg tgttgaggaa cgggcggttg gccaggcgta
    11281 agcggctggg ttgcctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg
    11341 gcgtgagcgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc
    11401 tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag gcagaagcac
    11461 gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc cggcaaccgc
    11521 cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa ccagattttt
    11581 tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg
    11641 ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac gagcttccag
    11701 acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg gattacgacc
    11761 tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg gaagggaagg
    11821 gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag ttctgccggc
    11881 gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg ttaaacacca
    11941 cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg acggtatccg
    12001 agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg cggccggagt
    12061 acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc aagaacccgg
    12121 acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc cgttttctct
    12181 accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc aagacgatct
    12241 acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg cgcaagctga
    12301 tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag gctggcccga
    12361 tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt tcctaatgta
    12421 cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa gcactctttc
    12481 ctgtgaatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg aacccgtaca
    12541 ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact gatataaaag
    12601 agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact cttaaaaccc
    12661 gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa aaagcgccta
    12721 cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg
    12781 gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga caagccgcgc
    12841 cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat
    12901 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
    12961 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
    13021 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
    13081 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
    13141 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
    13201 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
    13261 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
    13321 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
    13381 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
    13441 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
    13501 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
    13561 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
    13621 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
    13681 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
    13741 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
    13801 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
    13861 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
    13921 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
    13981 aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat tcatccagta
    14041 aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca aaaaatagct
    14101 cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag gcaatgtcat
    14161 accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact ttgccatctt
    14221 tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc tcttcgggct
    14281 tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg tcttcttccc
    14341 agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat tcggctaagc
    14401 ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga aagagcctga
    14461 tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca tactcttccg
    14521 agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca tgccgttcaa
    14581 agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg tccttttccc
    14641 gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa tataggtttt
    14701 cattttctcc caccagctta tataccttag caggagacat tccttccgta tcttttacgc
    14761 agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta gccatttatt
    14821 atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt ataacaagac
    14881 gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa cagctttttc
    14941 aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt tgaaaccgcg
    15001 gtgatcacag gcagcaacgc tctgtcatcg ttacaatca
//
