pBSE901

LOCUS       pBSE901           16987 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  CRISPR/Cas9-mediated base editing in dicots. Contains Cas9(D10A) 
            under CaMV 35S promoter and empty gRNA scaffold, Basta resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pBSE901
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16987)
  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 16987)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..16987
                     /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..9853
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             9898..10449
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     polyA_signal    10456..10630
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    10708..10732
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             11157..11951
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      12038..12626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12527..12546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12780..12797
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    12812..12952
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(12878..12897)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13038..13060
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      13296..13490
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13556..14629)
                     /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(15058..15687)
                     /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 cttcmaagca agtggattga
     9421 tgtgacagac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt
     9481 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     9541 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     9601 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga
     9661 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
     9721 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     9781 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
     9841 gaggacacgc tgaaatcacc agtctctctc tacaaatcta tctctctcga gtctaccatg
     9901 agcccagaac gacgcccggc cgacatccgc cgtgccaccg aggcggacat gccggcggtc
     9961 tgcaccatcg tcaaccacta catcgagaca agcacggtca acttccgtac cgagccgcag
    10021 gaaccgcagg agtggacgga cgacctcgtc cgtctgcggg agcgctatcc ctggctcgtc
    10081 gccgaggtgg acggcgaggt cgccggcatc gcctacgcgg gcccctggaa ggcacgcaac
    10141 gcctacgact ggacggccga gtcgaccgtg tacgtctccc cccgccacca gcggacggga
    10201 ctgggctcca cgctctacac ccacctgctg aagtccctgg aggcacaggg cttcaagagc
    10261 gtggtcgctg tcatcgggct gcccaacgac ccgagcgtgc gcatgcacga ggcgctcgga
    10321 tatgcccccc gcggcatgct gcgggcggcc ggcttcaagc acgggaactg gcatgacgtg
    10381 ggtttctggc agctggactt cagcctgccg gtaccgcccc gtccggtcct gcccgtcacc
    10441 gagatttgac tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag
    10501 ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt
    10561 atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa
    10621 aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt
    10681 gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag
    10741 ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc
    10801 agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc
    10861 gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg
    10921 agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca ccgcggtttc
    10981 aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt tgaaaaagct
    11041 gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt cgtcttgtta
    11101 taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa taataaatgg
    11161 ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag
    11221 atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat
    11281 atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg
    11341 acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc
    11401 atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt
    11461 atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct
    11521 ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg
    11581 aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag
    11641 acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag
    11701 aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca
    11761 aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg
    11821 ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc gagctatttt
    11881 ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg
    11941 aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt tcgttccact
    12001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    12061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    12121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    12181 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    12241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    12301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    12361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    12421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    12481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    12541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    12601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    12661 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    12721 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    12781 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
    12841 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
    12901 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
    12961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
    13021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
    13081 acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt
    13141 gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg ccagcggccg
    13201 cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt
    13261 tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg cgggttttaa
    13321 gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc tcttttatat
    13381 cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca atgtacgggt
    13441 tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca ggaaacagac
    13501 cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc gtacattagg
    13561 aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg atcgggccag
    13621 cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg atcagcttgc
    13681 gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg tagatcgtct
    13741 tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac
    13801 ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg tccgggttct
    13861 tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg tactccggcc
    13921 gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc tcggataccg
    13981 tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta
    14041 accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct cgccggcaga
    14101 acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct cccttccctt
    14161 cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc aggtcgtaat
    14221 cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg tctggaagct
    14281 cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa acggccacgt
    14341 ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg aaaaaatctg
    14401 gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc
    14461 gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg cgtgcttctg
    14521 cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc aggtcatcac
    14581 ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgctcacg ccgattcctc
    14641 gggcttgggg gttccagtgc cattgcaggg ccggcaggca acccagccgc ttacgcctgg
    14701 ccaaccgccc gttcctccac acatggggca ttccacggcg tcggtgcctg gttgttcttg
    14761 attttccatg ccgcctcctt tagccgctaa aattcatcta ctcatttatt catttgctca
    14821 tttactctgg tagctgcgcg atgtattcag atagcagctc ggtaatggtc ttgccttggc
    14881 gtaccgcgta catcttcagc ttggtgtgat cctccgccgg caactgaaag ttgacccgct
    14941 tcatggctgg cgtgtctgcc aggctggcca acgttgcagc cttgctgctg cgtgcgctcg
    15001 gacggccggc acttagcgtg tttgtgcttt tgctcatttt ctctttacct cattaactca
    15061 aatgagtttt gatttaattt cagcggccag cgcctggacc tcgcgggcag cgtcgccctc
    15121 gggttctgat tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc tcacgcgctg
    15181 cgtgatacgg gactcaagaa tgggcagctc gtacccggcc agcgcctcgg caacctcacc
    15241 gccgatgcgc gtgcctttga tcgcccgcga cacgacaaag gccgcttgta gccttccatc
    15301 cgtgacctca atgcgctgct taaccagctc caccaggtcg gcggtggccc atatgtcgta
    15361 agggcttggc tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg acacagccaa
    15421 gtccgccgcc tggggcgctc cgtcgatcac tacgaagtcg cgccggccga tggccttcac
    15481 gtcgcggtca atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat cttcccgcac
    15541 ggccgcccaa tcgcgggcac tgccctgggg atcggaatcg actaacagaa catcggcccc
    15601 ggcgagttgc agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg acccgccttt
    15661 ctggttaagt acagcgataa ccttcatgcg ttccccttgc gtatttgttt atttactcat
    15721 cgcatcatat acgcagcgac cgcatgacgc aagctgtttt actcaaatac acatcacctt
    15781 tttagacggc ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc cgccagcttg
    15841 gcatcagaca aaccggccag gatttcatgc agccgcacgg ttgagacgtg cgcgggcggc
    15901 tcgaacacgt acccggccgc gatcatctcc gcctcgatct cttcggtaat gaaaaacggt
    15961 tcgtcctggc cgtcctggtg cggtttcatg cttgttcctc ttggcgttca ttctcggcgg
    16021 ccgccagggc gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc cgcctggcct
    16081 cggtgggcgt cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag cgatgcacgc
    16141 caagcagtgc agccgcctct ttcacggtgc ggccttcctg gtcgatcagc tcgcgggcgt
    16201 gcgcgatctg tgccggggtg agggtagggc gggggccaaa cttcacgcct cgggccttgg
    16261 cggcctcgcg cccgctccgg gtgcggtcga tgattaggga acgctcgaac tcggcaatgc
    16321 cggcgaacac ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc cacggctctg
    16381 ccaggctacg caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc agtaggtcgc
    16441 gggtgctgcg ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca gggcgtaggt
    16501 ggtcaagcat cctggccagc tccgggcggt cgcgcctggt gccggtgatc ttctcggaaa
    16561 acagcttggt gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag tcctggtcgt
    16621 cggtgctgac gcgggcatag cccagcaggc cagcggcggc gctcttgttc atggcgtaat
    16681 gtctccggtt ctagtcgcaa gtattctact ttatgcgact aaaacacgcg acaagaaaac
    16741 gccaggaaaa gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc gacatgtcgt
    16801 tttcagaaga cggctgcact gaacgtcaga agccgactgc actatagcag cggaggggtt
    16861 ggatcaaagt actttgatcc cgaggggaac cctgtggttg gcatgcacat acaaatggac
    16921 gaacggataa accttttcac gcccttttaa atatccgatt attctaataa acgctctttt
    16981 ctcttag
//