pHEE401E

LOCUS       pHEE401E               17112 bp ds-DNA     circular SYN 12-DEC-2018
DEFINITION  Egg cell-specific promoter-controlled expression of 
            3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target
            sequence (U6-26 promoter), Hyg resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pHEE401E
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17112)
  AUTHORS   Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ
  TITLE     Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently 
            generates homozygous mutants for multiple target genes in 
            Arabidopsis in a single generation.
  JOURNAL   Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0.
  PUBMED    26193878
REFERENCE   2  (bases 1 to 17112)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 12, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17112
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     78..100
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     92..109
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     93..109
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             822..1613
                     /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        1765..1840
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3410..3475
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3482..3502
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3527..7627
                     /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             7540..7548
                     /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             7628..7675
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8332..8348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8332..8348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8345..8367)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8356..8372
                     /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(8380..8410)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8425..8446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8637..9313
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     9250..9270
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             9380..10405
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     polyA_signal    10446..10620
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    10698..10722
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             11147..11941
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12028..12616
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12517..12536
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12770..12787
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    12802..12942
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(12868..12887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13028..13050
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      13286..13480
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13546..14619)
                     /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(15048..15677)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    16978..17002
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
       61 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagcttcg
      121 acttgccttc cgcacaatac atcatttctt cttagctttt tttcttcttc ttcgttcata
      181 cagttttttt ttgtttatca gcttacattt tcttgaaccg tagctttcgt tttcttcttt
      241 ttaactttcc attcggagtt tttgtatctt gtttcatagt ttgtcccagg attagaatga
      301 ttaggcatcg aaccttcaag aatttgattg aataaaacat cttcattctt aagatatgaa
      361 gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca tttatatggg
      421 aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct tcaaaagtcc
      481 cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga
      541 ttgggagacc aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
      601 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
      661 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
      721 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
      781 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catgggggaa gcggtgatcg
      841 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
      901 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
      961 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     1021 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     1081 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     1141 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     1201 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     1261 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     1321 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     1381 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     1441 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     1501 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     1561 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taatgtctag
     1621 ctagaaattc gttcaagccg acgccgcttc gcggcgcggc ttaactcaag cgttagatgc
     1681 actaagcaca taattgctca cagccaaact atcaggtcaa gtctgctttt attattttta
     1741 agcgtgcata ataagccggt ctcggtttta gagctagaaa tagcaagtta aaataaggct
     1801 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttttgc aaaattttcc
     1861 agatcgattt cttcttcctc tgttcttcgg cgttcaattt ctggggtttt ctcttcgttt
     1921 tctgtaactg aaacctaaaa tttgacctaa aaaaaatctc aaataatatg attcagtggt
     1981 tttgtacttt tcagttagtt gagttttgca gttccgatga gataaaccaa taccatggtt
     2041 atactagtga ataaaagcat ttgcgtttgg tttatcattg cgtttataca aggacagaga
     2101 tccactgagc tggaatagct taaaaccatt atcagaacaa aataaaccat tttttgttaa
     2161 gaatcagagc atagtaaaca acagaaacaa cctaagagag gtaacttgtc caagaagata
     2221 gctaattata tctattttat aaaagttatc atagtttgta agtcacaaaa gatgcaaata
     2281 acagagaaac taggagactt gagaatatac attcttgtat atttgtattc gagattgtga
     2341 aaatttgacc ataagtttaa attcttaaaa agatatatct gatctaggtg atggttatag
     2401 actgtaattt taccacatgt ttaatgatgg atagtgacac acatgacaca tcgacaacac
     2461 tatagcatct tatttagatt acaacatgaa atttttctgt aatacatgtc tttgtacata
     2521 atttaaaagt aattcctaag aaatatattt atacaaggag tttaaagaaa acatagcata
     2581 aagttcaatg agtagtaaaa accatataca gtatatagca taaagttcaa tgagtttatt
     2641 acaaaagcat tggttcactt tctgtaacac gacgttaaac cttcgtctcc aataggagcg
     2701 ctactgattc aacatgccaa tatatactaa atacgtttct acagtcaaat gctttaacgt
     2761 ttcatgatta agtgactatt taccgtcaat cctttcccat tcctcccact aatccaactt
     2821 tttaattact cttaaatcac cactaagcta gtaacgccta tcatgaatta gctctactaa
     2881 atctagcaac ctttcaaatt tgcagtattg caggtgtctc tgtgtcttta aaatagttgc
     2941 cttatgattt cttcggtttc aagatgatca aatagttata gatttcatgc tcacacatgc
     3001 tcattagatg tgtacatact ttacttaccc aaatctattt tctcgcaaag attttgatgg
     3061 taaagctgat ttggttctat tgaactaaat caaacgagtt tcagactgag tgattctaat
     3121 ccggcccatt agcccctaaa cagacccact aattacgcag cttttaatag agtaattaca
     3181 cctagtttac ccactaaacc actaagcact aattatctca caatctaatg agcttccctc
     3241 gtaattactt gggctttcac tctaccattt atttgtaaca gtcaagtctc tactgtctct
     3301 atataaactc tctaaagtta acacacaatt ctcatcacaa acaaatcaac caaagcaact
     3361 tctactcttt cttctttcga ccttatcaat ctgttgagaa atctagatgg attacaagga
     3421 ccacgacggg gattacaagg accacgacat tgattacaag gatgatgatg acaagatggc
     3481 tccgaagaag aagaggaagg ttggcatcca cggggtgcca gctgctgaca agaagtactc
     3541 gatcggcctc gatattggga ctaactctgt tggctgggcc gtgatcaccg acgagtacaa
     3601 ggtgccctca aagaagttca aggtcctggg caacaccgat cggcattcca tcaagaagaa
     3661 tctcattggc gctctcctgt tcgacagcgg cgagacggct gaggctacgc ggctcaagcg
     3721 caccgcccgc aggcggtaca cgcgcaggaa gaatcgcatc tgctacctgc aggagatttt
     3781 ctccaacgag atggcgaagg ttgacgattc tttcttccac aggctggagg agtcattcct
     3841 cgtggaggag gataagaagc acgagcggca tccaatcttc ggcaacattg tcgacgaggt
     3901 tgcctaccac gagaagtacc ctacgatcta ccatctgcgg aagaagctcg tggactccac
     3961 agataaggcg gacctccgcc tgatctacct cgctctggcc cacatgatta agttcagggg
     4021 ccatttcctg atcgaggggg atctcaaccc ggacaatagc gatgttgaca agctgttcat
     4081 ccagctcgtg cagacgtaca accagctctt cgaggagaac cccattaatg cgtcaggcgt
     4141 cgacgcgaag gctatcctgt ccgctaggct ctcgaagtct cggcgcctcg agaacctgat
     4201 cgcccagctg ccgggcgaga agaagaacgg cctgttcggg aatctcattg cgctcagcct
     4261 ggggctcacg cccaacttca agtcgaattt cgatctcgct gaggacgcca agctgcagct
     4321 ctccaaggac acatacgacg atgacctgga taacctcctg gcccagatcg gcgatcagta
     4381 cgcggacctg ttcctcgctg ccaagaatct gtcggacgcc atcctcctgt ctgatattct
     4441 cagggtgaac accgagatta cgaaggctcc gctctcagcc tccatgatca agcgctacga
     4501 cgagcaccat caggatctga ccctcctgaa ggcgctggtc aggcagcagc tccccgagaa
     4561 gtacaaggag atcttcttcg atcagtcgaa gaacggctac gctgggtaca ttgacggcgg
     4621 ggcctctcag gaggagttct acaagttcat caagccgatt ctggagaaga tggacggcac
     4681 ggaggagctg ctggtgaagc tcaatcgcga ggacctcctg aggaagcagc ggacattcga
     4741 taacggcagc atcccacacc agattcatct cggggagctg cacgctatcc tgaggaggca
     4801 ggaggacttc taccctttcc tcaaggataa ccgcgagaag atcgagaaga ttctgacttt
     4861 caggatcccg tactacgtcg gcccactcgc taggggcaac tcccgcttcg cttggatgac
     4921 ccgcaagtca gaggagacga tcacgccgtg gaacttcgag gaggtggtcg acaagggcgc
     4981 tagcgctcag tcgttcatcg agaggatgac gaatttcgac aagaacctgc caaatgagaa
     5041 ggtgctccct aagcactcgc tcctgtacga gtacttcaca gtctacaacg agctgactaa
     5101 ggtgaagtat gtgaccgagg gcatgaggaa gccggctttc ctgtctgggg agcagaagaa
     5161 ggccatcgtg gacctcctgt tcaagaccaa ccggaaggtc acggttaagc agctcaagga
     5221 ggactacttc aagaagattg agtgcttcga ttcggtcgag atctctggcg ttgaggaccg
     5281 cttcaacgcc tccctgggga cctaccacga tctcctgaag atcattaagg ataaggactt
     5341 cctggacaac gaggagaatg aggatatcct cgaggacatt gtgctgacac tcactctgtt
     5401 cgaggaccgg gagatgatcg aggagcgcct gaagacttac gcccatctct tcgatgacaa
     5461 ggtcatgaag cagctcaaga ggaggaggta caccggctgg gggaggctga gcaggaagct
     5521 catcaacggc attcgggaca agcagtccgg gaagacgatc ctcgacttcc tgaagagcga
     5581 tggcttcgcg aaccgcaatt tcatgcagct gattcacgat gacagcctca cattcaagga
     5641 ggatatccag aaggctcagg tgagcggcca gggggactcg ctgcacgagc atatcgcgaa
     5701 cctcgctggc tcgccagcta tcaagaaggg gattctgcag accgtgaagg ttgtggacga
     5761 gctggtgaag gtcatgggca ggcacaagcc tgagaacatc gtcattgaga tggcccggga
     5821 gaatcagacc acgcagaagg gccagaagaa ctcacgcgag aggatgaaga ggatcgagga
     5881 gggcattaag gagctggggt cccagatcct caaggagcac ccggtggaga acacgcagct
     5941 gcagaatgag aagctctacc tgtactacct ccagaatggc cgcgatatgt atgtggacca
     6001 ggagctggat attaacaggc tcagcgatta cgacgtcgat catatcgttc cacagtcatt
     6061 cctgaaggat gactccattg acaacaaggt cctcaccagg tcggacaaga accggggcaa
     6121 gtctgataat gttccttcag aggaggtcgt taagaagatg aagaactact ggcgccagct
     6181 cctgaatgcc aagctgatca cgcagcggaa gttcgataac ctcacaaagg ctgagagggg
     6241 cgggctctct gagctggaca aggcgggctt catcaagagg cagctggtcg agacacggca
     6301 gatcactaag cacgttgcgc agattctcga ctcacggatg aacactaagt acgatgagaa
     6361 tgacaagctg atccgcgagg tgaaggtcat caccctgaag tcaaagctcg tctccgactt
     6421 caggaaggat ttccagttct acaaggttcg ggagatcaac aattaccacc atgcccatga
     6481 cgcgtacctg aacgcggtgg tcggcacagc tctgatcaag aagtacccaa agctcgagag
     6541 cgagttcgtg tacggggact acaaggttta cgatgtgagg aagatgatcg ccaagtcgga
     6601 gcaggagatt ggcaaggcta ccgccaagta cttcttctac tctaacatta tgaatttctt
     6661 caagacagag atcactctgg ccaatggcga gatccggaag cgccccctca tcgagacgaa
     6721 cggcgagacg ggggagatcg tgtgggacaa gggcagggat ttcgcgaccg tcaggaaggt
     6781 tctctccatg ccacaagtga atatcgtcaa gaagacagag gtccagactg gcgggttctc
     6841 taaggagtca attctgccta agcggaacag cgacaagctc atcgcccgca agaaggactg
     6901 ggatccgaag aagtacggcg ggttcgacag ccccactgtg gcctactcgg tcctggttgt
     6961 ggcgaaggtt gagaagggca agtccaagaa gctcaagagc gtgaaggagc tgctggggat
     7021 cacgattatg gagcgctcca gcttcgagaa gaacccgatc gatttcctgg aggcgaaggg
     7081 ctacaaggag gtgaagaagg acctgatcat taagctcccc aagtactcac tcttcgagct
     7141 ggagaacggc aggaagcgga tgctggcttc cgctggcgag ctgcagaagg ggaacgagct
     7201 ggctctgccg tccaagtatg tgaacttcct ctacctggcc tcccactacg agaagctcaa
     7261 gggcagcccc gaggacaacg agcagaagca gctgttcgtc gagcagcaca agcattacct
     7321 cgacgagatc attgagcaga tttccgagtt ctccaagcgc gtgatcctgg ccgacgcgaa
     7381 tctggataag gtcctctccg cgtacaacaa gcaccgcgac aagccaatca gggagcaggc
     7441 tgagaatatc attcatctct tcaccctgac gaacctcggc gcccctgctg ctttcaagta
     7501 cttcgacaca actatcgatc gcaagaggta cacaagcact aaggaggtcc tggacgcgac
     7561 cctcatccac cagtcgatta ccggcctcta cgagacgcgc atcgacctgt ctcagctcgg
     7621 gggcgacaag cggccagcgg cgacgaagaa ggcggggcag gcgaagaaga agaagtgagc
     7681 tcagagcttt cgttcgtatc atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt
     7741 tcattgcgca cacaccagaa tcctactgag tttgagtatt atggcattgg gaaaactgtt
     7801 tttcttgtac catttgttgt gcttgtaatt tactgtgttt tttattcggt tttcgctatc
     7861 gaactgtgaa atggaaatgg atggagaaga gttaatgaat gatatggtcc ttttgttcat
     7921 tctcaaatta atattatttg ttttttctct tatttgttgt gtgttgaatt tgaaattata
     7981 agagatatgc aaacattttg ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc
     8041 gaagttaata tgaggagtaa aacacttgta gttgtaccat tatgcttatt cactaggcaa
     8101 caaatatatt ttcagaccta gaaaagctgc aaatgttact gaatacaagt atgtcctctt
     8161 gtgttttaga catttatgaa ctttccttta tgtaattttc cagaatcctt gtcagattct
     8221 aatcattgct ttataattat agttatactc atggatttgt agttgagtat gaaaatattt
     8281 tttaatgcat tttatgactt gccaattgat tgacaacgaa ttcgtaatca tgtcatagct
     8341 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     8401 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     8461 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     8521 cgcggggaga ggcggtttgc gtattggcta gagcagcttg ccaacatggt ggagcacgac
     8581 actctcgtct actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag
     8641 acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt
     8701 cacttcatca aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat
     8761 aaaggaaagg ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca
     8821 cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat
     8881 tgatgtgaac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt
     8941 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     9001 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     9061 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga
     9121 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
     9181 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     9241 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
     9301 gaggacacgc tgaaatcacc agtctctctc tacaaatcta tctctctcga gctttcgcag
     9361 atccgggggg caatgagata tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     9421 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     9481 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     9541 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     9601 tccggaagtg cttgacattg gggagtttag cgagagcctg acctattgca tctcccgccg
     9661 ttcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctacaacc
     9721 ggtcgcggag gctatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     9781 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     9841 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     9901 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     9961 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
    10021 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
    10081 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
    10141 ggagcttgca ggatcgccac gactccgggc gtatatgctc cgcattggtc ttgaccaact
    10201 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
    10261 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
    10321 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
    10381 cactcgtccg agggcaaaga aatagagtag atgccgaccg ggatctgtcg atcgacaagc
    10441 tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata
    10501 gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa
    10561 tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc
    10621 ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt
    10681 tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc
    10741 cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac
    10801 ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc
    10861 ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat
    10921 gttgattgta acgatgacag agcgttgctg cctgtgatca ccgcggtttc aaaatcggct
    10981 ccgtcgatac tatgttatac gccaactttg aaaacaactt tgaaaaagct gttttctggt
    11041 atttaaggtt ttagaatgca aggaacagtg aattggagtt cgtcttgtta taattagctt
    11101 cttggggtat ctttaaatac tgtagaaaag aggaaggaaa taataaatgg ctaaaatgag
    11161 aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag atacggaagg
    11221 aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat atttaaaaat
    11281 gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg acatgatgct
    11341 atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc atgatggctg
    11401 gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt atgaagatga
    11461 acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct ttcactccat
    11521 cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg aattggatta
    11581 cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag acactccatt
    11641 taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag aggaacttgt
    11701 cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca aagtaagtgg
    11761 ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg ccttctgcgt
    11821 ccggtcgatc agggaggata tcggggaaga acagtatgtc gagctatttt ttgacttact
    11881 ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg aattgtttta
    11941 gtacctagaa tgcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
    12001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
    12061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
    12121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
    12181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
    12241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
    12301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
    12361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
    12421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
    12481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
    12541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
    12601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
    12661 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
    12721 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
    12781 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
    12841 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
    12901 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
    12961 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
    13021 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
    13081 cagggtgcct tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt gtccgcgccc
    13141 tggtagattg cctggccgta ggccagccat ttttgagcgg ccagcggccg cgataggccg
    13201 acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt tttgcagctc
    13261 ttcggctgtg cgctggccag acagttatgc acaggccagg cgggttttaa gagttttaat
    13321 aagttttaaa gagttttagg cggaaaaatc gccttttttc tcttttatat cagtcactta
    13381 catgtgtgac cggttcccaa tgtacggctt tgggttccca atgtacgggt tccggttccc
    13441 aatgtacggc tttgggttcc caatgtacgt gctatccaca ggaaacagac cttttcgacc
    13501 tttttcccct gctagggcaa tttgccctag catctgctcc gtacattagg aaccggcgga
    13561 tgcttcgccc tcgatcaggt tgcggtagcg catgactagg atcgggccag cctgccccgc
    13621 ctcctccttc aaatcgtact ccggcaggtc atttgacccg atcagcttgc gcacggtgaa
    13681 acagaacttc ttgaactctc cggcgctgcc actgcgttcg tagatcgtct tgaacaacca
    13741 tctggcttct gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac ggccgatgcc
    13801 gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg tccgggttct tgccttctgt
    13861 gatctcgcgg tacatccaat cagctagctc gatctcgatg tactccggcc gcccggtttc
    13921 gctctttacg atcttgtagc ggctaatcaa ggcttcaccc tcggataccg tcaccaggcg
    13981 gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta accgaatgca
    14041 ggtttctacc aggtcgtctt tctgctttcc gccatcggct cgccggcaga acttgagtac
    14101 gtccgcaacg tgtggacgga acacgcggcc gggcttgtct cccttccctt cccggtatcg
    14161 gttcatggat tcggttagat gggaaaccgc catcagtacc aggtcgtaat cccacacact
    14221 ggccatgccg gccggccctg cggaaacctc tacgtgcccg tctggaagct cgtagcggat
    14281 cacctcgcca gctcgtcggt cacgcttcga cagacggaaa acggccacgt ccatgatgct
    14341 gcgactatcg cgggtgccca cgtcatagag catcggaacg aaaaaatctg gttgctcgtc
    14401 gcccttgggc ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc gggattcttt
    14461 gcggattcga tcagcggccg cttgccacga ttcaccgggg cgtgcttctg cctcgatgcg
    14521 ttgccgctgg gcggcctgcg cggccttcaa cttctccacc aggtcatcac ccagcgccgc
    14581 gccgatttgt accgggccgg atggtttgcg accgctcacg ccgattcctc gggcttgggg
    14641 gttccagtgc cattgcaggg ccggcaggca acccagccgc ttacgcctgg ccaaccgccc
    14701 gttcctccac acatggggca ttccacggcg tcggtgcctg gttgttcttg attttccatg
    14761 ccgcctcctt tagccgctaa aattcatcta ctcatttatt catttgctca tttactctgg
    14821 tagctgcgcg atgtattcag atagcagctc ggtaatggtc ttgccttggc gtaccgcgta
    14881 catcttcagc ttggtgtgat cctccgccgg caactgaaag ttgacccgct tcatggctgg
    14941 cgtgtctgcc aggctggcca acgttgcagc cttgctgctg cgtgcgctcg gacggccggc
    15001 acttagcgtg tttgtgcttt tgctcatttt ctctttacct cattaactca aatgagtttt
    15061 gatttaattt cagcggccag cgcctggacc tcgcgggcag cgtcgccctc gggttctgat
    15121 tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc tcacgcgctg cgtgatacgg
    15181 gactcaagaa tgggcagctc gtacccggcc agcgcctcgg caacctcacc gccgatgcgc
    15241 gtgcctttga tcgcccgcga cacgacaaag gccgcttgta gccttccatc cgtgacctca
    15301 atgcgctgct taaccagctc caccaggtcg gcggtggccc atatgtcgta agggcttggc
    15361 tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg acacagccaa gtccgccgcc
    15421 tggggcgctc cgtcgatcac tacgaagtcg cgccggccga tggccttcac gtcgcggtca
    15481 atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat cttcccgcac ggccgcccaa
    15541 tcgcgggcac tgccctgggg atcggaatcg actaacagaa catcggcccc ggcgagttgc
    15601 agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg acccgccttt ctggttaagt
    15661 acagcgataa ccttcatgcg ttccccttgc gtatttgttt atttactcat cgcatcatat
    15721 acgcagcgac cgcatgacgc aagctgtttt actcaaatac acatcacctt tttagacggc
    15781 ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc cgccagcttg gcatcagaca
    15841 aaccggccag gatttcatgc agccgcacgg ttgagacgtg cgcgggcggc tcgaacacgt
    15901 acccggccgc gatcatctcc gcctcgatct cttcggtaat gaaaaacggt tcgtcctggc
    15961 cgtcctggtg cggtttcatg cttgttcctc ttggcgttca ttctcggcgg ccgccagggc
    16021 gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc cgcctggcct cggtgggcgt
    16081 cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag cgatgcacgc caagcagtgc
    16141 agccgcctct ttcacggtgc ggccttcctg gtcgatcagc tcgcgggcgt gcgcgatctg
    16201 tgccggggtg agggtagggc gggggccaaa cttcacgcct cgggccttgg cggcctcgcg
    16261 cccgctccgg gtgcggtcga tgattaggga acgctcgaac tcggcaatgc cggcgaacac
    16321 ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc cacggctctg ccaggctacg
    16381 caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc agtaggtcgc gggtgctgcg
    16441 ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca gggcgtaggt ggtcaagcat
    16501 cctggccagc tccgggcggt cgcgcctggt gccggtgatc ttctcggaaa acagcttggt
    16561 gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag tcctggtcgt cggtgctgac
    16621 gcgggcatag cccagcaggc cagcggcggc gctcttgttc atggcgtaat gtctccggtt
    16681 ctagtcgcaa gtattctact ttatgcgact aaaacacgcg acaagaaaac gccaggaaaa
    16741 gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc gacatgtcgt tttcagaaga
    16801 cggctgcact gaacgtcaga agccgactgc actatagcag cggaggggtt ggatcaaagt
    16861 actttgatcc cgaggggaac cctgtggttg gcatgcacat acaaatggac gaacggataa
    16921 accttttcac gcccttttaa atatccgatt attctaataa acgctctttt ctcttaggtt
    16981 tacccgccaa tatatcctgt caaacactga tagtttaaac tgaaggcggg aaacgacaat
    17041 ctgatccaag ctcaagctgc tctagcattc gccattcagg ctgcgcaact gttgggaagg
    17101 gcgatcggtg cg
//