LOCUS       sequence-182268-       15744 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: 
            35S:Csy4-P2A-AtCas9 + CmYLCV:gRNAs with Csy4 spacers , Plant 
            Selection: 2x35S:bar.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15744)
  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 15744)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_23C
FEATURES             Location/Qualifiers
     source          1..15744
                     /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(1793..2344)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(2389..3065)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     protein_bind    3256..3277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3292..3297,3298..3315,3316..3322)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3330..3346
                     /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     3335..3357
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3354..3370
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3354..3370
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3900..4245
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             4837..4893
                     /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             4894..8997
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4894..8997
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9010..9030
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      9041..9289
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    9794..9813
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        9814..9889
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    9890..9909
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             10229..10534
                     /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     10413..10432
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    10562..10736
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(10755..10772)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10755..10771)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10764..10786)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    10974..10998
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             12299..12928
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             13357..14430
                     /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      14496..14690
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(14926..14948)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    15034..15174
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     15089..15108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(15189..15206)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(15360..15744,1..204))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(15440..15459)
                     /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 agtcaaatct
     1801 cggtgacggg caggaccgga cggggcggta ccggcaggct gaagtccagc tgccagaaac
     1861 ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg cagcatgccg cggggggcat
     1921 atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg cagcccgatg acagcgacca
     1981 cgctcttgaa gccctgtgcc tccagggact tcagcagatg ggtgtagagc gtggagccca
     2041 gtcccgtccg ctggtggcgg gggctgacgt acacggtcga ctcggccgtc cagtcgtagg
     2101 cgttgcgtgc cttccagggg cccgcgtagg cgatgccggc gacctcgccg tccacctcgg
     2161 cgacgagcca gggatagcgc tcccgcagac ggacgaggtc gtccgtccac tcctgcggtt
     2221 cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat gtagtggttg acgatggtgc
     2281 agaccgccgg catgtccgcc tcggtggcac ggcggatgtc ggccgggcgt cgttctgggc
     2341 tcatggtaga ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct
     2401 ctccaaatga aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg
     2461 tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt
     2521 tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact
     2581 gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt
     2641 gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc
     2701 gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga
     2761 gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttcacatc
     2821 aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg
     2881 gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata gcctttcctt
     2941 tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt ttgatgaagt
     3001 gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt tgttgaaaag
     3061 tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag tagacgagag
     3121 tgtcgtgctc caccatgttg gcaagctgct ctagccaata cgcaaaccgc ctctccccgc
     3181 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3241 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     3301 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     3361 cagctatgac atgattacga attcgagctc ggtacccggg gatcggcgcg ccagatttgc
     3421 cttttcaatt tcagaaagaa tgctaaccca cagatggtta gagaggctta cgcagcaggt
     3481 atcatcaaga cgatctaccc gagcaataat ctccaggaaa tcaaatacct tcccaagaag
     3541 gttaaagatg cagtcaaaag attcaggact aactgcatca agaacacaga gaaagatata
     3601 tttctcaaga tcagaagtac tattccagta tggacgattc aaggcttgct tcacaaacca
     3661 aggcaagtaa tagagattgg agtctctaaa aaggtagttc ccactgaatc aaaggccatg
     3721 gagtcaaaga ttcaaataga ggacctaaca gaactcgccg taaagactgg cgaacagttc
     3781 atacagagtc tcttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac
     3841 gacacacttg tctactccaa aaatatcaaa gatacagtct cagaagacca aagggcaatt
     3901 gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg cccagctatc
     3961 tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc
     4021 gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc
     4081 ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg
     4141 gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa
     4201 gacccttcct ctatataagg aagttcattt catttggaga gaacacgggg gactcctgca
     4261 ggaaaaatgg atcattatct tgatattaga cttagacctg atccagaatt tccaccagct
     4321 caacttatgt ctgttctttt tggaaaactt catcaagctc ttgttgctca aggaggagat
     4381 agaattggag tttcttttcc tgatcttgat gaatcaagat caagacttgg agaaagactt
     4441 agaattcatg cttctgctga tgatcttaga gctttgcttg ctagaccttg gcttgaagga
     4501 cttagagatc atcttcaatt tggagaacca gctgttgttc cacatccaac tccttataga
     4561 caagtttcaa gagttcaagc taaatctaat ccagaaagac ttagaagaag acttatgaga
     4621 agacatgatc tttctgaaga agaagctaga aaaagaattc ctgatactgt tgctagagct
     4681 ttggatttgc cttttgttac acttagatca caatctactg gacaacattt tagacttttt
     4741 attagacatg gaccacttca agttactgct gaagaaggag gatttacttg ttatggactt
     4801 tctaagggag gttttgttcc ttggtttgga tctggagcta ctaatttttc tcttcttaag
     4861 caagctggag atgttgaaga aaatcctgga cccatggata agaagtactc tatcggactc
     4921 gatatcggaa ctaactctgt gggatgggct gtgatcaccg atgagtacaa ggtgccatct
     4981 aagaagttca aggttctcgg aaacaccgat aggcactcta tcaagaaaaa ccttatcggt
     5041 gctctcctct tcgattctgg tgaaactgct gaggctacca gactcaagag aaccgctaga
     5101 agaaggtaca ccagaagaaa gaacaggatc tgctacctcc aagagatctt ctctaacgag
     5161 atggctaaag tggatgattc attcttccac aggctcgaag agtcattcct cgtggaagaa
     5221 gataagaagc acgagaggca ccctatcttc ggaaacatcg ttgatgaggt ggcataccac
     5281 gagaagtacc ctactatcta ccacctcaga aagaagctcg ttgattctac tgataaggct
     5341 gatctcaggc tcatctacct cgctctcgct cacatgatca agttcagagg acacttcctc
     5401 atcgagggtg atctcaaccc tgataactct gatgtggata agttgttcat ccagctcgtg
     5461 cagacctaca accagctttt cgaagagaac cctatcaacg cttcaggtgt ggatgctaag
     5521 gctatcctct ctgctaggct ctctaagtca agaaggcttg agaacctcat tgctcagctc
     5581 cctggtgaga agaagaacgg acttttcgga aacttgatcg ctctctctct cggactcacc
     5641 cctaacttca agtctaactt cgatctcgct gaggatgcaa agctccagct ctcaaaggat
     5701 acctacgatg atgatctcga taacctcctc gctcagatcg gagatcagta cgctgatttg
     5761 ttcctcgctg ctaagaacct ctctgatgct atcctcctca gtgatatcct cagagtgaac
     5821 accgagatca ccaaggctcc actctcagct tctatgatca agagatacga tgagcaccac
     5881 caggatctca cacttctcaa ggctcttgtt agacagcagc tcccagagaa gtacaaagag
     5941 attttcttcg atcagtctaa gaacggatac gctggttaca tcgatggtgg tgcatctcaa
     6001 gaagagttct acaagttcat caagcctatc ctcgagaaga tggatggaac cgaggaactc
     6061 ctcgtgaagc tcaatagaga ggatcttctc agaaagcaga ggaccttcga taacggatct
     6121 atccctcatc agatccacct cggagagttg cacgctatcc ttagaaggca agaggatttc
     6181 tacccattcc tcaaggataa cagggaaaag attgagaaga ttctcacctt cagaatccct
     6241 tactacgtgg gacctctcgc tagaggaaac tcaagattcg cttggatgac cagaaagtct
     6301 gaggaaacca tcaccccttg gaacttcgaa gaggtggtgg ataagggtgc tagtgctcag
     6361 tctttcatcg agaggatgac caacttcgat aagaaccttc caaacgagaa ggtgctccct
     6421 aagcactctt tgctctacga gtacttcacc gtgtacaacg agttgaccaa ggttaagtac
     6481 gtgaccgagg gaatgaggaa gcctgctttt ttgtcaggtg agcaaaagaa ggctatcgtt
     6541 gatctcttgt tcaagaccaa cagaaaggtg accgtgaagc agctcaaaga ggattacttc
     6601 aagaaaatcg agtgcttcga ttcagttgag atttctggtg ttgaggatag gttcaacgca
     6661 tctctcggaa cctaccacga tctcctcaag atcattaagg ataaggattt cttggataac
     6721 gaggaaaacg aggatatctt ggaggatatc gttcttaccc tcaccctctt tgaagataga
     6781 gagatgattg aagaaaggct caagacctac gctcatctct tcgatgataa ggtgatgaag
     6841 cagttgaaga gaagaagata cactggttgg ggaaggctct caagaaagct cattaacgga
     6901 atcagggata agcagtctgg aaagacaatc cttgatttcc tcaagtctga tggattcgct
     6961 aacagaaact tcatgcagct catccacgat gattctctca cctttaaaga ggatatccag
     7021 aaggctcagg tttcaggaca gggtgatagt ctccatgagc atatcgctaa cctcgctgga
     7081 tctcctgcaa tcaagaaggg aatcctccag actgtgaagg ttgtggatga gttggtgaag
     7141 gtgatgggaa ggcataagcc tgagaacatc gtgatcgaaa tggctagaga gaaccagacc
     7201 actcagaagg gacagaagaa ctctagggaa aggatgaaga ggatcgagga aggtatcaaa
     7261 gagcttggat ctcagatcct caaagagcac cctgttgaga acactcagct ccagaatgag
     7321 aagctctacc tctactacct ccagaacgga agggatatgt atgtggatca agagttggat
     7381 atcaacaggc tctctgatta cgatgttgat catatcgtgc cacagtcatt cttgaaggat
     7441 gattctatcg ataacaaggt gctcaccagg tctgataaga acaggggtaa gagtgataac
     7501 gtgccaagtg aagaggttgt gaagaaaatg aagaactatt ggaggcagct cctcaacgct
     7561 aagctcatca ctcagagaaa gttcgataac ttgactaagg ctgagagggg aggactctct
     7621 gaattggata aggcaggatt catcaagagg cagcttgtgg aaaccaggca gatcactaag
     7681 cacgttgcac agatcctcga ttctaggatg aacaccaagt acgatgagaa cgataagttg
     7741 atcagggaag tgaaggttat caccctcaag tcaaagctcg tgtctgattt cagaaaggat
     7801 ttccaattct acaaggtgag ggaaatcaac aactaccacc acgctcacga tgcttacctt
     7861 aacgctgttg ttggaaccgc tctcatcaag aagtatccta agctcgagtc agagttcgtg
     7921 tacggtgatt acaaggtgta cgatgtgagg aagatgatcg ctaagtctga gcaagagatc
     7981 ggaaaggcta ccgctaagta tttcttctac tctaacatca tgaatttctt caagaccgag
     8041 attaccctcg ctaacggtga gatcagaaag aggccactca tcgagacaaa cggtgaaaca
     8101 ggtgagatcg tgtgggataa gggaagggat ttcgctaccg ttagaaaggt gctctctatg
     8161 ccacaggtga acatcgttaa gaaaaccgag gtgcagaccg gtggattctc taaagagtct
     8221 atcctcccta agaggaactc tgataagctc attgctagga agaaggattg ggaccctaag
     8281 aaatacggtg gtttcgattc tcctaccgtg gcttactctg ttctcgttgt ggctaaggtt
     8341 gagaagggaa agagtaagaa gctcaagtct gttaaggaac ttctcggaat cactatcatg
     8401 gaaaggtcat ctttcgagaa gaacccaatc gatttcctcg aggctaaggg atacaaagag
     8461 gttaagaagg atctcatcat caagctccca aagtactcac tcttcgaact cgagaacggt
     8521 agaaagagga tgctcgcttc tgctggtgag cttcaaaagg gaaacgagct tgctctccca
     8581 tctaagtacg ttaactttct ttacctcgct tctcactacg agaagttgaa gggatctcca
     8641 gaagataacg agcagaagca acttttcgtt gagcagcaca agcactactt ggatgagatc
     8701 atcgagcaga tctctgagtt ctctaaaagg gtgatcctcg ctgatgcaaa cctcgataag
     8761 gtgttgtctg cttacaacaa gcacagagat aagcctatca gggaacaggc agagaacatc
     8821 atccatctct tcacccttac caacctcggt gctcctgctg ctttcaagta cttcgataca
     8881 accatcgata ggaagagata cacctctacc aaagaagtgc tcgatgctac cctcatccat
     8941 cagtctatca ctggactcta cgagactagg atcgatctct cacagctcgg tggtgattca
     9001 agggctgatc ctaagaagaa gaggaaggtt tgactcgaga tatgaagatg aagatgaaat
     9061 atttggtgtg tcaaataaaa agcttgtgtg cttaagtttg tgtttttttc ttggcttgtt
     9121 gtgttatgaa tttgtggctt tttctaatat taaatgaatg taagatcaca ttataatgaa
     9181 taaacaaatg tttctataat ccattgtgaa tgttttgttg gatctcttct gcagcatata
     9241 actactgtat gtgctatggt atggactatg gaatatgatt aaagataagg agctccggtg
     9301 acggacggcg cgccgaagag ctggcagaca tactgtccca caaatgaaga tggaatctgt
     9361 aaaagaaaac gcgtgaaata atgcgtctga caaaggttag gtcggctgcc tttaatcaat
     9421 accaaagtgg tccctaccac gatggaaaaa ctgtgcagtc ggtttggctt tttctgacga
     9481 acaaataaga ttcgtggccg acaggtgggg gtccaccatg tgaaggcatc ttcagactcc
     9541 aataatggag caatgacgta agggcttacg aaataagtaa gggtagtttg ggaaatgtcc
     9601 actcacccgt cagtctataa atacttagcc cctccctcat tgttaaggga gcaaaatctc
     9661 agagagatag tcctagagag agaaagagag caagtagcct agaagtagtc aaggcggcga
     9721 agtattcagg cacgtggcca ggaagaagaa aagccaagac gacgaaaaca ggtaagagct
     9781 aagcttcctg caggttcact gccgtatagg caggttttag agctagaaat agcaagttaa
     9841 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg
     9901 tataggcaga agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa
     9961 tatatactga tatgtatacc cgaagtatgt caaaaggagg tatgctatga agcagcgtat
    10021 tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc
    10081 tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg
    10141 aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct
    10201 tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga
    10261 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
    10321 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
    10381 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
    10441 gccggtttcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
    10501 aaacgccatt aacctgatgt tctggggaat ataagctctt cagtcgatcg acaagctcga
    10561 gtttctccat aataatgtgt gagtagttcc cagataaggg aattagggtt cctatagggt
    10621 ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact
    10681 tctatcaata aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatccccc
    10741 gaattaccgg tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
    10801 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
    10861 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ctagagcagc
    10921 ttgagcttgg atcagattgt cgtttcccgc cttcagttta aactatcagt gtttgacagg
    10981 atatattggc gggtaaacct aagagaaaag agcgtttatt agaataatcg gatatttaaa
    11041 agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc acagggttcc
    11101 cctcgggatc aaagtacttt gatccaaccc ctccgctgct atagtgcagt cggcttctga
    11161 cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt tacgcgacag
    11221 gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg cataaagtag
    11281 aatacttgcg actagaaccg gagacattac gccatgaaca agagcgccgc cgctggcctg
    11341 ctgggctatg cccgcgtcag caccgacgac caggacttga ccaaccaacg ggccgaactg
    11401 cacgcggccg gctgcaccaa gctgttttcc gagaagatca ccggcaccag gcgcgaccgc
    11461 ccggagctgg ccaggatgct tgaccaccta cgccctggcg acgttgtgac agtgaccagg
    11521 ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg catccaggag
    11581 gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac gccggccggc
    11641 cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct aatcatcgac
    11701 cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg cccccgccct
    11761 accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga aggccgcacc
    11821 gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg cgcacttgag
    11881 cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg tgaggacgca
    11941 ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga acaagcatga
    12001 aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc gaggcggaga
    12061 tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgg ctcaaccgtg cggctgcatg
    12121 aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg
    12181 aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc
    12241 atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat
    12301 gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca
    12361 tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca
    12421 gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat
    12481 cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat
    12541 cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt
    12601 gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct
    12661 ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg
    12721 ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct
    12781 gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg
    12841 cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc aggcgctggc
    12901 cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc
    12961 acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc
    13021 agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac
    13081 accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa
    13141 tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc
    13201 ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc
    13261 catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgcct gccggccctg
    13321 caatggcact ggaaccccca agcccgagga atcggcgtga gcggtcgcaa accatccggc
    13381 ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa ggccgcgcag
    13441 gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc
    13501 gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg
    13561 aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta tgacgtgggc
    13621 acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga
    13681 cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt ttccgcaggg
    13741 ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt ttcccatcta
    13801 accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg cgtgttccgt
    13861 ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa gcagaaagac
    13921 gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca gcgtacgaag
    13981 aaggccaaga acggccgcct ggtgacggta tccgagggtg aagccttgat tagccgctac
    14041 aagatcgtaa agagcgaaac cgggcggccg gagtacatcg agatcgagct agctgattgg
    14101 atgtaccgcg agatcacaga aggcaagaac ccggacgtgc tgacggttca ccccgattac
    14161 tttttgatcg atcccggcat cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc
    14221 aaggcagaag ccagatggtt gttcaagacg atctacgaac gcagtggcag cgccggagag
    14281 ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct gccggagtac
    14341 gatttgaagg aggaggcggg gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg
    14401 atcgagggcg aagcatccgc cggttcctaa tgtacggagc agatgctagg gcaaattgcc
    14461 ctagcagggg aaaaaggtcg aaaaggcctc tttcctgtgg atagcacgta cattgggaac
    14521 ccaaagccgt acattgggaa ccggaacccg tacattggga acccaaagcc gtacattggg
    14581 aaccggtcac acatgtaagt gactgatata aaagagaaaa aaggcgattt ttccgcctaa
    14641 aactctttaa aacttattaa aactcttaaa acccgcctgg cctgtgcata actgtctggc
    14701 cagcgcacag cccaagagct gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc
    14761 cccgccgctt cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc tggcctacgg
    14821 ccaggcaatc taccagggcg cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca
    14881 catcaaggca ccctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
    14941 gctcccggaa acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
    15001 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
    15061 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
    15121 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggccctct
    15181 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    15241 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    15301 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    15361 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    15421 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    15481 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    15541 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    15601 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    15661 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    15721 aacaggatta gcagagcgag gtat
//
