pHSE901

LOCUS       pHSE901               17514 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR/Cas9-mediated base editing in dicots. Contains Cas9(D10A) 
            under CaMV 35S promoter and empty gRNA scaffold, HygR.
ACCESSION   .
VERSION     .
KEYWORDS    pHSE901
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17514)
  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 17514)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17514
                     /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"
     promoter        2589..2933
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3083..3769
                     /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             3818..7918
                     /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             5601..5609
                     /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             7931..8179
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             8192..8212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(8869..8885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8869..8885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8882..8904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8893..8909
                     /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(8917..8947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8962..8983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        9174..9850
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             9917..10942
                     /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    10983..11157
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    11235..11259
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             11684..12478
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12565..13153
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13054..13073
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13307..13324
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    13339..13479
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(13405..13424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13565..13587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      13823..14017
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(14083..15156)
                     /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(15585..16214)
                     /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 accaataagc ttgcatgcct gcaggtcaac atggtggagc acgacacact tgtctactcc
     2221 aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg
     2281 gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag
     2341 atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc
     2401 gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc
     2461 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgataacatg
     2521 gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa
     2581 agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc
     2641 ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc
     2701 catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa
     2761 gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca
     2821 aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat
     2881 ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gacctcgacc
     2941 tcaacacaac atatacaaaa caaacgaatc tcaagcaatc aagcattcta cttctattgc
     3001 agcaatttaa atcatttctt ttaaagcaaa agcaattttc tgaaaatttt caccatttac
     3061 gaacgatact cgagtaatct agatgtcttc cgagacagga ccggttgccg tcgaccctac
     3121 tcttagaagg cgcattgagc cacacgagtt cgaggtgttc ttcgatccga gagagctgag
     3181 gaaggagact tgcctccttt acgagatcaa ttggggcgga aggcactcta tttggcgcca
     3241 tacctcccag aacacaaata agcatgtgga ggttaatttc atcgagaagt tcactaccga
     3301 gaggtacttc tgcccaaaca cacgctgcag catcacttgg ttccttagct ggtcaccgtg
     3361 cggagagtgc tctcgcgcca ttacagagtt cctgtccaga tacccgcacg ttactctttt
     3421 catctacatt gccagactgt accaccatgc ggatcctcgc aacagacagg gtcttaggga
     3481 cctgatcagc tcaggcgtca ccatccagat tatgacagag caggagtctg gatactgctg
     3541 gcgcaacttc gtgaattact ctccttccaa tgaggctcac tggccaagat acccgcatct
     3601 gtgggtcagg ctctacgtgc tcgagcttta ctgcatcatt cttggtctgc ctccatgcct
     3661 caacatcctt agaaggaagc agccacagct cacattcttc actattgccc ttcagtcttg
     3721 ccactaccag aggcttccgc ctcatattct gtgggcgact ggcctcaaga gcggctcaga
     3781 gactccggga acatctgagt ccgctactcc tgagtctgac aagaagtact ccatcggact
     3841 cgccattggt actaactccg ttggatgggc ggtcatcacc gatgagtaca aggtgcctag
     3901 caagaagttc aaggttcttg gtaacacaga cagacactca atcaagaaga atctgattgg
     3961 tgctctgctc ttcgattctg gagagactgc cgaggctacc aggctcaaga gaaccgcccg
     4021 cagaaggtac acacgcagaa agaataggat ctgctacctt caggagattt tctctaacga
     4081 gatggctaag gttgatgaca gcttcttcca tcgccttgag gagtcattcc tggtcgagga
     4141 ggacaagaag cacgagagac atcctatctt cggtaacatt gtcgatgagg tggcctacca
     4201 cgagaagtac ccaactatct accatcttag gaagaagctg gtggatagca ccgacaaggc
     4261 ggatctccgc cttatctacc tggctctcgc ccacatgatt aagttcagag gccatttcct
     4321 catcgagggc gatctcaacc cagataattc agacgtcgat aagctcttca tccagcttgt
     4381 gcagacatac aatcagcttt tcgaggagaa cccgattaat gcgagcggtg ttgatgcgaa
     4441 ggctatcctg tcagctagac tcagcaagtc aaggcgcctg gagaacctca tcgcccagct
     4501 gccaggcgag aagaagaacg gtcttttcgg caatctgatt gcgctttctc tgggactcac
     4561 cccgaacttc aagtccaatt tcgacctggc tgaggatgcc aagctccagc tgtctaagga
     4621 tacatacgat gacgatctcg acaaccttct ggctcagatc ggcgaccagt acgccgatct
     4681 cttccttgct gccaagaatc ttagcgatgc catcctcctt tcagacattc tgagagttaa
     4741 cactgagatt accaaggctc cgctgtctgc ctccatgatc aagagatacg atgagcacca
     4801 tcaggacctc actctgctca aggcgctggt ccgccagcag ctccctgaga agtacaagga
     4861 gatcttcttc gaccagtcta agaacggcta cgcgggttac attgatggtg gcgctagcca
     4921 ggaggagttc tacaagttca tcaagccaat tctggagaag atggatggca ctgaggagct
     4981 tctggtcaag ctcaataggg aggatctcct taggaagcag cgcaccttcg acaacggatc
     5041 tatccctcac cagattcatc ttggagagct gcacgccatc ctcagaaggc aggaggattt
     5101 ctacccattc cttaaggaca accgcgagaa gatcgagaag attctgactt tcagaatccc
     5161 ttactacgtt ggcccgctcg ctagaggcaa ctctaggttc gcgtggatga ccaggaagtc
     5221 agaggagact atcacccctt ggaacttcga ggaggtggtt gacaagggag ccagcgcgca
     5281 gtcattcatt gagcgcatga ctaatttcga taagaacctg cctaatgaga aggtcctccc
     5341 aaagcatagc ctgctctacg agtacttcac tgtgtacaac gagcttacca aggtgaagta
     5401 tgtgacagag ggcatgcgca agccggcttt cctttcagga gagcagaaga aggccatcgt
     5461 ggatcttctg ttcaagacta atagaaaggt caccgtgaag cagctgaagg aggattactt
     5521 caagaagatt gagtgcttcg actctgttga gatctccggt gtcgaggata ggttcaacgc
     5581 ttccctcggc acctaccacg acctccttaa gatcattaag gacaaggatt tcctggataa
     5641 cgaggagaat gaggacatcc tcgaggatat tgtgctgaca ctcactcttt tcgaggacag
     5701 ggagatgatc gaggagcgcc ttaagacata cgcgcatctg ttcgacgata aggttatgaa
     5761 gcagctcaag cgcagaaggt acactggatg gggtagactc tctaggaagc tcatcaacgg
     5821 catcagagat aagcagtctg gcaagactat tctcgatttc cttaagtccg acggcttcgc
     5881 taacaggaat ttcatgcagc tcattcacga cgattctctt actttcaagg aggacatcca
     5941 gaaggcgcag gttagcggcc agggagattc actgcacgag catatcgcga acctcgctgg
     6001 ctcccctgct atcaagaagg gcatcctcca gaccgttaag gtcgtggatg agctggttaa
     6061 ggtcatgggc agacataagc cagagaacat cgtcattgag atggccaggg agaatcagac
     6121 aactcagaag ggacagaaga actctaggga gcgcatgaag agaatcgagg agggtattaa
     6181 ggagcttggc tcccagatcc tgaaggagca cccggtggag aacacacagc tgcagaatga
     6241 gaagctgtac ctctactacc tccagaatgg ccgcgacatg tatgtggatc aggagcttga
     6301 cattaacaga ctttctgact acgatgtgga ccatatcgtt ccacagtctt tccttaagga
     6361 cgattccatt gataataagg tgctgactag atccgataag aacaggggaa agtctgacaa
     6421 tgttccgtcc gaggaggttg tcaagaagat gaagaactac tggaggcagc tgctcaatgc
     6481 taagctcatc acccagagga agttcgacaa ccttacaaag gccgagcgcg gaggtctgag
     6541 cgagcttgat aaggcgggtt tcattaagag acagctcgtt gagacaaggc agatcactaa
     6601 gcacgtcgcc cagattcttg actcaaggat gaacaccaag tacgacgaga atgataagct
     6661 gatccgcgag gtgaaggtta ttacactgaa gagcaagctc gtttcagatt tcagaaagga
     6721 cttccagttc tacaaggtca gggagatcaa caattaccac catgcccatg atgcgtacct
     6781 caacgcggtg gttggtactg ctcttattaa gaagtacccg aagctggagt ctgagttcgt
     6841 gtacggcgat tacaaggtgt acgacgttag aaagatgatc gctaagagcg agcaggagat
     6901 tggcaaggct accgccaagt acttcttcta ctcaaacatt atgaatttct tcaagacaga
     6961 gatcactctc gcgaacggcg agatcagaaa gaggccactt attgagacta acggcgagac
     7021 aggagagatc gtctgggata agggtcgcga cttcgctact gtcagaaagg tgctctctat
     7081 gccgcaggtt aatattgtca agaagactga ggtgcagacc ggcggattct ctaaggagtc
     7141 cattctccct aagaggaact ccgacaagct catcgcccgc aagaaggatt gggaccctaa
     7201 gaagtacggt ggcttcgata gcccaaccgt cgcttactca gtgcttgtcg tggccaaggt
     7261 cgagaaggga aagagcaaga agctgaagtc agtgaaggag cttctgggta tcacaattat
     7321 ggagaggtct tccttcgaga agaatcctat cgacttcctc gaggcgaagg gctacaagga
     7381 ggttaagaag gatcttatca ttaagctgcc aaagtactca cttttcgagc tggagaacgg
     7441 acgcaagaga atgctggcgt ctgctggaga gcttcagaag ggtaatgagc ttgctctgcc
     7501 gtctaagtat gtgaacttcc tctaccttgc ctctcattac gagaagctca agggctcccc
     7561 tgaggacaac gagcagaagc agctgttcgt cgagcagcac aagcattacc tcgatgagat
     7621 cattgagcag attagcgagt tctcaaagag agtgatcctc gccgatgcga atctcgacaa
     7681 ggttcttagc gcgtacaaca agcaccgcga taagccaatc agagagcagg ctgagaatat
     7741 cattcatctc ttcaccctta caaacctggg tgctccggcg gctttcaagt acttcgatac
     7801 cacaattgac aggaagcgct acacttcaac caaggaggtg ctggacgcca ccctcatcca
     7861 ccagtctatt actggcctct acgagactag gatcgatctc tcccagcttg gtggtgactc
     7921 tggcggatcc accaacctca gcgatatcat tgagaaggag acaggcaagc agcttgttat
     7981 ccaggagtca attctgatgc tcccggagga ggtggaggag gttattggca ataagcctga
     8041 gtctgatatc ctcgtgcata ctgcctacga tgagagcacc gacgagaacg ttatgctcct
     8101 tacatcagac gcgcctgagt acaagccttg ggctctcgtc attcaggatt ccaacggaga
     8161 gaataagatc aagatgctta gcggtggctc tcctaagaag aagagaaagg tgtgagctca
     8221 gagctttcgt tcgtatcatc ggtttcgaca acgttcgtca agttcaatgc atcagtttca
     8281 ttgcgcacac accagaatcc tactgagttt gagtattatg gcattgggaa aactgttttt
     8341 cttgtaccat ttgttgtgct tgtaatttac tgtgtttttt attcggtttt cgctatcgaa
     8401 ctgtgaaatg gaaatggatg gagaagagtt aatgaatgat atggtccttt tgttcattct
     8461 caaattaata ttatttgttt tttctcttat ttgttgtgtg ttgaatttga aattataaga
     8521 gatatgcaaa cattttgttt tgagtaaaaa tgtgtcaaat cgtggcctct aatgaccgaa
     8581 gttaatatga ggagtaaaac acttgtagtt gtaccattat gcttattcac taggcaacaa
     8641 atatattttc agacctagaa aagctgcaaa tgttactgaa tacaagtatg tcctcttgtg
     8701 ttttagacat ttatgaactt tcctttatgt aattttccag aatccttgtc agattctaat
     8761 cattgcttta taattatagt tatactcatg gatttgtagt tgagtatgaa aatatttttt
     8821 aatgcatttt atgacttgcc aattgattga caacgaattc gtaatcatgt catagctgtt
     8881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     8941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     9001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     9061 ggggagaggc ggtttgcgta ttggctagag cagcttgcca acatggtgga gcacgacact
     9121 ctcgtctact ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact
     9181 tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac
     9241 ttcatcaaaa ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa
     9301 ggaaaggcta tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc
     9361 acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga
     9421 tgtgaacatg gtggagcacg acactctcgt ctactccaag aatatcaaag atacagtctc
     9481 agaagaccaa agggctattg agacttttca acaaagggta atatcgggaa acctcctcgg
     9541 attccattgc ccagctatct gtcacttcat caaaaggaca gtagaaaagg aaggtggcac
     9601 ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag
     9661 tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac
     9721 cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca
     9781 atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag
     9841 gacacgctga aatcaccagt ctctctctac aaatctatct ctctcgagct ttcgcagatc
     9901 cggggggcaa tgagatatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     9961 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
    10021 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
    10081 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
    10141 ggaagtgctt gacattgggg agtttagcga gagcctgacc tattgcatct cccgccgttc
    10201 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tacaaccggt
    10261 cgcggaggct atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
    10321 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
    10381 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
    10441 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
    10501 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
    10561 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
    10621 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
    10681 gcttgcagga tcgccacgac tccgggcgta tatgctccgc attggtcttg accaactcta
    10741 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
    10801 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
    10861 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
    10921 tcgtccgagg gcaaagaaat agagtagatg ccgaccggga tctgtcgatc gacaagctcg
    10981 agtttctcca taataatgtg tgagtagttc ccagataagg gaattagggt tcctataggg
    11041 tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac
    11101 ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat ccagatcccc
    11161 cgaattaatt cggcgttaat tcagtacatt aaaaacgtcc gcaatgtgtt attaagttgt
    11221 ctaagcgtca atttgtttac accacaatat atcctgccac cagccagcca acagctcccc
    11281 gaccggcagc tcggcacaaa atcaccactc gatacaggca gcccatcagt ccgggacggc
    11341 gtcagcggga gagccgttgt aaggcggcag actttgctca tgttaccgat gctattcgga
    11401 agaacggcaa ctaagctgcc gggtttgaaa cacggatgat ctcgcggagg gtagcatgtt
    11461 gattgtaacg atgacagagc gttgctgcct gtgatcaccg cggtttcaaa atcggctccg
    11521 tcgatactat gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt
    11581 taaggtttta gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt
    11641 ggggtatctt taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat
    11701 atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat
    11761 gtctcctgct aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac
    11821 ggacagccgg tataaaggga ccacctatga tgtggaacgg gaaaaggaca tgatgctatg
    11881 gctggaagga aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag
    11941 caatctgctc atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca
    12001 aagccctgaa aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga
    12061 catatcggat tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt
    12121 actgaataac gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa
    12181 agatccgcgc gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt
    12241 ttcccacggc gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt
    12301 tattgatctt gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg
    12361 gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag ctattttttg acttactggg
    12421 gatcaagcct gattgggaga aaataaaata ttatatttta ctggatgaat tgttttagta
    12481 cctagaatgc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
    12541 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
    12601 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
    12661 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
    12721 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
    12781 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
    12841 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
    12901 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
    12961 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
    13021 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
    13081 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
    13141 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
    13201 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
    13261 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
    13321 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
    13381 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
    13441 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
    13501 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
    13561 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
    13621 ggtgccttga tgtgggcgcc ggcggtcgag tggcgacggc gcggcttgtc cgcgccctgg
    13681 tagattgcct ggccgtaggc cagccatttt tgagcggcca gcggccgcga taggccgacg
    13741 cgaagcggcg gggcgtaggg agcgcagcga ccgaagggta ggcgcttttt gcagctcttc
    13801 ggctgtgcgc tggccagaca gttatgcaca ggccaggcgg gttttaagag ttttaataag
    13861 ttttaaagag ttttaggcgg aaaaatcgcc ttttttctct tttatatcag tcacttacat
    13921 gtgtgaccgg ttcccaatgt acggctttgg gttcccaatg tacgggttcc ggttcccaat
    13981 gtacggcttt gggttcccaa tgtacgtgct atccacagga aacagacctt ttcgaccttt
    14041 ttcccctgct agggcaattt gccctagcat ctgctccgta cattaggaac cggcggatgc
    14101 ttcgccctcg atcaggttgc ggtagcgcat gactaggatc gggccagcct gccccgcctc
    14161 ctccttcaaa tcgtactccg gcaggtcatt tgacccgatc agcttgcgca cggtgaaaca
    14221 gaacttcttg aactctccgg cgctgccact gcgttcgtag atcgtcttga acaaccatct
    14281 ggcttctgcc ttgcctgcgg cgcggcgtgc caggcggtag agaaaacggc cgatgccggg
    14341 atcgatcaaa aagtaatcgg ggtgaaccgt cagcacgtcc gggttcttgc cttctgtgat
    14401 ctcgcggtac atccaatcag ctagctcgat ctcgatgtac tccggccgcc cggtttcgct
    14461 ctttacgatc ttgtagcggc taatcaaggc ttcaccctcg gataccgtca ccaggcggcc
    14521 gttcttggcc ttcttcgtac gctgcatggc aacgtgcgtg gtgtttaacc gaatgcaggt
    14581 ttctaccagg tcgtctttct gctttccgcc atcggctcgc cggcagaact tgagtacgtc
    14641 cgcaacgtgt ggacggaaca cgcggccggg cttgtctccc ttcccttccc ggtatcggtt
    14701 catggattcg gttagatggg aaaccgccat cagtaccagg tcgtaatccc acacactggc
    14761 catgccggcc ggccctgcgg aaacctctac gtgcccgtct ggaagctcgt agcggatcac
    14821 ctcgccagct cgtcggtcac gcttcgacag acggaaaacg gccacgtcca tgatgctgcg
    14881 actatcgcgg gtgcccacgt catagagcat cggaacgaaa aaatctggtt gctcgtcgcc
    14941 cttgggcggc ttcctaatcg acggcgcacc ggctgccggc ggttgccggg attctttgcg
    15001 gattcgatca gcggccgctt gccacgattc accggggcgt gcttctgcct cgatgcgttg
    15061 ccgctgggcg gcctgcgcgg ccttcaactt ctccaccagg tcatcaccca gcgccgcgcc
    15121 gatttgtacc gggccggatg gtttgcgacc gctcacgccg attcctcggg cttgggggtt
    15181 ccagtgccat tgcagggccg gcaggcaacc cagccgctta cgcctggcca accgcccgtt
    15241 cctccacaca tggggcattc cacggcgtcg gtgcctggtt gttcttgatt ttccatgccg
    15301 cctcctttag ccgctaaaat tcatctactc atttattcat ttgctcattt actctggtag
    15361 ctgcgcgatg tattcagata gcagctcggt aatggtcttg ccttggcgta ccgcgtacat
    15421 cttcagcttg gtgtgatcct ccgccggcaa ctgaaagttg acccgcttca tggctggcgt
    15481 gtctgccagg ctggccaacg ttgcagcctt gctgctgcgt gcgctcggac ggccggcact
    15541 tagcgtgttt gtgcttttgc tcattttctc tttacctcat taactcaaat gagttttgat
    15601 ttaatttcag cggccagcgc ctggacctcg cgggcagcgt cgccctcggg ttctgattca
    15661 agaacggttg tgccggcggc ggcagtgcct gggtagctca cgcgctgcgt gatacgggac
    15721 tcaagaatgg gcagctcgta cccggccagc gcctcggcaa cctcaccgcc gatgcgcgtg
    15781 cctttgatcg cccgcgacac gacaaaggcc gcttgtagcc ttccatccgt gacctcaatg
    15841 cgctgcttaa ccagctccac caggtcggcg gtggcccata tgtcgtaagg gcttggctgc
    15901 accggaatca gcacgaagtc ggctgccttg atcgcggaca cagccaagtc cgccgcctgg
    15961 ggcgctccgt cgatcactac gaagtcgcgc cggccgatgg ccttcacgtc gcggtcaatc
    16021 gtcgggcggt cgatgccgac aacggttagc ggttgatctt cccgcacggc cgcccaatcg
    16081 cgggcactgc cctggggatc ggaatcgact aacagaacat cggccccggc gagttgcagg
    16141 gcgcgggcta gatgggttgc gatggtcgtc ttgcctgacc cgcctttctg gttaagtaca
    16201 gcgataacct tcatgcgttc cccttgcgta tttgtttatt tactcatcgc atcatatacg
    16261 cagcgaccgc atgacgcaag ctgttttact caaatacaca tcaccttttt agacggcggc
    16321 gctcggtttc ttcagcggcc aagctggccg gccaggccgc cagcttggca tcagacaaac
    16381 cggccaggat ttcatgcagc cgcacggttg agacgtgcgc gggcggctcg aacacgtacc
    16441 cggccgcgat catctccgcc tcgatctctt cggtaatgaa aaacggttcg tcctggccgt
    16501 cctggtgcgg tttcatgctt gttcctcttg gcgttcattc tcggcggccg ccagggcgtc
    16561 ggcctcggtc aatgcgtcct cacggaaggc accgcgccgc ctggcctcgg tgggcgtcac
    16621 ttcctcgctg cgctcaagtg cgcggtacag ggtcgagcga tgcacgccaa gcagtgcagc
    16681 cgcctctttc acggtgcggc cttcctggtc gatcagctcg cgggcgtgcg cgatctgtgc
    16741 cggggtgagg gtagggcggg ggccaaactt cacgcctcgg gccttggcgg cctcgcgccc
    16801 gctccgggtg cggtcgatga ttagggaacg ctcgaactcg gcaatgccgg cgaacacggt
    16861 caacaccatg cggccggccg gcgtggtggt gtcggcccac ggctctgcca ggctacgcag
    16921 gcccgcgccg gcctcctgga tgcgctcggc aatgtccagt aggtcgcggg tgctgcgggc
    16981 caggcggtct agcctggtca ctgtcacaac gtcgccaggg cgtaggtggt caagcatcct
    17041 ggccagctcc gggcggtcgc gcctggtgcc ggtgatcttc tcggaaaaca gcttggtgca
    17101 gccggccgcg tgcagttcgg cccgttggtt ggtcaagtcc tggtcgtcgg tgctgacgcg
    17161 ggcatagccc agcaggccag cggcggcgct cttgttcatg gcgtaatgtc tccggttcta
    17221 gtcgcaagta ttctacttta tgcgactaaa acacgcgaca agaaaacgcc aggaaaaggg
    17281 cagggcggca gcctgtcgcg taacttagga cttgtgcgac atgtcgtttt cagaagacgg
    17341 ctgcactgaa cgtcagaagc cgactgcact atagcagcgg aggggttgga tcaaagtact
    17401 ttgatcccga ggggaaccct gtggttggca tgcacataca aatggacgaa cggataaacc
    17461 ttttcacgcc cttttaaata tccgattatt ctaataaacg ctcttttctc ttag
//