LOCUS       Exported               17973 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR/Cas9-mediated base editing in Arabidopsis. Contains 
            Cas9(D10A) and empty gRNA scaffold, HygR.
ACCESSION   .
VERSION     .
KEYWORDS    pHEE901
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17973)
  AUTHORS   Chen Y, Wang Z, Ni H, Xu Y, Chen Q, Jiang L
  TITLE     CRISPR/Cas9-mediated base-editing system efficiently generates 
            gain-of-function mutations in Arabidopsis.
  JOURNAL   Sci China Life Sci. 2017 Mar 16. doi: 10.1007/s11427-017-9021-5.
  PUBMED    28303459
REFERENCE   2  (bases 1 to 17973)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17973
                     /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             3542..4228
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4277..8377
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             6060..6068
                     /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             8390..8638
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8651..8671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(9328..9344)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9328..9344)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9341..9363)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9352..9368
                     /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(9376..9406)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9421..9442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9633..10309
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     10246..10266
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             10376..11401
                     /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    11442..11616
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11694..11718
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             12143..12937
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      13024..13612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13513..13532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13766..13783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13798..13938
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13864..13883)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     14024..14046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      14282..14476
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(14542..15615)
                     /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(16044..16673)
                     /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 gatgtcttcc gagacaggac cggttgccgt cgaccctact cttagaaggc gcattgagcc
     3601 acacgagttc gaggtgttct tcgatccgag agagctgagg aaggagactt gcctccttta
     3661 cgagatcaat tggggcggaa ggcactctat ttggcgccat acctcccaga acacaaataa
     3721 gcatgtggag gttaatttca tcgagaagtt cactaccgag aggtacttct gcccaaacac
     3781 acgctgcagc atcacttggt tccttagctg gtcaccgtgc ggagagtgct ctcgcgccat
     3841 tacagagttc ctgtccagat acccgcacgt tactcttttc atctacattg ccagactgta
     3901 ccaccatgcg gatcctcgca acagacaggg tcttagggac ctgatcagct caggcgtcac
     3961 catccagatt atgacagagc aggagtctgg atactgctgg cgcaacttcg tgaattactc
     4021 tccttccaat gaggctcact ggccaagata cccgcatctg tgggtcaggc tctacgtgct
     4081 cgagctttac tgcatcattc ttggtctgcc tccatgcctc aacatcctta gaaggaagca
     4141 gccacagctc acattcttca ctattgccct tcagtcttgc cactaccaga ggcttccgcc
     4201 tcatattctg tgggcgactg gcctcaagag cggctcagag actccgggaa catctgagtc
     4261 cgctactcct gagtctgaca agaagtactc catcggactc gccattggta ctaactccgt
     4321 tggatgggcg gtcatcaccg atgagtacaa ggtgcctagc aagaagttca aggttcttgg
     4381 taacacagac agacactcaa tcaagaagaa tctgattggt gctctgctct tcgattctgg
     4441 agagactgcc gaggctacca ggctcaagag aaccgcccgc agaaggtaca cacgcagaaa
     4501 gaataggatc tgctaccttc aggagatttt ctctaacgag atggctaagg ttgatgacag
     4561 cttcttccat cgccttgagg agtcattcct ggtcgaggag gacaagaagc acgagagaca
     4621 tcctatcttc ggtaacattg tcgatgaggt ggcctaccac gagaagtacc caactatcta
     4681 ccatcttagg aagaagctgg tggatagcac cgacaaggcg gatctccgcc ttatctacct
     4741 ggctctcgcc cacatgatta agttcagagg ccatttcctc atcgagggcg atctcaaccc
     4801 agataattca gacgtcgata agctcttcat ccagcttgtg cagacataca atcagctttt
     4861 cgaggagaac ccgattaatg cgagcggtgt tgatgcgaag gctatcctgt cagctagact
     4921 cagcaagtca aggcgcctgg agaacctcat cgcccagctg ccaggcgaga agaagaacgg
     4981 tcttttcggc aatctgattg cgctttctct gggactcacc ccgaacttca agtccaattt
     5041 cgacctggct gaggatgcca agctccagct gtctaaggat acatacgatg acgatctcga
     5101 caaccttctg gctcagatcg gcgaccagta cgccgatctc ttccttgctg ccaagaatct
     5161 tagcgatgcc atcctccttt cagacattct gagagttaac actgagatta ccaaggctcc
     5221 gctgtctgcc tccatgatca agagatacga tgagcaccat caggacctca ctctgctcaa
     5281 ggcgctggtc cgccagcagc tccctgagaa gtacaaggag atcttcttcg accagtctaa
     5341 gaacggctac gcgggttaca ttgatggtgg cgctagccag gaggagttct acaagttcat
     5401 caagccaatt ctggagaaga tggatggcac tgaggagctt ctggtcaagc tcaataggga
     5461 ggatctcctt aggaagcagc gcaccttcga caacggatct atccctcacc agattcatct
     5521 tggagagctg cacgccatcc tcagaaggca ggaggatttc tacccattcc ttaaggacaa
     5581 ccgcgagaag atcgagaaga ttctgacttt cagaatccct tactacgttg gcccgctcgc
     5641 tagaggcaac tctaggttcg cgtggatgac caggaagtca gaggagacta tcaccccttg
     5701 gaacttcgag gaggtggttg acaagggagc cagcgcgcag tcattcattg agcgcatgac
     5761 taatttcgat aagaacctgc ctaatgagaa ggtcctccca aagcatagcc tgctctacga
     5821 gtacttcact gtgtacaacg agcttaccaa ggtgaagtat gtgacagagg gcatgcgcaa
     5881 gccggctttc ctttcaggag agcagaagaa ggccatcgtg gatcttctgt tcaagactaa
     5941 tagaaaggtc accgtgaagc agctgaagga ggattacttc aagaagattg agtgcttcga
     6001 ctctgttgag atctccggtg tcgaggatag gttcaacgct tccctcggca cctaccacga
     6061 cctccttaag atcattaagg acaaggattt cctggataac gaggagaatg aggacatcct
     6121 cgaggatatt gtgctgacac tcactctttt cgaggacagg gagatgatcg aggagcgcct
     6181 taagacatac gcgcatctgt tcgacgataa ggttatgaag cagctcaagc gcagaaggta
     6241 cactggatgg ggtagactct ctaggaagct catcaacggc atcagagata agcagtctgg
     6301 caagactatt ctcgatttcc ttaagtccga cggcttcgct aacaggaatt tcatgcagct
     6361 cattcacgac gattctctta ctttcaagga ggacatccag aaggcgcagg ttagcggcca
     6421 gggagattca ctgcacgagc atatcgcgaa cctcgctggc tcccctgcta tcaagaaggg
     6481 catcctccag accgttaagg tcgtggatga gctggttaag gtcatgggca gacataagcc
     6541 agagaacatc gtcattgaga tggccaggga gaatcagaca actcagaagg gacagaagaa
     6601 ctctagggag cgcatgaaga gaatcgagga gggtattaag gagcttggct cccagatcct
     6661 gaaggagcac ccggtggaga acacacagct gcagaatgag aagctgtacc tctactacct
     6721 ccagaatggc cgcgacatgt atgtggatca ggagcttgac attaacagac tttctgacta
     6781 cgatgtggac catatcgttc cacagtcttt ccttaaggac gattccattg ataataaggt
     6841 gctgactaga tccgataaga acaggggaaa gtctgacaat gttccgtccg aggaggttgt
     6901 caagaagatg aagaactact ggaggcagct gctcaatgct aagctcatca cccagaggaa
     6961 gttcgacaac cttacaaagg ccgagcgcgg aggtctgagc gagcttgata aggcgggttt
     7021 cattaagaga cagctcgttg agacaaggca gatcactaag cacgtcgccc agattcttga
     7081 ctcaaggatg aacaccaagt acgacgagaa tgataagctg atccgcgagg tgaaggttat
     7141 tacactgaag agcaagctcg tttcagattt cagaaaggac ttccagttct acaaggtcag
     7201 ggagatcaac aattaccacc atgcccatga tgcgtacctc aacgcggtgg ttggtactgc
     7261 tcttattaag aagtacccga agctggagtc tgagttcgtg tacggcgatt acaaggtgta
     7321 cgacgttaga aagatgatcg ctaagagcga gcaggagatt ggcaaggcta ccgccaagta
     7381 cttcttctac tcaaacatta tgaatttctt caagacagag atcactctcg cgaacggcga
     7441 gatcagaaag aggccactta ttgagactaa cggcgagaca ggagagatcg tctgggataa
     7501 gggtcgcgac ttcgctactg tcagaaaggt gctctctatg ccgcaggtta atattgtcaa
     7561 gaagactgag gtgcagaccg gcggattctc taaggagtcc attctcccta agaggaactc
     7621 cgacaagctc atcgcccgca agaaggattg ggaccctaag aagtacggtg gcttcgatag
     7681 cccaaccgtc gcttactcag tgcttgtcgt ggccaaggtc gagaagggaa agagcaagaa
     7741 gctgaagtca gtgaaggagc ttctgggtat cacaattatg gagaggtctt ccttcgagaa
     7801 gaatcctatc gacttcctcg aggcgaaggg ctacaaggag gttaagaagg atcttatcat
     7861 taagctgcca aagtactcac ttttcgagct ggagaacgga cgcaagagaa tgctggcgtc
     7921 tgctggagag cttcagaagg gtaatgagct tgctctgccg tctaagtatg tgaacttcct
     7981 ctaccttgcc tctcattacg agaagctcaa gggctcccct gaggacaacg agcagaagca
     8041 gctgttcgtc gagcagcaca agcattacct cgatgagatc attgagcaga ttagcgagtt
     8101 ctcaaagaga gtgatcctcg ccgatgcgaa tctcgacaag gttcttagcg cgtacaacaa
     8161 gcaccgcgat aagccaatca gagagcaggc tgagaatatc attcatctct tcacccttac
     8221 aaacctgggt gctccggcgg ctttcaagta cttcgatacc acaattgaca ggaagcgcta
     8281 cacttcaacc aaggaggtgc tggacgccac cctcatccac cagtctatta ctggcctcta
     8341 cgagactagg atcgatctct cccagcttgg tggtgactct ggcggatcca ccaacctcag
     8401 cgatatcatt gagaaggaga caggcaagca gcttgttatc caggagtcaa ttctgatgct
     8461 cccggaggag gtggaggagg ttattggcaa taagcctgag tctgatatcc tcgtgcatac
     8521 tgcctacgat gagagcaccg acgagaacgt tatgctcctt acatcagacg cgcctgagta
     8581 caagccttgg gctctcgtca ttcaggattc caacggagag aataagatca agatgcttag
     8641 cggtggctct cctaagaaga agagaaaggt gtgagctcag agctttcgtt cgtatcatcg
     8701 gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat tgcgcacaca ccagaatcct
     8761 actgagtttg agtattatgg cattgggaaa actgtttttc ttgtaccatt tgttgtgctt
     8821 gtaatttact gtgtttttta ttcggttttc gctatcgaac tgtgaaatgg aaatggatgg
     8881 agaagagtta atgaatgata tggtcctttt gttcattctc aaattaatat tatttgtttt
     8941 ttctcttatt tgttgtgtgt tgaatttgaa attataagag atatgcaaac attttgtttt
     9001 gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag ttaatatgag gagtaaaaca
     9061 cttgtagttg taccattatg cttattcact aggcaacaaa tatattttca gacctagaaa
     9121 agctgcaaat gttactgaat acaagtatgt cctcttgtgt tttagacatt tatgaacttt
     9181 cctttatgta attttccaga atccttgtca gattctaatc attgctttat aattatagtt
     9241 atactcatgg atttgtagtt gagtatgaaa atatttttta atgcatttta tgacttgcca
     9301 attgattgac aacgaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc
     9361 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     9421 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     9481 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     9541 tggctagagc agcttgccaa catggtggag cacgacactc tcgtctactc caagaatatc
     9601 aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag ggtaatatcg
     9661 ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcaaaag gacagtagaa
     9721 aaggaaggtg gcacctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat
     9781 gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa
     9841 gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgaacatgg tggagcacga
     9901 cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga
     9961 gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg
    10021 tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga
    10081 taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc
    10141 acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga
    10201 ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga
    10261 cccttcctct atataaggaa gttcatttca tttggagagg acacgctgaa atcaccagtc
    10321 tctctctaca aatctatctc tctcgagctt tcgcagatcc ggggggcaat gagatatgaa
    10381 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
    10441 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
    10501 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
    10561 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
    10621 gtttagcgag agcctgacct attgcatctc ccgccgttca cagggtgtca cgttgcaaga
    10681 cctgcctgaa accgaactgc ccgctgttct acaaccggtc gcggaggcta tggatgcgat
    10741 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
    10801 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
    10861 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
    10921 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
    10981 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
    11041 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
    11101 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccacgact
    11161 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
    11221 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
    11281 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
    11341 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaagaaata
    11401 gagtagatgc cgaccgggat ctgtcgatcg acaagctcga gtttctccat aataatgtgt
    11461 gagtagttcc cagataaggg aattagggtt cctatagggt ttcgctcatg tgttgagcat
    11521 ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa
    11581 ttcctaaaac caaaatccag tactaaaatc cagatccccc gaattaattc ggcgttaatt
    11641 cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca
    11701 ccacaatata tcctgccacc agccagccaa cagctccccg accggcagct cggcacaaaa
    11761 tcaccactcg atacaggcag cccatcagtc cgggacggcg tcagcgggag agccgttgta
    11821 aggcggcaga ctttgctcat gttaccgatg ctattcggaa gaacggcaac taagctgccg
    11881 ggtttgaaac acggatgatc tcgcggaggg tagcatgttg attgtaacga tgacagagcg
    11941 ttgctgcctg tgatcaccgc ggtttcaaaa tcggctccgt cgatactatg ttatacgcca
    12001 actttgaaaa caactttgaa aaagctgttt tctggtattt aaggttttag aatgcaagga
    12061 acagtgaatt ggagttcgtc ttgttataat tagcttcttg gggtatcttt aaatactgta
    12121 gaaaagagga aggaaataat aaatggctaa aatgagaata tcaccggaat tgaaaaaact
    12181 gatcgaaaaa taccgctgcg taaaagatac ggaaggaatg tctcctgcta aggtatataa
    12241 gctggtggga gaaaatgaaa acctatattt aaaaatgacg gacagccggt ataaagggac
    12301 cacctatgat gtggaacggg aaaaggacat gatgctatgg ctggaaggaa agctgcctgt
    12361 tccaaaggtc ctgcactttg aacggcatga tggctggagc aatctgctca tgagtgaggc
    12421 cgatggcgtc ctttgctcgg aagagtatga agatgaacaa agccctgaaa agattatcga
    12481 gctgtatgcg gagtgcatca ggctctttca ctccatcgac atatcggatt gtccctatac
    12541 gaatagctta gacagccgct tagccgaatt ggattactta ctgaataacg atctggccga
    12601 tgtggattgc gaaaactggg aagaagacac tccatttaaa gatccgcgcg agctgtatga
    12661 ttttttaaag acggaaaagc ccgaagagga acttgtcttt tcccacggcg acctgggaga
    12721 cagcaacatc tttgtgaaag atggcaaagt aagtggcttt attgatcttg ggagaagcgg
    12781 cagggcggac aagtggtatg acattgcctt ctgcgtccgg tcgatcaggg aggatatcgg
    12841 ggaagaacag tatgtcgagc tattttttga cttactgggg atcaagcctg attgggagaa
    12901 aataaaatat tatattttac tggatgaatt gttttagtac ctagaatgca tgaccaaaat
    12961 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
    13021 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
    13081 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
    13141 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
    13201 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
    13261 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
    13321 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
    13381 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
    13441 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
    13501 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
    13561 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
    13621 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
    13681 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
    13741 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
    13801 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
    13861 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
    13921 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
    13981 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
    14041 cagaggtttt caccgtcatc accgaaacgc gcgaggcagg gtgccttgat gtgggcgccg
    14101 gcggtcgagt ggcgacggcg cggcttgtcc gcgccctggt agattgcctg gccgtaggcc
    14161 agccattttt gagcggccag cggccgcgat aggccgacgc gaagcggcgg ggcgtaggga
    14221 gcgcagcgac cgaagggtag gcgctttttg cagctcttcg gctgtgcgct ggccagacag
    14281 ttatgcacag gccaggcggg ttttaagagt tttaataagt tttaaagagt tttaggcgga
    14341 aaaatcgcct tttttctctt ttatatcagt cacttacatg tgtgaccggt tcccaatgta
    14401 cggctttggg ttcccaatgt acgggttccg gttcccaatg tacggctttg ggttcccaat
    14461 gtacgtgcta tccacaggaa acagaccttt tcgacctttt tcccctgcta gggcaatttg
    14521 ccctagcatc tgctccgtac attaggaacc ggcggatgct tcgccctcga tcaggttgcg
    14581 gtagcgcatg actaggatcg ggccagcctg ccccgcctcc tccttcaaat cgtactccgg
    14641 caggtcattt gacccgatca gcttgcgcac ggtgaaacag aacttcttga actctccggc
    14701 gctgccactg cgttcgtaga tcgtcttgaa caaccatctg gcttctgcct tgcctgcggc
    14761 gcggcgtgcc aggcggtaga gaaaacggcc gatgccggga tcgatcaaaa agtaatcggg
    14821 gtgaaccgtc agcacgtccg ggttcttgcc ttctgtgatc tcgcggtaca tccaatcagc
    14881 tagctcgatc tcgatgtact ccggccgccc ggtttcgctc tttacgatct tgtagcggct
    14941 aatcaaggct tcaccctcgg ataccgtcac caggcggccg ttcttggcct tcttcgtacg
    15001 ctgcatggca acgtgcgtgg tgtttaaccg aatgcaggtt tctaccaggt cgtctttctg
    15061 ctttccgcca tcggctcgcc ggcagaactt gagtacgtcc gcaacgtgtg gacggaacac
    15121 gcggccgggc ttgtctccct tcccttcccg gtatcggttc atggattcgg ttagatggga
    15181 aaccgccatc agtaccaggt cgtaatccca cacactggcc atgccggccg gccctgcgga
    15241 aacctctacg tgcccgtctg gaagctcgta gcggatcacc tcgccagctc gtcggtcacg
    15301 cttcgacaga cggaaaacgg ccacgtccat gatgctgcga ctatcgcggg tgcccacgtc
    15361 atagagcatc ggaacgaaaa aatctggttg ctcgtcgccc ttgggcggct tcctaatcga
    15421 cggcgcaccg gctgccggcg gttgccggga ttctttgcgg attcgatcag cggccgcttg
    15481 ccacgattca ccggggcgtg cttctgcctc gatgcgttgc cgctgggcgg cctgcgcggc
    15541 cttcaacttc tccaccaggt catcacccag cgccgcgccg atttgtaccg ggccggatgg
    15601 tttgcgaccg ctcacgccga ttcctcgggc ttgggggttc cagtgccatt gcagggccgg
    15661 caggcaaccc agccgcttac gcctggccaa ccgcccgttc ctccacacat ggggcattcc
    15721 acggcgtcgg tgcctggttg ttcttgattt tccatgccgc ctcctttagc cgctaaaatt
    15781 catctactca tttattcatt tgctcattta ctctggtagc tgcgcgatgt attcagatag
    15841 cagctcggta atggtcttgc cttggcgtac cgcgtacatc ttcagcttgg tgtgatcctc
    15901 cgccggcaac tgaaagttga cccgcttcat ggctggcgtg tctgccaggc tggccaacgt
    15961 tgcagccttg ctgctgcgtg cgctcggacg gccggcactt agcgtgtttg tgcttttgct
    16021 cattttctct ttacctcatt aactcaaatg agttttgatt taatttcagc ggccagcgcc
    16081 tggacctcgc gggcagcgtc gccctcgggt tctgattcaa gaacggttgt gccggcggcg
    16141 gcagtgcctg ggtagctcac gcgctgcgtg atacgggact caagaatggg cagctcgtac
    16201 ccggccagcg cctcggcaac ctcaccgccg atgcgcgtgc ctttgatcgc ccgcgacacg
    16261 acaaaggccg cttgtagcct tccatccgtg acctcaatgc gctgcttaac cagctccacc
    16321 aggtcggcgg tggcccatat gtcgtaaggg cttggctgca ccggaatcag cacgaagtcg
    16381 gctgccttga tcgcggacac agccaagtcc gccgcctggg gcgctccgtc gatcactacg
    16441 aagtcgcgcc ggccgatggc cttcacgtcg cggtcaatcg tcgggcggtc gatgccgaca
    16501 acggttagcg gttgatcttc ccgcacggcc gcccaatcgc gggcactgcc ctggggatcg
    16561 gaatcgacta acagaacatc ggccccggcg agttgcaggg cgcgggctag atgggttgcg
    16621 atggtcgtct tgcctgaccc gcctttctgg ttaagtacag cgataacctt catgcgttcc
    16681 ccttgcgtat ttgtttattt actcatcgca tcatatacgc agcgaccgca tgacgcaagc
    16741 tgttttactc aaatacacat caccttttta gacggcggcg ctcggtttct tcagcggcca
    16801 agctggccgg ccaggccgcc agcttggcat cagacaaacc ggccaggatt tcatgcagcc
    16861 gcacggttga gacgtgcgcg ggcggctcga acacgtaccc ggccgcgatc atctccgcct
    16921 cgatctcttc ggtaatgaaa aacggttcgt cctggccgtc ctggtgcggt ttcatgcttg
    16981 ttcctcttgg cgttcattct cggcggccgc cagggcgtcg gcctcggtca atgcgtcctc
    17041 acggaaggca ccgcgccgcc tggcctcggt gggcgtcact tcctcgctgc gctcaagtgc
    17101 gcggtacagg gtcgagcgat gcacgccaag cagtgcagcc gcctctttca cggtgcggcc
    17161 ttcctggtcg atcagctcgc gggcgtgcgc gatctgtgcc ggggtgaggg tagggcgggg
    17221 gccaaacttc acgcctcggg ccttggcggc ctcgcgcccg ctccgggtgc ggtcgatgat
    17281 tagggaacgc tcgaactcgg caatgccggc gaacacggtc aacaccatgc ggccggccgg
    17341 cgtggtggtg tcggcccacg gctctgccag gctacgcagg cccgcgccgg cctcctggat
    17401 gcgctcggca atgtccagta ggtcgcgggt gctgcgggcc aggcggtcta gcctggtcac
    17461 tgtcacaacg tcgccagggc gtaggtggtc aagcatcctg gccagctccg ggcggtcgcg
    17521 cctggtgccg gtgatcttct cggaaaacag cttggtgcag ccggccgcgt gcagttcggc
    17581 ccgttggttg gtcaagtcct ggtcgtcggt gctgacgcgg gcatagccca gcaggccagc
    17641 ggcggcgctc ttgttcatgg cgtaatgtct ccggttctag tcgcaagtat tctactttat
    17701 gcgactaaaa cacgcgacaa gaaaacgcca ggaaaagggc agggcggcag cctgtcgcgt
    17761 aacttaggac ttgtgcgaca tgtcgttttc agaagacggc tgcactgaac gtcagaagcc
    17821 gactgcacta tagcagcgga ggggttggat caaagtactt tgatcccgag gggaaccctg
    17881 tggttggcat gcacatacaa atggacgaac ggataaacct tttcacgccc ttttaaatat
    17941 ccgattattc taataaacgc tcttttctct tag
//
