LOCUS       Exported               17112 bp ds-DNA     circular SYN 12-MAY-2021
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 May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17112
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..25
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     213..235
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     227..244
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     228..244
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             957..1748
                     /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        1900..1975
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             3545..3610
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3617..3637
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3662..7762
                     /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             7675..7683
                     /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             7763..7810
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8467..8483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8467..8483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8480..8502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8491..8507
                     /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(8515..8545)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8560..8581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8772..9449
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             9517..10542
                     /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    10582..10756
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    10834..10858
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             11283..12077
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12164..12752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12653..12672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12906..12923
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    12938..13078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13004..13023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13164..13186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      13422..13616
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13682..14755)
                     /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(15184..15813)
                     /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 gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac
       61 aatctgatcc aagctcaagc tgctctagca ttcgccattc aggctgcgca actgttggga
      121 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
      181 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
      241 cagtgccaag cttcgacttg ccttccgcac aatacatcat ttcttcttag ctttttttct
      301 tcttcttcgt tcatacagtt tttttttgtt tatcagctta cattttcttg aaccgtagct
      361 ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc atagtttgtc
      421 ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa aacatcttca
      481 ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga agagaagcag
      541 gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta agttgaaaac
      601 aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta tatacagcta
      661 gagtcgaagt agtgattggg agaccaaccc agtggacata agcctgttcg gttcgtaagc
      721 tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg
      781 gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt
      841 ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat
      901 ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatgg
      961 gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc
     1021 gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc
     1081 tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa
     1141 cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga
     1201 ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc
     1261 cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct
     1321 tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata
     1381 gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc
     1441 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
     1501 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
     1561 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
     1621 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
     1681 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
     1741 gcaaataatg tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac
     1801 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtctg
     1861 cttttattat ttttaagcgt gcataataag ccggtctcgg ttttagagct agaaatagca
     1921 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1981 tttgcaaaat tttccagatc gatttcttct tcctctgttc ttcggcgttc aatttctggg
     2041 gttttctctt cgttttctgt aactgaaacc taaaatttga cctaaaaaaa atctcaaata
     2101 atatgattca gtggttttgt acttttcagt tagttgagtt ttgcagttcc gatgagataa
     2161 accaatacca tggttatact agtgaataaa agcatttgcg tttggtttat cattgcgttt
     2221 atacaaggac agagatccac tgagctggaa tagcttaaaa ccattatcag aacaaaataa
     2281 accatttttt gttaagaatc agagcatagt aaacaacaga aacaacctaa gagaggtaac
     2341 ttgtccaaga agatagctaa ttatatctat tttataaaag ttatcatagt ttgtaagtca
     2401 caaaagatgc aaataacaga gaaactagga gacttgagaa tatacattct tgtatatttg
     2461 tattcgagat tgtgaaaatt tgaccataag tttaaattct taaaaagata tatctgatct
     2521 aggtgatggt tatagactgt aattttacca catgtttaat gatggatagt gacacacatg
     2581 acacatcgac aacactatag catcttattt agattacaac atgaaatttt tctgtaatac
     2641 atgtctttgt acataattta aaagtaattc ctaagaaata tatttataca aggagtttaa
     2701 agaaaacata gcataaagtt caatgagtag taaaaaccat atacagtata tagcataaag
     2761 ttcaatgagt ttattacaaa agcattggtt cactttctgt aacacgacgt taaaccttcg
     2821 tctccaatag gagcgctact gattcaacat gccaatatat actaaatacg tttctacagt
     2881 caaatgcttt aacgtttcat gattaagtga ctatttaccg tcaatccttt cccattcctc
     2941 ccactaatcc aactttttaa ttactcttaa atcaccacta agctagtaac gcctatcatg
     3001 aattagctct actaaatcta gcaacctttc aaatttgcag tattgcaggt gtctctgtgt
     3061 ctttaaaata gttgccttat gatttcttcg gtttcaagat gatcaaatag ttatagattt
     3121 catgctcaca catgctcatt agatgtgtac atactttact tacccaaatc tattttctcg
     3181 caaagatttt gatggtaaag ctgatttggt tctattgaac taaatcaaac gagtttcaga
     3241 ctgagtgatt ctaatccggc ccattagccc ctaaacagac ccactaatta cgcagctttt
     3301 aatagagtaa ttacacctag tttacccact aaaccactaa gcactaatta tctcacaatc
     3361 taatgagctt ccctcgtaat tacttgggct ttcactctac catttatttg taacagtcaa
     3421 gtctctactg tctctatata aactctctaa agttaacaca caattctcat cacaaacaaa
     3481 tcaaccaaag caacttctac tctttcttct ttcgacctta tcaatctgtt gagaaatcta
     3541 gatggattac aaggaccacg acggggatta caaggaccac gacattgatt acaaggatga
     3601 tgatgacaag atggctccga agaagaagag gaaggttggc atccacgggg tgccagctgc
     3661 tgacaagaag tactcgatcg gcctcgatat tgggactaac tctgttggct gggccgtgat
     3721 caccgacgag tacaaggtgc cctcaaagaa gttcaaggtc ctgggcaaca ccgatcggca
     3781 ttccatcaag aagaatctca ttggcgctct cctgttcgac agcggcgaga cggctgaggc
     3841 tacgcggctc aagcgcaccg cccgcaggcg gtacacgcgc aggaagaatc gcatctgcta
     3901 cctgcaggag attttctcca acgagatggc gaaggttgac gattctttct tccacaggct
     3961 ggaggagtca ttcctcgtgg aggaggataa gaagcacgag cggcatccaa tcttcggcaa
     4021 cattgtcgac gaggttgcct accacgagaa gtaccctacg atctaccatc tgcggaagaa
     4081 gctcgtggac tccacagata aggcggacct ccgcctgatc tacctcgctc tggcccacat
     4141 gattaagttc aggggccatt tcctgatcga gggggatctc aacccggaca atagcgatgt
     4201 tgacaagctg ttcatccagc tcgtgcagac gtacaaccag ctcttcgagg agaaccccat
     4261 taatgcgtca ggcgtcgacg cgaaggctat cctgtccgct aggctctcga agtctcggcg
     4321 cctcgagaac ctgatcgccc agctgccggg cgagaagaag aacggcctgt tcgggaatct
     4381 cattgcgctc agcctggggc tcacgcccaa cttcaagtcg aatttcgatc tcgctgagga
     4441 cgccaagctg cagctctcca aggacacata cgacgatgac ctggataacc tcctggccca
     4501 gatcggcgat cagtacgcgg acctgttcct cgctgccaag aatctgtcgg acgccatcct
     4561 cctgtctgat attctcaggg tgaacaccga gattacgaag gctccgctct cagcctccat
     4621 gatcaagcgc tacgacgagc accatcagga tctgaccctc ctgaaggcgc tggtcaggca
     4681 gcagctcccc gagaagtaca aggagatctt cttcgatcag tcgaagaacg gctacgctgg
     4741 gtacattgac ggcggggcct ctcaggagga gttctacaag ttcatcaagc cgattctgga
     4801 gaagatggac ggcacggagg agctgctggt gaagctcaat cgcgaggacc tcctgaggaa
     4861 gcagcggaca ttcgataacg gcagcatccc acaccagatt catctcgggg agctgcacgc
     4921 tatcctgagg aggcaggagg acttctaccc tttcctcaag gataaccgcg agaagatcga
     4981 gaagattctg actttcagga tcccgtacta cgtcggccca ctcgctaggg gcaactcccg
     5041 cttcgcttgg atgacccgca agtcagagga gacgatcacg ccgtggaact tcgaggaggt
     5101 ggtcgacaag ggcgctagcg ctcagtcgtt catcgagagg atgacgaatt tcgacaagaa
     5161 cctgccaaat gagaaggtgc tccctaagca ctcgctcctg tacgagtact tcacagtcta
     5221 caacgagctg actaaggtga agtatgtgac cgagggcatg aggaagccgg ctttcctgtc
     5281 tggggagcag aagaaggcca tcgtggacct cctgttcaag accaaccgga aggtcacggt
     5341 taagcagctc aaggaggact acttcaagaa gattgagtgc ttcgattcgg tcgagatctc
     5401 tggcgttgag gaccgcttca acgcctccct ggggacctac cacgatctcc tgaagatcat
     5461 taaggataag gacttcctgg acaacgagga gaatgaggat atcctcgagg acattgtgct
     5521 gacactcact ctgttcgagg accgggagat gatcgaggag cgcctgaaga cttacgccca
     5581 tctcttcgat gacaaggtca tgaagcagct caagaggagg aggtacaccg gctgggggag
     5641 gctgagcagg aagctcatca acggcattcg ggacaagcag tccgggaaga cgatcctcga
     5701 cttcctgaag agcgatggct tcgcgaaccg caatttcatg cagctgattc acgatgacag
     5761 cctcacattc aaggaggata tccagaaggc tcaggtgagc ggccaggggg actcgctgca
     5821 cgagcatatc gcgaacctcg ctggctcgcc agctatcaag aaggggattc tgcagaccgt
     5881 gaaggttgtg gacgagctgg tgaaggtcat gggcaggcac aagcctgaga acatcgtcat
     5941 tgagatggcc cgggagaatc agaccacgca gaagggccag aagaactcac gcgagaggat
     6001 gaagaggatc gaggagggca ttaaggagct ggggtcccag atcctcaagg agcacccggt
     6061 ggagaacacg cagctgcaga atgagaagct ctacctgtac tacctccaga atggccgcga
     6121 tatgtatgtg gaccaggagc tggatattaa caggctcagc gattacgacg tcgatcatat
     6181 cgttccacag tcattcctga aggatgactc cattgacaac aaggtcctca ccaggtcgga
     6241 caagaaccgg ggcaagtctg ataatgttcc ttcagaggag gtcgttaaga agatgaagaa
     6301 ctactggcgc cagctcctga atgccaagct gatcacgcag cggaagttcg ataacctcac
     6361 aaaggctgag aggggcgggc tctctgagct ggacaaggcg ggcttcatca agaggcagct
     6421 ggtcgagaca cggcagatca ctaagcacgt tgcgcagatt ctcgactcac ggatgaacac
     6481 taagtacgat gagaatgaca agctgatccg cgaggtgaag gtcatcaccc tgaagtcaaa
     6541 gctcgtctcc gacttcagga aggatttcca gttctacaag gttcgggaga tcaacaatta
     6601 ccaccatgcc catgacgcgt acctgaacgc ggtggtcggc acagctctga tcaagaagta
     6661 cccaaagctc gagagcgagt tcgtgtacgg ggactacaag gtttacgatg tgaggaagat
     6721 gatcgccaag tcggagcagg agattggcaa ggctaccgcc aagtacttct tctactctaa
     6781 cattatgaat ttcttcaaga cagagatcac tctggccaat ggcgagatcc ggaagcgccc
     6841 cctcatcgag acgaacggcg agacggggga gatcgtgtgg gacaagggca gggatttcgc
     6901 gaccgtcagg aaggttctct ccatgccaca agtgaatatc gtcaagaaga cagaggtcca
     6961 gactggcggg ttctctaagg agtcaattct gcctaagcgg aacagcgaca agctcatcgc
     7021 ccgcaagaag gactgggatc cgaagaagta cggcgggttc gacagcccca ctgtggccta
     7081 ctcggtcctg gttgtggcga aggttgagaa gggcaagtcc aagaagctca agagcgtgaa
     7141 ggagctgctg gggatcacga ttatggagcg ctccagcttc gagaagaacc cgatcgattt
     7201 cctggaggcg aagggctaca aggaggtgaa gaaggacctg atcattaagc tccccaagta
     7261 ctcactcttc gagctggaga acggcaggaa gcggatgctg gcttccgctg gcgagctgca
     7321 gaaggggaac gagctggctc tgccgtccaa gtatgtgaac ttcctctacc tggcctccca
     7381 ctacgagaag ctcaagggca gccccgagga caacgagcag aagcagctgt tcgtcgagca
     7441 gcacaagcat tacctcgacg agatcattga gcagatttcc gagttctcca agcgcgtgat
     7501 cctggccgac gcgaatctgg ataaggtcct ctccgcgtac aacaagcacc gcgacaagcc
     7561 aatcagggag caggctgaga atatcattca tctcttcacc ctgacgaacc tcggcgcccc
     7621 tgctgctttc aagtacttcg acacaactat cgatcgcaag aggtacacaa gcactaagga
     7681 ggtcctggac gcgaccctca tccaccagtc gattaccggc ctctacgaga cgcgcatcga
     7741 cctgtctcag ctcgggggcg acaagcggcc agcggcgacg aagaaggcgg ggcaggcgaa
     7801 gaagaagaag tgagctcaga gctttcgttc gtatcatcgg tttcgacaac gttcgtcaag
     7861 ttcaatgcat cagtttcatt gcgcacacac cagaatccta ctgagtttga gtattatggc
     7921 attgggaaaa ctgtttttct tgtaccattt gttgtgcttg taatttactg tgttttttat
     7981 tcggttttcg ctatcgaact gtgaaatgga aatggatgga gaagagttaa tgaatgatat
     8041 ggtccttttg ttcattctca aattaatatt atttgttttt tctcttattt gttgtgtgtt
     8101 gaatttgaaa ttataagaga tatgcaaaca ttttgttttg agtaaaaatg tgtcaaatcg
     8161 tggcctctaa tgaccgaagt taatatgagg agtaaaacac ttgtagttgt accattatgc
     8221 ttattcacta ggcaacaaat atattttcag acctagaaaa gctgcaaatg ttactgaata
     8281 caagtatgtc ctcttgtgtt ttagacattt atgaactttc ctttatgtaa ttttccagaa
     8341 tccttgtcag attctaatca ttgctttata attatagtta tactcatgga tttgtagttg
     8401 agtatgaaaa tattttttaa tgcattttat gacttgccaa ttgattgaca acgaattcgt
     8461 aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     8521 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     8581 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     8641 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt ggctagagca gcttgccaac
     8701 atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac
     8761 caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat
     8821 tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa
     8881 tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc
     8941 aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct
     9001 tcaaagcaag tggattgatg tgataacatg gtggagcacg acactctcgt ctactccaag
     9061 aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta
     9121 atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca
     9181 gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt
     9241 caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg
     9301 gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact
     9361 gacgtaaggg atgacgcaca atcccactat ccttcgcaag accttcctct atataaggaa
     9421 gttcatttca tttggagagg acacgctgaa atcaccagtc tctctctaca aatctatctc
     9481 tctcgagctt tcgcagatcc cggggggcaa tgagatatga aaaagcctga actcaccgcg
     9541 acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
     9601 tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
     9661 cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
     9721 tcggccgcgc tcccgattcc ggaagtgctt gacattgggg agtttagcga gagcctgacc
     9781 tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg
     9841 cccgctgttc tacaaccggt cgcggaggct atggatgcga tcgctgcggc cgatcttagc
     9901 cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt
     9961 gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
    10021 accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc
    10081 cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat
    10141 ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag
    10201 gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac
    10261 ttcgagcgga ggcatccgga gcttgcagga tcgccacgac tccgggcgta tatgctccgc
    10321 attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg
    10381 gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
    10441 atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt
    10501 ggaaaccgac gccccagcac tcgtccgagg gcaaagaaat agagtagatg ccgaccggat
    10561 ctgtcgatcg acaagctcga gtttctccat aataatgtgt gagtagttcc cagataaggg
    10621 aattagggtt cctatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt
    10681 atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag
    10741 tactaaaatc cagatccccc gaattaattc ggcgttaatt cagtacatta aaaacgtccg
    10801 caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgccacc
    10861 agccagccaa cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag
    10921 cccatcagtc cgggacggcg tcagcgggag agccgttgta aggcggcaga ctttgctcat
    10981 gttaccgatg ctattcggaa gaacggcaac taagctgccg ggtttgaaac acggatgatc
    11041 tcgcggaggg tagcatgttg attgtaacga tgacagagcg ttgctgcctg tgatcaccgc
    11101 ggtttcaaaa tcggctccgt cgatactatg ttatacgcca actttgaaaa caactttgaa
    11161 aaagctgttt tctggtattt aaggttttag aatgcaagga acagtgaatt ggagttcgtc
    11221 ttgttataat tagcttcttg gggtatcttt aaatactgta gaaaagagga aggaaataat
    11281 aaatggctaa aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg
    11341 taaaagatac ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa
    11401 acctatattt aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg
    11461 aaaaggacat gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg
    11521 aacggcatga tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg
    11581 aagagtatga agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca
    11641 ggctctttca ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct
    11701 tagccgaatt ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg
    11761 aagaagacac tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc
    11821 ccgaagagga acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag
    11881 atggcaaagt aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg
    11941 acattgcctt ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc
    12001 tattttttga cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac
    12061 tggatgaatt gttttagtac ctagaatgca tgaccaaaat cccttaacgt gagttttcgt
    12121 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    12181 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    12241 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    12301 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    12361 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    12421 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    12481 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    12541 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    12601 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    12661 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    12721 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    12781 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    12841 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    12901 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
    12961 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
    13021 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
    13081 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
    13141 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
    13201 accgaaacgc gcgaggcagg gtgccttgat gtgggcgccg gcggtcgagt ggcgacggcg
    13261 cggcttgtcc gcgccctggt agattgcctg gccgtaggcc agccattttt gagcggccag
    13321 cggccgcgat aggccgacgc gaagcggcgg ggcgtaggga gcgcagcgac cgaagggtag
    13381 gcgctttttg cagctcttcg gctgtgcgct ggccagacag ttatgcacag gccaggcggg
    13441 ttttaagagt tttaataagt tttaaagagt tttaggcgga aaaatcgcct tttttctctt
    13501 ttatatcagt cacttacatg tgtgaccggt tcccaatgta cggctttggg ttcccaatgt
    13561 acgggttccg gttcccaatg tacggctttg ggttcccaat gtacgtgcta tccacaggaa
    13621 acagaccttt tcgacctttt tcccctgcta gggcaatttg ccctagcatc tgctccgtac
    13681 attaggaacc ggcggatgct tcgccctcga tcaggttgcg gtagcgcatg actaggatcg
    13741 ggccagcctg ccccgcctcc tccttcaaat cgtactccgg caggtcattt gacccgatca
    13801 gcttgcgcac ggtgaaacag aacttcttga actctccggc gctgccactg cgttcgtaga
    13861 tcgtcttgaa caaccatctg gcttctgcct tgcctgcggc gcggcgtgcc aggcggtaga
    13921 gaaaacggcc gatgccggga tcgatcaaaa agtaatcggg gtgaaccgtc agcacgtccg
    13981 ggttcttgcc ttctgtgatc tcgcggtaca tccaatcagc tagctcgatc tcgatgtact
    14041 ccggccgccc ggtttcgctc tttacgatct tgtagcggct aatcaaggct tcaccctcgg
    14101 ataccgtcac caggcggccg ttcttggcct tcttcgtacg ctgcatggca acgtgcgtgg
    14161 tgtttaaccg aatgcaggtt tctaccaggt cgtctttctg ctttccgcca tcggctcgcc
    14221 ggcagaactt gagtacgtcc gcaacgtgtg gacggaacac gcggccgggc ttgtctccct
    14281 tcccttcccg gtatcggttc atggattcgg ttagatggga aaccgccatc agtaccaggt
    14341 cgtaatccca cacactggcc atgccggccg gccctgcgga aacctctacg tgcccgtctg
    14401 gaagctcgta gcggatcacc tcgccagctc gtcggtcacg cttcgacaga cggaaaacgg
    14461 ccacgtccat gatgctgcga ctatcgcggg tgcccacgtc atagagcatc ggaacgaaaa
    14521 aatctggttg ctcgtcgccc ttgggcggct tcctaatcga cggcgcaccg gctgccggcg
    14581 gttgccggga ttctttgcgg attcgatcag cggccgcttg ccacgattca ccggggcgtg
    14641 cttctgcctc gatgcgttgc cgctgggcgg cctgcgcggc cttcaacttc tccaccaggt
    14701 catcacccag cgccgcgccg atttgtaccg ggccggatgg tttgcgaccg ctcacgccga
    14761 ttcctcgggc ttgggggttc cagtgccatt gcagggccgg cagacaaccc agccgcttac
    14821 gcctggccaa ccgcccgttc ctccacacat ggggcattcc acggcgtcgg tgcctggttg
    14881 ttcttgattt tccatgccgc ctcctttagc cgctaaaatt catctactca tttattcatt
    14941 tgctcattta ctctggtagc tgcgcgatgt attcagatag cagctcggta atggtcttgc
    15001 cttggcgtac cgcgtacatc ttcagcttgg tgtgatcctc cgccggcaac tgaaagttga
    15061 cccgcttcat ggctggcgtg tctgccaggc tggccaacgt tgcagccttg ctgctgcgtg
    15121 cgctcggacg gccggcactt agcgtgtttg tgcttttgct cattttctct ttacctcatt
    15181 aactcaaatg agttttgatt taatttcagc ggccagcgcc tggacctcgc gggcagcgtc
    15241 gccctcgggt tctgattcaa gaacggttgt gccggcggcg gcagtgcctg ggtagctcac
    15301 gcgctgcgtg atacgggact caagaatggg cagctcgtac ccggccagcg cctcggcaac
    15361 ctcaccgccg atgcgcgtgc ctttgatcgc ccgcgacacg acaaaggccg cttgtagcct
    15421 tccatccgtg acctcaatgc gctgcttaac cagctccacc aggtcggcgg tggcccatat
    15481 gtcgtaaggg cttggctgca ccggaatcag cacgaagtcg gctgccttga tcgcggacac
    15541 agccaagtcc gccgcctggg gcgctccgtc gatcactacg aagtcgcgcc ggccgatggc
    15601 cttcacgtcg cggtcaatcg tcgggcggtc gatgccgaca acggttagcg gttgatcttc
    15661 ccgcacggcc gcccaatcgc gggcactgcc ctggggatcg gaatcgacta acagaacatc
    15721 ggccccggcg agttgcaggg cgcgggctag atgggttgcg atggtcgtct tgcctgaccc
    15781 gcctttctgg ttaagtacag cgataacctt catgcgttcc ccttgcgtat ttgtttattt
    15841 actcatcgca tcatatacgc agcgaccgca tgacgcaagc tgttttactc aaatacacat
    15901 caccttttta gacggcggcg ctcggtttct tcagcggcca agctggccgg ccaggccgcc
    15961 agcttggcat cagacaaacc ggccaggatt tcatgcagcc gcacggttga gacgtgcgcg
    16021 ggcggctcga acacgtaccc ggccgcgatc atctccgcct cgatctcttc ggtaatgaaa
    16081 aacggttcgt cctggccgtc ctggtgcggt ttcatgcttg ttcctcttgg cgttcattct
    16141 cggcggccgc cagggcgtcg gcctcggtca atgcgtcctc acggaaggca ccgcgccgcc
    16201 tggcctcggt gggcgtcact tcctcgctgc gctcaagtgc gcggtacagg gtcgagcgat
    16261 gcacgccaag cagtgcagcc gcctctttca cggtgcggcc ttcctggtcg atcagctcgc
    16321 gggcgtgcgc gatctgtgcc ggggtgaggg tagggcgggg gccaaacttc acgcctcggg
    16381 ccttggcggc ctcgcgcccg ctccgggtgc ggtcgatgat tagggaacgc tcgaactcgg
    16441 caatgccggc gaacacggtc aacaccatgc ggccggccgg cgtggtggtg tcggcccacg
    16501 gctctgccag gctacgcagg cccgcgccgg cctcctggat gcgctcggca atgtccagta
    16561 ggtcgcgggt gctgcgggcc aggcggtcta gcctggtcac tgtcacaacg tcgccagggc
    16621 gtaggtggtc aagcatcctg gccagctccg ggcggtcgcg cctggtgccg gtgatcttct
    16681 cggaaaacag cttggtgcag ccggccgcgt gcagttcggc ccgttggttg gtcaagtcct
    16741 ggtcgtcggt gctgacgcgg gcatagccca gcaggccagc ggcggcgctc ttgttcatgg
    16801 cgtaatgtct ccggttctag tcgcaagtat tctactttat gcgactaaaa cacgcgacaa
    16861 gaaaacgcca ggaaaagggc agggcggcag cctgtcgcgt aacttaggac ttgtgcgaca
    16921 tgtcgttttc agaagacggc tgcactgaac gtcagaagcc gactgcacta tagcagcgga
    16981 ggggttggat caaagtactt tgatcccgag gggaaccctg tggttggcat gcacatacaa
    17041 atggacgaac ggataaacct tttcacgccc ttttaaatat ccgttattct aataaacgct
    17101 cttttctctt ag
//
