LOCUS       sequence-182255-       16274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: 
            35S:Csy4-P2A-AtCas9_dead + CmYLCV:gRNAs with Csy4 spacers, Plant 
            Selection: 2x35S:hpt II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16274)
  AUTHORS   Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, 
            Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF
  TITLE     A multi-purpose toolkit to enable advanced genome engineering in 
            plants.
  JOURNAL   Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 
            10.1105/tpc.16.00922.
  PUBMED    28522548
REFERENCE   2  (bases 1 to 16274)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant Cell.
            2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922."
COMMENT     Sequence Label: pDIRECT_21D
FEATURES             Location/Qualifiers
     source          1..16274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(291..1085)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    1510..1534
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    1612..1786
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(1827..2852)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     CDS             complement(1827..2852)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(2919..3595)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     protein_bind    3786..3807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3822..3827,3828..3845,3846..3852)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3860..3876
                     /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)."
     primer_bind     3865..3887
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3884..3900
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3884..3900
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4430..4775
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             5367..5423
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5424..5450,5451..5453,5454..7940,7941..7943,
                     7944..9527)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9540..9560
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      9571..9819
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    10324..10343
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        10344..10419
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    10420..10439
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             10759..11064
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     10943..10962
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    11092..11266
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(11285..11302)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11285..11301)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11294..11316)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    11504..11528
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             12829..13458
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             13887..14960
                     /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"
     rep_origin      15026..15220
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(15456..15478)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    15564..15704
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     15619..15638
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(15719..15736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(15890..16274,1..204))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(15970..15989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
       61 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      241 cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat
      301 tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca
      361 aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag
      421 gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact
      481 ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc
      541 tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg
      601 tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat
      661 tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga
      721 aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca
      781 tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca
      841 tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg
      901 tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa
      961 tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta
     1021 tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta
     1081 gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt
     1141 ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa
     1201 cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt
     1261 tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc
     1321 tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat
     1381 cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac
     1441 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg
     1501 gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa
     1561 cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat
     1621 tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa
     1681 tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac
     1741 cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga
     1801 tcgacagatc ccggtcggca tctactctat ttctttgccc tcggacgagt gctggggcgt
     1861 cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg
     1921 cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga
     1981 ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca
     2041 agaccaatgc ggagcatata cgcccggagt cgtggcgatc ctgcaagctc cggatgcctc
     2101 cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg
     2161 ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt
     2221 atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg
     2281 acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca
     2341 ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat
     2401 atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg
     2461 ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga
     2521 acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag
     2581 atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
     2641 gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta
     2701 tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
     2761 ccctccgaga gctgcatcag gtcggacacg ctgtcgaact tttcgatcag aaacttctcg
     2821 acagacgtcg cggtgagttc aggctttttc atatctcatt gccccccgga tctgcgaaag
     2881 ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga
     2941 aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc
     3001 cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct
     3061 tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag
     3121 gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt gccaccttcc
     3181 ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc gaggaggttt
     3241 cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga gactgtatct
     3301 ttgatattct tggagtagac gagagtgtcg tgctccacca tgttcacatc aatccacttg
     3361 ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc
     3421 atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg
     3481 atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc
     3541 tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc
     3601 cctttggtct tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc
     3661 caccatgttg gcaagctgct ctagccaata cgcaaaccgc ctctccccgc gcgttggccg
     3721 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3781 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3841 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3901 atgattacga attcgagctc ggtacccggg gatcggcgcg ccagatttgc cttttcaatt
     3961 tcagaaagaa tgctaaccca cagatggtta gagaggctta cgcagcaggt atcatcaaga
     4021 cgatctaccc gagcaataat ctccaggaaa tcaaatacct tcccaagaag gttaaagatg
     4081 cagtcaaaag attcaggact aactgcatca agaacacaga gaaagatata tttctcaaga
     4141 tcagaagtac tattccagta tggacgattc aaggcttgct tcacaaacca aggcaagtaa
     4201 tagagattgg agtctctaaa aaggtagttc ccactgaatc aaaggccatg gagtcaaaga
     4261 ttcaaataga ggacctaaca gaactcgccg taaagactgg cgaacagttc atacagagtc
     4321 tcttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac gacacacttg
     4381 tctactccaa aaatatcaaa gatacagtct cagaagacca aagggcaatt gagacttttc
     4441 aacaaagggt aatatccgga aacctcctcg gattccattg cccagctatc tgtcacttta
     4501 ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc gataaaggaa
     4561 aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga
     4621 ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg
     4681 atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa gacccttcct
     4741 ctatataagg aagttcattt catttggaga gaacacgggg gactcctgca ggaaaaatgg
     4801 atcattatct tgatattaga cttagacctg atccagaatt tccaccagct caacttatgt
     4861 ctgttctttt tggaaaactt catcaagctc ttgttgctca aggaggagat agaattggag
     4921 tttcttttcc tgatcttgat gaatcaagat caagacttgg agaaagactt agaattcatg
     4981 cttctgctga tgatcttaga gctttgcttg ctagaccttg gcttgaagga cttagagatc
     5041 atcttcaatt tggagaacca gctgttgttc cacatccaac tccttataga caagtttcaa
     5101 gagttcaagc taaatctaat ccagaaagac ttagaagaag acttatgaga agacatgatc
     5161 tttctgaaga agaagctaga aaaagaattc ctgatactgt tgctagagct ttggatttgc
     5221 cttttgttac acttagatca caatctactg gacaacattt tagacttttt attagacatg
     5281 gaccacttca agttactgct gaagaaggag gatttacttg ttatggactt tctaagggag
     5341 gttttgttcc ttggtttgga tctggagcta ctaatttttc tcttcttaag caagctggag
     5401 atgttgaaga aaatcctgga cccatggata agaagtactc tatcggactc gctatcggaa
     5461 ctaactctgt gggatgggct gtgatcaccg atgagtacaa ggtgccatct aagaagttca
     5521 aggttctcgg aaacaccgat aggcactcta tcaagaaaaa ccttatcggt gctctcctct
     5581 tcgattctgg tgaaactgct gaggctacca gactcaagag aaccgctaga agaaggtaca
     5641 ccagaagaaa gaacaggatc tgctacctcc aagagatctt ctctaacgag atggctaaag
     5701 tggatgattc attcttccac aggctcgaag agtcattcct cgtggaagaa gataagaagc
     5761 acgagaggca ccctatcttc ggaaacatcg ttgatgaggt ggcataccac gagaagtacc
     5821 ctactatcta ccacctcaga aagaagctcg ttgattctac tgataaggct gatctcaggc
     5881 tcatctacct cgctctcgct cacatgatca agttcagagg acacttcctc atcgagggtg
     5941 atctcaaccc tgataactct gatgtggata agttgttcat ccagctcgtg cagacctaca
     6001 accagctttt cgaagagaac cctatcaacg cttcaggtgt ggatgctaag gctatcctct
     6061 ctgctaggct ctctaagtca agaaggcttg agaacctcat tgctcagctc cctggtgaga
     6121 agaagaacgg acttttcgga aacttgatcg ctctctctct cggactcacc cctaacttca
     6181 agtctaactt cgatctcgct gaggatgcaa agctccagct ctcaaaggat acctacgatg
     6241 atgatctcga taacctcctc gctcagatcg gagatcagta cgctgatttg ttcctcgctg
     6301 ctaagaacct ctctgatgct atcctcctca gtgatatcct cagagtgaac accgagatca
     6361 ccaaggctcc actctcagct tctatgatca agagatacga tgagcaccac caggatctca
     6421 cacttctcaa ggctcttgtt agacagcagc tcccagagaa gtacaaagag attttcttcg
     6481 atcagtctaa gaacggatac gctggttaca tcgatggtgg tgcatctcaa gaagagttct
     6541 acaagttcat caagcctatc ctcgagaaga tggatggaac cgaggaactc ctcgtgaagc
     6601 tcaatagaga ggatcttctc agaaagcaga ggaccttcga taacggatct atccctcatc
     6661 agatccacct cggagagttg cacgctatcc ttagaaggca agaggatttc tacccattcc
     6721 tcaaggataa cagggaaaag attgagaaga ttctcacctt cagaatccct tactacgtgg
     6781 gacctctcgc tagaggaaac tcaagattcg cttggatgac cagaaagtct gaggaaacca
     6841 tcaccccttg gaacttcgaa gaggtggtgg ataagggtgc tagtgctcag tctttcatcg
     6901 agaggatgac caacttcgat aagaaccttc caaacgagaa ggtgctccct aagcactctt
     6961 tgctctacga gtacttcacc gtgtacaacg agttgaccaa ggttaagtac gtgaccgagg
     7021 gaatgaggaa gcctgctttt ttgtcaggtg agcaaaagaa ggctatcgtt gatctcttgt
     7081 tcaagaccaa cagaaaggtg accgtgaagc agctcaaaga ggattacttc aagaaaatcg
     7141 agtgcttcga ttcagttgag atttctggtg ttgaggatag gttcaacgca tctctcggaa
     7201 cctaccacga tctcctcaag atcattaagg ataaggattt cttggataac gaggaaaacg
     7261 aggatatctt ggaggatatc gttcttaccc tcaccctctt tgaagataga gagatgattg
     7321 aagaaaggct caagacctac gctcatctct tcgatgataa ggtgatgaag cagttgaaga
     7381 gaagaagata cactggttgg ggaaggctct caagaaagct cattaacgga atcagggata
     7441 agcagtctgg aaagacaatc cttgatttcc tcaagtctga tggattcgct aacagaaact
     7501 tcatgcagct catccacgat gattctctca cctttaaaga ggatatccag aaggctcagg
     7561 tttcaggaca gggtgatagt ctccatgagc atatcgctaa cctcgctgga tctcctgcaa
     7621 tcaagaaggg aatcctccag actgtgaagg ttgtggatga gttggtgaag gtgatgggaa
     7681 ggcataagcc tgagaacatc gtgatcgaaa tggctagaga gaaccagacc actcagaagg
     7741 gacagaagaa ctctagggaa aggatgaaga ggatcgagga aggtatcaaa gagcttggat
     7801 ctcagatcct caaagagcac cctgttgaga acactcagct ccagaatgag aagctctacc
     7861 tctactacct ccagaacgga agggatatgt atgtggatca agagttggat atcaacaggc
     7921 tctctgatta cgatgttgat gctatcgtgc cacagtcatt cttgaaggat gattctatcg
     7981 ataacaaggt gctcaccagg tctgataaga acaggggtaa gagtgataac gtgccaagtg
     8041 aagaggttgt gaagaaaatg aagaactatt ggaggcagct cctcaacgct aagctcatca
     8101 ctcagagaaa gttcgataac ttgactaagg ctgagagggg aggactctct gaattggata
     8161 aggcaggatt catcaagagg cagcttgtgg aaaccaggca gatcactaag cacgttgcac
     8221 agatcctcga ttctaggatg aacaccaagt acgatgagaa cgataagttg atcagggaag
     8281 tgaaggttat caccctcaag tcaaagctcg tgtctgattt cagaaaggat ttccaattct
     8341 acaaggtgag ggaaatcaac aactaccacc acgctcacga tgcttacctt aacgctgttg
     8401 ttggaaccgc tctcatcaag aagtatccta agctcgagtc agagttcgtg tacggtgatt
     8461 acaaggtgta cgatgtgagg aagatgatcg ctaagtctga gcaagagatc ggaaaggcta
     8521 ccgctaagta tttcttctac tctaacatca tgaatttctt caagaccgag attaccctcg
     8581 ctaacggtga gatcagaaag aggccactca tcgagacaaa cggtgaaaca ggtgagatcg
     8641 tgtgggataa gggaagggat ttcgctaccg ttagaaaggt gctctctatg ccacaggtga
     8701 acatcgttaa gaaaaccgag gtgcagaccg gtggattctc taaagagtct atcctcccta
     8761 agaggaactc tgataagctc attgctagga agaaggattg ggaccctaag aaatacggtg
     8821 gtttcgattc tcctaccgtg gcttactctg ttctcgttgt ggctaaggtt gagaagggaa
     8881 agagtaagaa gctcaagtct gttaaggaac ttctcggaat cactatcatg gaaaggtcat
     8941 ctttcgagaa gaacccaatc gatttcctcg aggctaaggg atacaaagag gttaagaagg
     9001 atctcatcat caagctccca aagtactcac tcttcgaact cgagaacggt agaaagagga
     9061 tgctcgcttc tgctggtgag cttcaaaagg gaaacgagct tgctctccca tctaagtacg
     9121 ttaactttct ttacctcgct tctcactacg agaagttgaa gggatctcca gaagataacg
     9181 agcagaagca acttttcgtt gagcagcaca agcactactt ggatgagatc atcgagcaga
     9241 tctctgagtt ctctaaaagg gtgatcctcg ctgatgcaaa cctcgataag gtgttgtctg
     9301 cttacaacaa gcacagagat aagcctatca gggaacaggc agagaacatc atccatctct
     9361 tcacccttac caacctcggt gctcctgctg ctttcaagta cttcgataca accatcgata
     9421 ggaagagata cacctctacc aaagaagtgc tcgatgctac cctcatccat cagtctatca
     9481 ctggactcta cgagactagg atcgatctct cacagctcgg tggtgattca agggctgatc
     9541 ctaagaagaa gaggaaggtt tgactcgaga tatgaagatg aagatgaaat atttggtgtg
     9601 tcaaataaaa agcttgtgtg cttaagtttg tgtttttttc ttggcttgtt gtgttatgaa
     9661 tttgtggctt tttctaatat taaatgaatg taagatcaca ttataatgaa taaacaaatg
     9721 tttctataat ccattgtgaa tgttttgttg gatctcttct gcagcatata actactgtat
     9781 gtgctatggt atggactatg gaatatgatt aaagataagg agctccggtg acggacggcg
     9841 cgccgaagag ctggcagaca tactgtccca caaatgaaga tggaatctgt aaaagaaaac
     9901 gcgtgaaata atgcgtctga caaaggttag gtcggctgcc tttaatcaat accaaagtgg
     9961 tccctaccac gatggaaaaa ctgtgcagtc ggtttggctt tttctgacga acaaataaga
    10021 ttcgtggccg acaggtgggg gtccaccatg tgaaggcatc ttcagactcc aataatggag
    10081 caatgacgta agggcttacg aaataagtaa gggtagtttg ggaaatgtcc actcacccgt
    10141 cagtctataa atacttagcc cctccctcat tgttaaggga gcaaaatctc agagagatag
    10201 tcctagagag agaaagagag caagtagcct agaagtagtc aaggcggcga agtattcagg
    10261 cacgtggcca ggaagaagaa aagccaagac gacgaaaaca ggtaagagct aagcttcctg
    10321 caggttcact gccgtatagg caggttttag agctagaaat agcaagttaa aataaggcta
    10381 gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg tataggcaga
    10441 agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga
    10501 tatgtatacc cgaagtatgt caaaaggagg tatgctatga agcagcgtat tacagtgaca
    10561 gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg
    10621 taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa
    10681 atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg
    10741 agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat
    10801 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc
    10861 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
    10921 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtttcc
    10981 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
    11041 aacctgatgt tctggggaat ataagctctt cagtcgatcg acaagctcga gtttctccat
    11101 aataatgtgt gagtagttcc cagataaggg aattagggtt cctatagggt ttcgctcatg
    11161 tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata
    11221 aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatccccc gaattaccgg
    11281 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
    11341 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
    11401 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ctagagcagc ttgagcttgg
    11461 atcagattgt cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggc
    11521 gggtaaacct aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaa
    11581 aaggtttatc cgttcgtcca tttgtatgtg catgccaacc acagggttcc cctcgggatc
    11641 aaagtacttt gatccaaccc ctccgctgct atagtgcagt cggcttctga cgttcagtgc
    11701 agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt tacgcgacag gctgccgccc
    11761 tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg cataaagtag aatacttgcg
    11821 actagaaccg gagacattac gccatgaaca agagcgccgc cgctggcctg ctgggctatg
    11881 cccgcgtcag caccgacgac caggacttga ccaaccaacg ggccgaactg cacgcggccg
    11941 gctgcaccaa gctgttttcc gagaagatca ccggcaccag gcgcgaccgc ccggagctgg
    12001 ccaggatgct tgaccaccta cgccctggcg acgttgtgac agtgaccagg ctagaccgcc
    12061 tggcccgcag cacccgcgac ctactggaca ttgccgagcg catccaggag gccggcgcgg
    12121 gcctgcgtag cctggcagag ccgtgggccg acaccaccac gccggccggc cgcatggtgt
    12181 tgaccgtgtt cgccggcatt gccgagttcg agcgttccct aatcatcgac cgcacccgga
    12241 gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg cccccgccct accctcaccc
    12301 cggcacagat cgcgcacgcc cgcgagctga tcgaccagga aggccgcacc gtgaaagagg
    12361 cggctgcact gcttggcgtg catcgctcga ccctgtaccg cgcacttgag cgcagcgagg
    12421 aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg tgaggacgca ttgaccgagg
    12481 ccgacgccct ggcggccgcc gagaatgaac gccaagagga acaagcatga aaccgcacca
    12541 ggacggccag gacgaaccgt ttttcattac cgaagagatc gaggcggaga tgatcgcggc
    12601 cgggtacgtg ttcgagccgc ccgcgcacgg ctcaaccgtg cggctgcatg aaatcctggc
    12661 cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg aagaaaccga
    12721 gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc atgcggtcgc
    12781 tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat gaaggttatc
    12841 gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca tctagcccgc
    12901 gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca gggcagtgcc
    12961 cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat cgaccgcccg
    13021 acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat cgacggagcg
    13081 ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt gctgattccg
    13141 gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct ggttaagcag
    13201 cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg ggcgatcaaa
    13261 ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct gcccattctt
    13321 gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg cacaaccgtt
    13381 cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc aggcgctggc cgctgaaatt
    13441 aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc acaaacacgc
    13501 taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc agcctggcag
    13561 acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac accaagctga
    13621 agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa tacatcgcgc
    13681 agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc ggctaaagga
    13741 ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc catgtgtgga
    13801 ggaacgggcg gttggccagg cgtaagcggc tgggttgcct gccggccctg caatggcact
    13861 ggaaccccca agcccgagga atcggcgtga gcggtcgcaa accatccggc ccggtacaaa
    13921 tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc
    13981 ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa
    14041 tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg aagccgccca
    14101 agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata
    14161 gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg
    14221 aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca
    14281 tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca
    14341 tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg
    14401 cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag
    14461 aaacctgcat tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga
    14521 acggccgcct ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa
    14581 agagcgaaac cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg
    14641 agatcacaga aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg
    14701 atcccggcat cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag
    14761 ccagatggtt gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt
    14821 tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg
    14881 aggaggcggg gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg
    14941 aagcatccgc cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg
    15001 aaaaaggtcg aaaaggcctc tttcctgtgg atagcacgta cattgggaac ccaaagccgt
    15061 acattgggaa ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac
    15121 acatgtaagt gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa
    15181 aacttattaa aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag
    15241 cccaagagct gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc cccgccgctt
    15301 cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc tggcctacgg ccaggcaatc
    15361 taccagggcg cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca catcaaggca
    15421 ccctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggaa
    15481 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
    15541 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
    15601 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
    15661 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggccctct tccgcttcct
    15721 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
    15781 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
    15841 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
    15901 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
    15961 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
    16021 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
    16081 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
    16141 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
    16201 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
    16261 gcagagcgag gtat
//
