pBUE411

LOCUS       pBUE411               17340 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  CRISPR/Cas based plant genome editing and gene regulation; expresses
            3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence
            (OsU3 promoter), Bar resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pBUE411
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17340)
  AUTHORS   Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
  TITLE     A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
  JOURNAL   BMC Plant Biol. 2014 Nov 29;14(1):327.
  PUBMED    25432517
REFERENCE   2  (bases 1 to 17340)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17340
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    21..45
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     233..255
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     247..264
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     248..264
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        331..710
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     CDS             990..1781
                     /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        1933..2008
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2312..4303
                     /label=Ubi promoter
                     /note="maize polyubiquitin gene promoter"
     CDS             4322..4387
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4394..4414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4439..8539
                     /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             8452..8460
                     /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             8540..8587
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(9245..9261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9245..9261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9258..9280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9269..9285
                     /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(9293..9323)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9338..9359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9550..10227
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             10272..10823
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     polyA_signal    10830..11004
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11082..11106
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             11531..12325
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12412..13000
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12901..12920
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13154..13171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13186..13326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13252..13271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13412..13434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      13670..13864
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13930..15003)
                     /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(15432..16061)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta
       61 aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc
      121 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      181 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      241 cgacgttgta aaacgacggc cagtgccaag cttagtaatt catccaggtc accaagttct
      301 aggattttca gaactgcaac ttattttatc aaggaatctt taaacatacg aacagatcac
      361 ttaaagttct tctgaagcaa cttaaagtta tcaggcatgc atggatcttg gaggaatcag
      421 atgtgcagtc agggaccata gcacaagaca ggcgtcttct actggtgcta ccagcaaatg
      481 ctggaagccg ggaacactgg gtacgttgga aaccacgtga tgtgaagaag taagataaac
      541 tgtaggagaa aagcatttcg tagtgggcca tgaagccttt caggacatgt attgcagtat
      601 gggccggccc attacgcaat tggacgacaa caaagactag tattagtacc acctcggcta
      661 tccacataga tcaaagctga tttaaaagag ttgtgcagat gatccgtggc gtgagaccaa
      721 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
      781 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
      841 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
      901 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
      961 gcagtcgccc taaaacaaag ttaaacatca tgggggaagc ggtgatcgcc gaagtatcga
     1021 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     1081 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     1141 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     1201 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     1261 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     1321 aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc
     1381 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     1441 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     1501 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     1561 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     1621 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     1681 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     1741 actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctagct agaaattcgt
     1801 tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg ttagatgcac taagcacata
     1861 attgctcaca gccaaactat caggtcaagt ctgcttttat tatttttaag cgtgcataat
     1921 aagccggtct cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     1981 acttgaaaaa gtggcaccga gtcggtgctt ttttttttcg ttttgcattg agttttctcc
     2041 gtcgcatgtt tgcagtttta ttttccgttt tgcattgaaa tttctccgtc tcatgtttgc
     2101 agcgtgttca aaaagtacgc agctgtattt cacttattta cggcgccaca ttttcatgcc
     2161 gtttgtgcca actatcccga gctagtgaat acagcttggc ttcacacaac actggtgacc
     2221 cgctgacctg ctcgtacctc gtaccgtcgt acggcacagc atttggaatt aaagggtgtg
     2281 atcgatactg cttgctgcta agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc
     2341 ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt
     2401 tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa actttactct
     2461 acgaataata taatctatag tactacaata atatcagtgt tttagagaat catataaatg
     2521 aacagttaga catggtctaa aggacaattg agtattttga caacaggact ctacagtttt
     2581 atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac
     2641 ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa
     2701 tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct
     2761 attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa
     2821 attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag
     2881 tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc
     2941 agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg
     3001 acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt
     3061 gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc
     3121 acggcagcta cgggggattc ctttcccacc gctccttcgc tttcccttcc tcgcccgccg
     3181 taataaatag acaccccctc cacaccctct ttccccaacc tcgtgttgtt cggagcgcac
     3241 acacacacaa ccagatctcc cccaaatcca cccgtcggca cctccgcttc aaggtacgcc
     3301 gctcgtcctc cccccccccc cctctctacc ttctctagat cggcgttccg gtccatggtt
     3361 agggcccggt agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc
     3421 gtgctgctag cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac
     3481 ttgccagtgt ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc
     3541 gatttcatga ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc
     3601 aatatatgcc gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg
     3661 tgatgatgtg gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta
     3721 cctggtggat ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga
     3781 attgaagatg atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt
     3841 actgatgcat atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg
     3901 gcggtcgttc attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt
     3961 attaattttg gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga
     4021 tggaaatatc gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca
     4081 tgatggcata tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata
     4141 aacaagtatg ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca
     4201 gctatatgtg gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt
     4261 cttttgtcga tgctcaccct gttgtttggt gttacttctg cagccctagg cctactagat
     4321 ggattacaag gaccacgacg gggattacaa ggaccacgac attgattaca aggatgatga
     4381 tgacaagatg gctccgaaga agaagaggaa ggttggcatc cacggggtgc cagctgctga
     4441 caagaagtac tcgatcggcc tcgatattgg gactaactct gttggctggg ccgtgatcac
     4501 cgacgagtac aaggtgccct caaagaagtt caaggtcctg ggcaacaccg atcggcattc
     4561 catcaagaag aatctcattg gcgctctcct gttcgacagc ggcgagacgg ctgaggctac
     4621 gcggctcaag cgcaccgccc gcaggcggta cacgcgcagg aagaatcgca tctgctacct
     4681 gcaggagatt ttctccaacg agatggcgaa ggttgacgat tctttcttcc acaggctgga
     4741 ggagtcattc ctcgtggagg aggataagaa gcacgagcgg catccaatct tcggcaacat
     4801 tgtcgacgag gttgcctacc acgagaagta ccctacgatc taccatctgc ggaagaagct
     4861 cgtggactcc acagataagg cggacctccg cctgatctac ctcgctctgg cccacatgat
     4921 taagttcagg ggccatttcc tgatcgaggg ggatctcaac ccggacaata gcgatgttga
     4981 caagctgttc atccagctcg tgcagacgta caaccagctc ttcgaggaga accccattaa
     5041 tgcgtcaggc gtcgacgcga aggctatcct gtccgctagg ctctcgaagt ctcggcgcct
     5101 cgagaacctg atcgcccagc tgccgggcga gaagaagaac ggcctgttcg ggaatctcat
     5161 tgcgctcagc ctggggctca cgcccaactt caagtcgaat ttcgatctcg ctgaggacgc
     5221 caagctgcag ctctccaagg acacatacga cgatgacctg gataacctcc tggcccagat
     5281 cggcgatcag tacgcggacc tgttcctcgc tgccaagaat ctgtcggacg ccatcctcct
     5341 gtctgatatt ctcagggtga acaccgagat tacgaaggct ccgctctcag cctccatgat
     5401 caagcgctac gacgagcacc atcaggatct gaccctcctg aaggcgctgg tcaggcagca
     5461 gctccccgag aagtacaagg agatcttctt cgatcagtcg aagaacggct acgctgggta
     5521 cattgacggc ggggcctctc aggaggagtt ctacaagttc atcaagccga ttctggagaa
     5581 gatggacggc acggaggagc tgctggtgaa gctcaatcgc gaggacctcc tgaggaagca
     5641 gcggacattc gataacggca gcatcccaca ccagattcat ctcggggagc tgcacgctat
     5701 cctgaggagg caggaggact tctacccttt cctcaaggat aaccgcgaga agatcgagaa
     5761 gattctgact ttcaggatcc cgtactacgt cggcccactc gctaggggca actcccgctt
     5821 cgcttggatg acccgcaagt cagaggagac gatcacgccg tggaacttcg aggaggtggt
     5881 cgacaagggc gctagcgctc agtcgttcat cgagaggatg acgaatttcg acaagaacct
     5941 gccaaatgag aaggtgctcc ctaagcactc gctcctgtac gagtacttca cagtctacaa
     6001 cgagctgact aaggtgaagt atgtgaccga gggcatgagg aagccggctt tcctgtctgg
     6061 ggagcagaag aaggccatcg tggacctcct gttcaagacc aaccggaagg tcacggttaa
     6121 gcagctcaag gaggactact tcaagaagat tgagtgcttc gattcggtcg agatctctgg
     6181 cgttgaggac cgcttcaacg cctccctggg gacctaccac gatctcctga agatcattaa
     6241 ggataaggac ttcctggaca acgaggagaa tgaggatatc ctcgaggaca ttgtgctgac
     6301 actcactctg ttcgaggacc gggagatgat cgaggagcgc ctgaagactt acgcccatct
     6361 cttcgatgac aaggtcatga agcagctcaa gaggaggagg tacaccggct gggggaggct
     6421 gagcaggaag ctcatcaacg gcattcggga caagcagtcc gggaagacga tcctcgactt
     6481 cctgaagagc gatggcttcg cgaaccgcaa tttcatgcag ctgattcacg atgacagcct
     6541 cacattcaag gaggatatcc agaaggctca ggtgagcggc cagggggact cgctgcacga
     6601 gcatatcgcg aacctcgctg gctcgccagc tatcaagaag gggattctgc agaccgtgaa
     6661 ggttgtggac gagctggtga aggtcatggg caggcacaag cctgagaaca tcgtcattga
     6721 gatggcccgg gagaatcaga ccacgcagaa gggccagaag aactcacgcg agaggatgaa
     6781 gaggatcgag gagggcatta aggagctggg gtcccagatc ctcaaggagc acccggtgga
     6841 gaacacgcag ctgcagaatg agaagctcta cctgtactac ctccagaatg gccgcgatat
     6901 gtatgtggac caggagctgg atattaacag gctcagcgat tacgacgtcg atcatatcgt
     6961 tccacagtca ttcctgaagg atgactccat tgacaacaag gtcctcacca ggtcggacaa
     7021 gaaccggggc aagtctgata atgttccttc agaggaggtc gttaagaaga tgaagaacta
     7081 ctggcgccag ctcctgaatg ccaagctgat cacgcagcgg aagttcgata acctcacaaa
     7141 ggctgagagg ggcgggctct ctgagctgga caaggcgggc ttcatcaaga ggcagctggt
     7201 cgagacacgg cagatcacta agcacgttgc gcagattctc gactcacgga tgaacactaa
     7261 gtacgatgag aatgacaagc tgatccgcga ggtgaaggtc atcaccctga agtcaaagct
     7321 cgtctccgac ttcaggaagg atttccagtt ctacaaggtt cgggagatca acaattacca
     7381 ccatgcccat gacgcgtacc tgaacgcggt ggtcggcaca gctctgatca agaagtaccc
     7441 aaagctcgag agcgagttcg tgtacgggga ctacaaggtt tacgatgtga ggaagatgat
     7501 cgccaagtcg gagcaggaga ttggcaaggc taccgccaag tacttcttct actctaacat
     7561 tatgaatttc ttcaagacag agatcactct ggccaatggc gagatccgga agcgccccct
     7621 catcgagacg aacggcgaga cgggggagat cgtgtgggac aagggcaggg atttcgcgac
     7681 cgtcaggaag gttctctcca tgccacaagt gaatatcgtc aagaagacag aggtccagac
     7741 tggcgggttc tctaaggagt caattctgcc taagcggaac agcgacaagc tcatcgcccg
     7801 caagaaggac tgggatccga agaagtacgg cgggttcgac agccccactg tggcctactc
     7861 ggtcctggtt gtggcgaagg ttgagaaggg caagtccaag aagctcaaga gcgtgaagga
     7921 gctgctgggg atcacgatta tggagcgctc cagcttcgag aagaacccga tcgatttcct
     7981 ggaggcgaag ggctacaagg aggtgaagaa ggacctgatc attaagctcc ccaagtactc
     8041 actcttcgag ctggagaacg gcaggaagcg gatgctggct tccgctggcg agctgcagaa
     8101 ggggaacgag ctggctctgc cgtccaagta tgtgaacttc ctctacctgg cctcccacta
     8161 cgagaagctc aagggcagcc ccgaggacaa cgagcagaag cagctgttcg tcgagcagca
     8221 caagcattac ctcgacgaga tcattgagca gatttccgag ttctccaagc gcgtgatcct
     8281 ggccgacgcg aatctggata aggtcctctc cgcgtacaac aagcaccgcg acaagccaat
     8341 cagggagcag gctgagaata tcattcatct cttcaccctg acgaacctcg gcgcccctgc
     8401 tgctttcaag tacttcgaca caactatcga tcgcaagagg tacacaagca ctaaggaggt
     8461 cctggacgcg accctcatcc accagtcgat taccggcctc tacgagacgc gcatcgacct
     8521 gtctcagctc gggggcgaca agcggccagc ggcgacgaag aaggcggggc aggcgaagaa
     8581 gaagaagtga gctcagagct ttcgttcgta tcatcggttt cgacaacgtt cgtcaagttc
     8641 aatgcatcag tttcattgcg cacacaccag aatcctactg agtttgagta ttatggcatt
     8701 gggaaaactg tttttcttgt accatttgtt gtgcttgtaa tttactgtgt tttttattcg
     8761 gttttcgcta tcgaactgtg aaatggaaat ggatggagaa gagttaatga atgatatggt
     8821 ccttttgttc attctcaaat taatattatt tgttttttct cttatttgtt gtgtgttgaa
     8881 tttgaaatta taagagatat gcaaacattt tgttttgagt aaaaatgtgt caaatcgtgg
     8941 cctctaatga ccgaagttaa tatgaggagt aaaacacttg tagttgtacc attatgctta
     9001 ttcactaggc aacaaatata ttttcagacc tagaaaagct gcaaatgtta ctgaatacaa
     9061 gtatgtcctc ttgtgtttta gacatttatg aactttcctt tatgtaattt tccagaatcc
     9121 ttgtcagatt ctaatcattg ctttataatt atagttatac tcatggattt gtagttgagt
     9181 atgaaaatat tttttaatgc attttatgac ttgccaattg attgacaacg aattcgtaat
     9241 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     9301 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     9361 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     9421 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg ctagagcagc ttgccaacat
     9481 ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca
     9541 aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg
     9601 cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca cctacaaatg
     9661 ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca gtggtcccaa
     9721 agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc
     9781 aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct actccaagaa
     9841 tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat
     9901 atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca aaaggacagt
     9961 agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca
    10021 agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga
    10081 aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga
    10141 cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat ataaggaagt
    10201 tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa tctatctctc
    10261 tcgagtctac catgagccca gaacgacgcc cggccgacat ccgccgtgcc accgaggcgg
    10321 acatgccggc ggtctgcacc atcgtcaacc actacatcga gacaagcacg gtcaacttcc
    10381 gtaccgagcc gcaggaaccg caggagtgga cggacgacct cgtccgtctg cgggagcgct
    10441 atccctggct cgtcgccgag gtggacggcg aggtcgccgg catcgcctac gcgggcccct
    10501 ggaaggcacg caacgcctac gactggacgg ccgagtcgac cgtgtacgtc tccccccgcc
    10561 accagcggac gggactgggc tccacgctct acacccacct gctgaagtcc ctggaggcac
    10621 agggcttcaa gagcgtggtc gctgtcatcg ggctgcccaa cgacccgagc gtgcgcatgc
    10681 acgaggcgct cggatatgcc ccccgcggca tgctgcgggc ggccggcttc aagcacggga
    10741 actggcatga cgtgggtttc tggcagctgg acttcagcct gccggtaccg ccccgtccgg
    10801 tcctgcccgt caccgagatt tgactcgagt ttctccataa taatgtgtga gtagttccca
    10861 gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct
    10921 tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca
    10981 aaatccagta ctaaaatcca gatcccccga attaattcgg cgttaattca gtacattaaa
    11041 aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc
    11101 ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat
    11161 acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact
    11221 ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac
    11281 ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg
    11341 atcaccgcgg tttcaaaatc ggctccgtcg atactatgtt atacgccaac tttgaaaaca
    11401 actttgaaaa agctgttttc tggtatttaa ggttttagaa tgcaaggaac agtgaattgg
    11461 agttcgtctt gttataatta gcttcttggg gtatctttaa atactgtaga aaagaggaag
    11521 gaaataataa atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata
    11581 ccgctgcgta aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga
    11641 aaatgaaaac ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt
    11701 ggaacgggaa aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct
    11761 gcactttgaa cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct
    11821 ttgctcggaa gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga
    11881 gtgcatcagg ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga
    11941 cagccgctta gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga
    12001 aaactgggaa gaagacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac
    12061 ggaaaagccc gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt
    12121 tgtgaaagat ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa
    12181 gtggtatgac attgccttct gcgtccggtc gatcagggag gatatcgggg aagaacagta
    12241 tgtcgagcta ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta
    12301 tattttactg gatgaattgt tttagtacct agaatgcatg accaaaatcc cttaacgtga
    12361 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
    12421 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
    12481 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
    12541 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
    12601 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
    12661 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
    12721 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
    12781 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
    12841 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
    12901 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
    12961 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
    13021 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
    13081 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
    13141 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
    13201 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
    13261 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
    13321 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
    13381 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
    13441 ccgtcatcac cgaaacgcgc gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg
    13501 cgacggcgcg gcttgtccgc gccctggtag attgcctggc cgtaggccag ccatttttga
    13561 gcggccagcg gccgcgatag gccgacgcga agcggcgggg cgtagggagc gcagcgaccg
    13621 aagggtaggc gctttttgca gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc
    13681 caggcgggtt ttaagagttt taataagttt taaagagttt taggcggaaa aatcgccttt
    13741 tttctctttt atatcagtca cttacatgtg tgaccggttc ccaatgtacg gctttgggtt
    13801 cccaatgtac gggttccggt tcccaatgta cggctttggg ttcccaatgt acgtgctatc
    13861 cacaggaaac agaccttttc gacctttttc ccctgctagg gcaatttgcc ctagcatctg
    13921 ctccgtacat taggaaccgg cggatgcttc gccctcgatc aggttgcggt agcgcatgac
    13981 taggatcggg ccagcctgcc ccgcctcctc cttcaaatcg tactccggca ggtcatttga
    14041 cccgatcagc ttgcgcacgg tgaaacagaa cttcttgaac tctccggcgc tgccactgcg
    14101 ttcgtagatc gtcttgaaca accatctggc ttctgccttg cctgcggcgc ggcgtgccag
    14161 gcggtagaga aaacggccga tgccgggatc gatcaaaaag taatcggggt gaaccgtcag
    14221 cacgtccggg ttcttgcctt ctgtgatctc gcggtacatc caatcagcta gctcgatctc
    14281 gatgtactcc ggccgcccgg tttcgctctt tacgatcttg tagcggctaa tcaaggcttc
    14341 accctcggat accgtcacca ggcggccgtt cttggccttc ttcgtacgct gcatggcaac
    14401 gtgcgtggtg tttaaccgaa tgcaggtttc taccaggtcg tctttctgct ttccgccatc
    14461 ggctcgccgg cagaacttga gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt
    14521 gtctcccttc ccttcccggt atcggttcat ggattcggtt agatgggaaa ccgccatcag
    14581 taccaggtcg taatcccaca cactggccat gccggccggc cctgcggaaa cctctacgtg
    14641 cccgtctgga agctcgtagc ggatcacctc gccagctcgt cggtcacgct tcgacagacg
    14701 gaaaacggcc acgtccatga tgctgcgact atcgcgggtg cccacgtcat agagcatcgg
    14761 aacgaaaaaa tctggttgct cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc
    14821 tgccggcggt tgccgggatt ctttgcggat tcgatcagcg gccgcttgcc acgattcacc
    14881 ggggcgtgct tctgcctcga tgcgttgccg ctgggcggcc tgcgcggcct tcaacttctc
    14941 caccaggtca tcacccagcg ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgct
    15001 cacgccgatt cctcgggctt gggggttcca gtgccattgc agggccggca gacaacccag
    15061 ccgcttacgc ctggccaacc gcccgttcct ccacacatgg ggcattccac ggcgtcggtg
    15121 cctggttgtt cttgattttc catgccgcct cctttagccg ctaaaattca tctactcatt
    15181 tattcatttg ctcatttact ctggtagctg cgcgatgtat tcagatagca gctcggtaat
    15241 ggtcttgcct tggcgtaccg cgtacatctt cagcttggtg tgatcctccg ccggcaactg
    15301 aaagttgacc cgcttcatgg ctggcgtgtc tgccaggctg gccaacgttg cagccttgct
    15361 gctgcgtgcg ctcggacggc cggcacttag cgtgtttgtg cttttgctca ttttctcttt
    15421 acctcattaa ctcaaatgag ttttgattta atttcagcgg ccagcgcctg gacctcgcgg
    15481 gcagcgtcgc cctcgggttc tgattcaaga acggttgtgc cggcggcggc agtgcctggg
    15541 tagctcacgc gctgcgtgat acgggactca agaatgggca gctcgtaccc ggccagcgcc
    15601 tcggcaacct caccgccgat gcgcgtgcct ttgatcgccc gcgacacgac aaaggccgct
    15661 tgtagccttc catccgtgac ctcaatgcgc tgcttaacca gctccaccag gtcggcggtg
    15721 gcccatatgt cgtaagggct tggctgcacc ggaatcagca cgaagtcggc tgccttgatc
    15781 gcggacacag ccaagtccgc cgcctggggc gctccgtcga tcactacgaa gtcgcgccgg
    15841 ccgatggcct tcacgtcgcg gtcaatcgtc gggcggtcga tgccgacaac ggttagcggt
    15901 tgatcttccc gcacggccgc ccaatcgcgg gcactgccct ggggatcgga atcgactaac
    15961 agaacatcgg ccccggcgag ttgcagggcg cgggctagat gggttgcgat ggtcgtcttg
    16021 cctgacccgc ctttctggtt aagtacagcg ataaccttca tgcgttcccc ttgcgtattt
    16081 gtttatttac tcatcgcatc atatacgcag cgaccgcatg acgcaagctg ttttactcaa
    16141 atacacatca cctttttaga cggcggcgct cggtttcttc agcggccaag ctggccggcc
    16201 aggccgccag cttggcatca gacaaaccgg ccaggatttc atgcagccgc acggttgaga
    16261 cgtgcgcggg cggctcgaac acgtacccgg ccgcgatcat ctccgcctcg atctcttcgg
    16321 taatgaaaaa cggttcgtcc tggccgtcct ggtgcggttt catgcttgtt cctcttggcg
    16381 ttcattctcg gcggccgcca gggcgtcggc ctcggtcaat gcgtcctcac ggaaggcacc
    16441 gcgccgcctg gcctcggtgg gcgtcacttc ctcgctgcgc tcaagtgcgc ggtacagggt
    16501 cgagcgatgc acgccaagca gtgcagccgc ctctttcacg gtgcggcctt cctggtcgat
    16561 cagctcgcgg gcgtgcgcga tctgtgccgg ggtgagggta gggcgggggc caaacttcac
    16621 gcctcgggcc ttggcggcct cgcgcccgct ccgggtgcgg tcgatgatta gggaacgctc
    16681 gaactcggca atgccggcga acacggtcaa caccatgcgg ccggccggcg tggtggtgtc
    16741 ggcccacggc tctgccaggc tacgcaggcc cgcgccggcc tcctggatgc gctcggcaat
    16801 gtccagtagg tcgcgggtgc tgcgggccag gcggtctagc ctggtcactg tcacaacgtc
    16861 gccagggcgt aggtggtcaa gcatcctggc cagctccggg cggtcgcgcc tggtgccggt
    16921 gatcttctcg gaaaacagct tggtgcagcc ggccgcgtgc agttcggccc gttggttggt
    16981 caagtcctgg tcgtcggtgc tgacgcgggc atagcccagc aggccagcgg cggcgctctt
    17041 gttcatggcg taatgtctcc ggttctagtc gcaagtattc tactttatgc gactaaaaca
    17101 cgcgacaaga aaacgccagg aaaagggcag ggcggcagcc tgtcgcgtaa cttaggactt
    17161 gtgcgacatg tcgttttcag aagacggctg cactgaacgt cagaagccga ctgcactata
    17221 gcagcggagg ggttggatca aagtactttg atcccgaggg gaaccctgtg gttggcatgc
    17281 acatacaaat ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gttattctaa
//