LOCUS       Exported               17870 bp ds-DNA     circular SYN 12-MAY-2021
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), Hyg resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pHUE411
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17870)
  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 17870)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17870
                     /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(9244..9260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9244..9260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9257..9279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9268..9284
                     /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(9292..9322)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9337..9358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9549..10226
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             10294..11319
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     polyA_signal    11360..11534
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11612..11636
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             12061..12855
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12942..13530
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13431..13450
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13684..13701
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13716..13856
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13782..13801)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13942..13964
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      14200..14394
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(14460..15533)
                     /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(15962..16591)
                     /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 catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     9301 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     9361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     9421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggc tagagcagct tgccaacatg
     9481 gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc agaagaccaa
     9541 agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc
     9601 ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc
     9661 catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag tggtcccaaa
     9721 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca
     9781 aagcaagtgg attgatgtga taacatggtg gagcacgaca ctctcgtcta ctccaagaat
     9841 atcaaagata cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata
     9901 tcgggaaacc tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta
     9961 gaaaaggaag gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa
    10021 gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa
    10081 aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac
    10141 gtaagggatg acgcacaatc ccactatcct tcgcaagacc ttcctctata taaggaagtt
    10201 catttcattt ggagaggaca cgctgaaatc accagtctct ctctacaaat ctatctctct
    10261 cgagctttcg cagatcccgg ggggcaatga gatatgaaaa agcctgaact caccgcgacg
    10321 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
    10381 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
    10441 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
    10501 gccgcgctcc cgattccgga agtgcttgac attggggagt ttagcgagag cctgacctat
    10561 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
    10621 gctgttctac aaccggtcgc ggaggctatg gatgcgatcg ctgcggccga tcttagccag
    10681 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
    10741 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
    10801 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
    10861 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
    10921 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
    10981 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
    11041 gagcggaggc atccggagct tgcaggatcg ccacgactcc gggcgtatat gctccgcatt
    11101 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
    11161 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
    11221 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
    11281 aaccgacgcc ccagcactcg tccgagggca aagaaataga gtagatgccg accgggatct
    11341 gtcgatcgac aagctcgagt ttctccataa taatgtgtga gtagttccca gataagggaa
    11401 ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat
    11461 ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagta
    11521 ctaaaatcca gatcccccga attaattcgg cgttaattca gtacattaaa aacgtccgca
    11581 atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag
    11641 ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc
    11701 catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt
    11761 taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac ggatgatctc
    11821 gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg atcaccgcgg
    11881 tttcaaaatc ggctccgtcg atactatgtt atacgccaac tttgaaaaca actttgaaaa
    11941 agctgttttc tggtatttaa ggttttagaa tgcaaggaac agtgaattgg agttcgtctt
    12001 gttataatta gcttcttggg gtatctttaa atactgtaga aaagaggaag gaaataataa
    12061 atggctaaaa tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta
    12121 aaagatacgg aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac
    12181 ctatatttaa aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa
    12241 aaggacatga tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa
    12301 cggcatgatg gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa
    12361 gagtatgaag atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg
    12421 ctctttcact ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta
    12481 gccgaattgg attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa
    12541 gaagacactc catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc
    12601 gaagaggaac ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat
    12661 ggcaaagtaa gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac
    12721 attgccttct gcgtccggtc gatcagggag gatatcgggg aagaacagta tgtcgagcta
    12781 ttttttgact tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg
    12841 gatgaattgt tttagtacct agaatgcatg accaaaatcc cttaacgtga gttttcgttc
    12901 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
    12961 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
    13021 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    13081 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    13141 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    13201 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    13261 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    13321 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    13381 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    13441 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    13501 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    13561 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
    13621 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
    13681 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
    13741 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
    13801 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
    13861 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
    13921 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
    13981 cgaaacgcgc gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg
    14041 gcttgtccgc gccctggtag attgcctggc cgtaggccag ccatttttga gcggccagcg
    14101 gccgcgatag gccgacgcga agcggcgggg cgtagggagc gcagcgaccg aagggtaggc
    14161 gctttttgca gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt
    14221 ttaagagttt taataagttt taaagagttt taggcggaaa aatcgccttt tttctctttt
    14281 atatcagtca cttacatgtg tgaccggttc ccaatgtacg gctttgggtt cccaatgtac
    14341 gggttccggt tcccaatgta cggctttggg ttcccaatgt acgtgctatc cacaggaaac
    14401 agaccttttc gacctttttc ccctgctagg gcaatttgcc ctagcatctg ctccgtacat
    14461 taggaaccgg cggatgcttc gccctcgatc aggttgcggt agcgcatgac taggatcggg
    14521 ccagcctgcc ccgcctcctc cttcaaatcg tactccggca ggtcatttga cccgatcagc
    14581 ttgcgcacgg tgaaacagaa cttcttgaac tctccggcgc tgccactgcg ttcgtagatc
    14641 gtcttgaaca accatctggc ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga
    14701 aaacggccga tgccgggatc gatcaaaaag taatcggggt gaaccgtcag cacgtccggg
    14761 ttcttgcctt ctgtgatctc gcggtacatc caatcagcta gctcgatctc gatgtactcc
    14821 ggccgcccgg tttcgctctt tacgatcttg tagcggctaa tcaaggcttc accctcggat
    14881 accgtcacca ggcggccgtt cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg
    14941 tttaaccgaa tgcaggtttc taccaggtcg tctttctgct ttccgccatc ggctcgccgg
    15001 cagaacttga gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt gtctcccttc
    15061 ccttcccggt atcggttcat ggattcggtt agatgggaaa ccgccatcag taccaggtcg
    15121 taatcccaca cactggccat gccggccggc cctgcggaaa cctctacgtg cccgtctgga
    15181 agctcgtagc ggatcacctc gccagctcgt cggtcacgct tcgacagacg gaaaacggcc
    15241 acgtccatga tgctgcgact atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa
    15301 tctggttgct cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt
    15361 tgccgggatt ctttgcggat tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct
    15421 tctgcctcga tgcgttgccg ctgggcggcc tgcgcggcct tcaacttctc caccaggtca
    15481 tcacccagcg ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt
    15541 cctcgggctt gggggttcca gtgccattgc agggccggca gacaacccag ccgcttacgc
    15601 ctggccaacc gcccgttcct ccacacatgg ggcattccac ggcgtcggtg cctggttgtt
    15661 cttgattttc catgccgcct cctttagccg ctaaaattca tctactcatt tattcatttg
    15721 ctcatttact ctggtagctg cgcgatgtat tcagatagca gctcggtaat ggtcttgcct
    15781 tggcgtaccg cgtacatctt cagcttggtg tgatcctccg ccggcaactg aaagttgacc
    15841 cgcttcatgg ctggcgtgtc tgccaggctg gccaacgttg cagccttgct gctgcgtgcg
    15901 ctcggacggc cggcacttag cgtgtttgtg cttttgctca ttttctcttt acctcattaa
    15961 ctcaaatgag ttttgattta atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc
    16021 cctcgggttc tgattcaaga acggttgtgc cggcggcggc agtgcctggg tagctcacgc
    16081 gctgcgtgat acgggactca agaatgggca gctcgtaccc ggccagcgcc tcggcaacct
    16141 caccgccgat gcgcgtgcct ttgatcgccc gcgacacgac aaaggccgct tgtagccttc
    16201 catccgtgac ctcaatgcgc tgcttaacca gctccaccag gtcggcggtg gcccatatgt
    16261 cgtaagggct tggctgcacc ggaatcagca cgaagtcggc tgccttgatc gcggacacag
    16321 ccaagtccgc cgcctggggc gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct
    16381 tcacgtcgcg gtcaatcgtc gggcggtcga tgccgacaac ggttagcggt tgatcttccc
    16441 gcacggccgc ccaatcgcgg gcactgccct ggggatcgga atcgactaac agaacatcgg
    16501 ccccggcgag ttgcagggcg cgggctagat gggttgcgat ggtcgtcttg cctgacccgc
    16561 ctttctggtt aagtacagcg ataaccttca tgcgttcccc ttgcgtattt gtttatttac
    16621 tcatcgcatc atatacgcag cgaccgcatg acgcaagctg ttttactcaa atacacatca
    16681 cctttttaga cggcggcgct cggtttcttc agcggccaag ctggccggcc aggccgccag
    16741 cttggcatca gacaaaccgg ccaggatttc atgcagccgc acggttgaga cgtgcgcggg
    16801 cggctcgaac acgtacccgg ccgcgatcat ctccgcctcg atctcttcgg taatgaaaaa
    16861 cggttcgtcc tggccgtcct ggtgcggttt catgcttgtt cctcttggcg ttcattctcg
    16921 gcggccgcca gggcgtcggc ctcggtcaat gcgtcctcac ggaaggcacc gcgccgcctg
    16981 gcctcggtgg gcgtcacttc ctcgctgcgc tcaagtgcgc ggtacagggt cgagcgatgc
    17041 acgccaagca gtgcagccgc ctctttcacg gtgcggcctt cctggtcgat cagctcgcgg
    17101 gcgtgcgcga tctgtgccgg ggtgagggta gggcgggggc caaacttcac gcctcgggcc
    17161 ttggcggcct cgcgcccgct ccgggtgcgg tcgatgatta gggaacgctc gaactcggca
    17221 atgccggcga acacggtcaa caccatgcgg ccggccggcg tggtggtgtc ggcccacggc
    17281 tctgccaggc tacgcaggcc cgcgccggcc tcctggatgc gctcggcaat gtccagtagg
    17341 tcgcgggtgc tgcgggccag gcggtctagc ctggtcactg tcacaacgtc gccagggcgt
    17401 aggtggtcaa gcatcctggc cagctccggg cggtcgcgcc tggtgccggt gatcttctcg
    17461 gaaaacagct tggtgcagcc ggccgcgtgc agttcggccc gttggttggt caagtcctgg
    17521 tcgtcggtgc tgacgcgggc atagcccagc aggccagcgg cggcgctctt gttcatggcg
    17581 taatgtctcc ggttctagtc gcaagtattc tactttatgc gactaaaaca cgcgacaaga
    17641 aaacgccagg aaaagggcag ggcggcagcc tgtcgcgtaa cttaggactt gtgcgacatg
    17701 tcgttttcag aagacggctg cactgaacgt cagaagccga ctgcactata gcagcggagg
    17761 ggttggatca aagtactttg atcccgaggg gaaccctgtg gttggcatgc acatacaaat
    17821 ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gttattctaa
//
