LOCUS       sequence-182257-       14990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: 35S:AtCas9 + 
            AtU6:gRNA, Plant Selection: 2x35S:npt II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14990)
  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 14990)
  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_22A
FEATURES             Location/Qualifiers
     source          1..14990
                     /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(1843..2640)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1954..1973)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2564..2583
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2703..3379)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     protein_bind    3570..3591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3606..3611,3612..3629,3630..3636)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3644..3660
                     /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     3668..3684
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4214..4559
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             4593..8696
                     /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             4593..8696
                     /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             8709..8729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8740..8988
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     CDS             complement(9455..9760)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGTCL"
     primer_bind     9625..9641
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9651..9670
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9651..9669
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9717..9737)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9717..9735)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9756..9772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    9780..9796
                     /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(join(9804..9810,9811..9828,9829..9834))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9849..9870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        9909..9984
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10001..10017)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    10220..10244
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             11545..12174
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             12603..13676
                     /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      13742..13936
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(14172..14194)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    14280..14420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     14335..14354
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(14435..14452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(14606..14990,1..204))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14686..14705)
                     /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 tcgactctag ctagaggatc gatccgaacc ccagagtccc gctcagaaga actcgtcaag
     1861 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     1921 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     1981 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2041 ttccaccatg atattcggca agcaggcatc gccatgtgtc acgacgagat cctcgccgtc
     2101 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     2161 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2221 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2281 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2341 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2401 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctggag
     2461 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2521 cagccgaaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2581 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatccccat
     2641 ggtcgatcga cagatctgcg aaagctcgag agagatagat ttgtagagag agactggtga
     2701 tttcagcgtg tcctctccaa atgaaatgaa cttccttata tagaggaagg gtcttgcgaa
     2761 ggatagtggg attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc
     2821 tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca
     2881 tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga
     2941 tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct
     3001 gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc
     3061 ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc
     3121 accatgttca catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac
     3181 gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac
     3241 gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt
     3301 ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac
     3361 cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt
     3421 ggagtagacg agagtgtcgt gctccaccat gttggcaagc tgctctagcc aatacgcaaa
     3481 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3541 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     3601 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     3661 tttcacacag gaaacagcta tgacatgatt acgaattcga gctcggtacc cggggatcgg
     3721 cgcgccagat ttgccttttc aatttcagaa agaatgctaa cccacagatg gttagagagg
     3781 cttacgcagc aggtatcatc aagacgatct acccgagcaa taatctccag gaaatcaaat
     3841 accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc atcaagaaca
     3901 cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg attcaaggct
     3961 tgcttcacaa accaaggcaa gtaatagaga ttggagtctc taaaaaggta gttcccactg
     4021 aatcaaaggc catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga
     4081 ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca
     4141 acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag
     4201 accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc
     4261 attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca
     4321 aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc
     4381 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt
     4441 cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc
     4501 actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac
     4561 gggggactcc tgcaggtaga tcgctcgtcg acatggataa gaagtactct atcggactcg
     4621 atatcggaac taactctgtg ggatgggctg tgatcaccga tgagtacaag gtgccatcta
     4681 agaagttcaa ggttctcgga aacaccgata ggcactctat caagaaaaac cttatcggtg
     4741 ctctcctctt cgattctggt gaaactgctg aggctaccag actcaagaga accgctagaa
     4801 gaaggtacac cagaagaaag aacaggatct gctacctcca agagatcttc tctaacgaga
     4861 tggctaaagt ggatgattca ttcttccaca ggctcgaaga gtcattcctc gtggaagaag
     4921 ataagaagca cgagaggcac cctatcttcg gaaacatcgt tgatgaggtg gcataccacg
     4981 agaagtaccc tactatctac cacctcagaa agaagctcgt tgattctact gataaggctg
     5041 atctcaggct catctacctc gctctcgctc acatgatcaa gttcagagga cacttcctca
     5101 tcgagggtga tctcaaccct gataactctg atgtggataa gttgttcatc cagctcgtgc
     5161 agacctacaa ccagcttttc gaagagaacc ctatcaacgc ttcaggtgtg gatgctaagg
     5221 ctatcctctc tgctaggctc tctaagtcaa gaaggcttga gaacctcatt gctcagctcc
     5281 ctggtgagaa gaagaacgga cttttcggaa acttgatcgc tctctctctc ggactcaccc
     5341 ctaacttcaa gtctaacttc gatctcgctg aggatgcaaa gctccagctc tcaaaggata
     5401 cctacgatga tgatctcgat aacctcctcg ctcagatcgg agatcagtac gctgatttgt
     5461 tcctcgctgc taagaacctc tctgatgcta tcctcctcag tgatatcctc agagtgaaca
     5521 ccgagatcac caaggctcca ctctcagctt ctatgatcaa gagatacgat gagcaccacc
     5581 aggatctcac acttctcaag gctcttgtta gacagcagct cccagagaag tacaaagaga
     5641 ttttcttcga tcagtctaag aacggatacg ctggttacat cgatggtggt gcatctcaag
     5701 aagagttcta caagttcatc aagcctatcc tcgagaagat ggatggaacc gaggaactcc
     5761 tcgtgaagct caatagagag gatcttctca gaaagcagag gaccttcgat aacggatcta
     5821 tccctcatca gatccacctc ggagagttgc acgctatcct tagaaggcaa gaggatttct
     5881 acccattcct caaggataac agggaaaaga ttgagaagat tctcaccttc agaatccctt
     5941 actacgtggg acctctcgct agaggaaact caagattcgc ttggatgacc agaaagtctg
     6001 aggaaaccat caccccttgg aacttcgaag aggtggtgga taagggtgct agtgctcagt
     6061 ctttcatcga gaggatgacc aacttcgata agaaccttcc aaacgagaag gtgctcccta
     6121 agcactcttt gctctacgag tacttcaccg tgtacaacga gttgaccaag gttaagtacg
     6181 tgaccgaggg aatgaggaag cctgcttttt tgtcaggtga gcaaaagaag gctatcgttg
     6241 atctcttgtt caagaccaac agaaaggtga ccgtgaagca gctcaaagag gattacttca
     6301 agaaaatcga gtgcttcgat tcagttgaga tttctggtgt tgaggatagg ttcaacgcat
     6361 ctctcggaac ctaccacgat ctcctcaaga tcattaagga taaggatttc ttggataacg
     6421 aggaaaacga ggatatcttg gaggatatcg ttcttaccct caccctcttt gaagatagag
     6481 agatgattga agaaaggctc aagacctacg ctcatctctt cgatgataag gtgatgaagc
     6541 agttgaagag aagaagatac actggttggg gaaggctctc aagaaagctc attaacggaa
     6601 tcagggataa gcagtctgga aagacaatcc ttgatttcct caagtctgat ggattcgcta
     6661 acagaaactt catgcagctc atccacgatg attctctcac ctttaaagag gatatccaga
     6721 aggctcaggt ttcaggacag ggtgatagtc tccatgagca tatcgctaac ctcgctggat
     6781 ctcctgcaat caagaaggga atcctccaga ctgtgaaggt tgtggatgag ttggtgaagg
     6841 tgatgggaag gcataagcct gagaacatcg tgatcgaaat ggctagagag aaccagacca
     6901 ctcagaaggg acagaagaac tctagggaaa ggatgaagag gatcgaggaa ggtatcaaag
     6961 agcttggatc tcagatcctc aaagagcacc ctgttgagaa cactcagctc cagaatgaga
     7021 agctctacct ctactacctc cagaacggaa gggatatgta tgtggatcaa gagttggata
     7081 tcaacaggct ctctgattac gatgttgatc atatcgtgcc acagtcattc ttgaaggatg
     7141 attctatcga taacaaggtg ctcaccaggt ctgataagaa caggggtaag agtgataacg
     7201 tgccaagtga agaggttgtg aagaaaatga agaactattg gaggcagctc ctcaacgcta
     7261 agctcatcac tcagagaaag ttcgataact tgactaaggc tgagagggga ggactctctg
     7321 aattggataa ggcaggattc atcaagaggc agcttgtgga aaccaggcag atcactaagc
     7381 acgttgcaca gatcctcgat tctaggatga acaccaagta cgatgagaac gataagttga
     7441 tcagggaagt gaaggttatc accctcaagt caaagctcgt gtctgatttc agaaaggatt
     7501 tccaattcta caaggtgagg gaaatcaaca actaccacca cgctcacgat gcttacctta
     7561 acgctgttgt tggaaccgct ctcatcaaga agtatcctaa gctcgagtca gagttcgtgt
     7621 acggtgatta caaggtgtac gatgtgagga agatgatcgc taagtctgag caagagatcg
     7681 gaaaggctac cgctaagtat ttcttctact ctaacatcat gaatttcttc aagaccgaga
     7741 ttaccctcgc taacggtgag atcagaaaga ggccactcat cgagacaaac ggtgaaacag
     7801 gtgagatcgt gtgggataag ggaagggatt tcgctaccgt tagaaaggtg ctctctatgc
     7861 cacaggtgaa catcgttaag aaaaccgagg tgcagaccgg tggattctct aaagagtcta
     7921 tcctccctaa gaggaactct gataagctca ttgctaggaa gaaggattgg gaccctaaga
     7981 aatacggtgg tttcgattct cctaccgtgg cttactctgt tctcgttgtg gctaaggttg
     8041 agaagggaaa gagtaagaag ctcaagtctg ttaaggaact tctcggaatc actatcatgg
     8101 aaaggtcatc tttcgagaag aacccaatcg atttcctcga ggctaaggga tacaaagagg
     8161 ttaagaagga tctcatcatc aagctcccaa agtactcact cttcgaactc gagaacggta
     8221 gaaagaggat gctcgcttct gctggtgagc ttcaaaaggg aaacgagctt gctctcccat
     8281 ctaagtacgt taactttctt tacctcgctt ctcactacga gaagttgaag ggatctccag
     8341 aagataacga gcagaagcaa cttttcgttg agcagcacaa gcactacttg gatgagatca
     8401 tcgagcagat ctctgagttc tctaaaaggg tgatcctcgc tgatgcaaac ctcgataagg
     8461 tgttgtctgc ttacaacaag cacagagata agcctatcag ggaacaggca gagaacatca
     8521 tccatctctt cacccttacc aacctcggtg ctcctgctgc tttcaagtac ttcgatacaa
     8581 ccatcgatag gaagagatac acctctacca aagaagtgct cgatgctacc ctcatccatc
     8641 agtctatcac tggactctac gagactagga tcgatctctc acagctcggt ggtgattcaa
     8701 gggctgatcc taagaagaag aggaaggttt gactcgagat atgaagatga agatgaaata
     8761 tttggtgtgt caaataaaaa gcttgtgtgc ttaagtttgt gtttttttct tggcttgttg
     8821 tgttatgaat ttgtggcttt ttctaatatt aaatgaatgt aagatcacat tataatgaat
     8881 aaacaaatgt ttctataatc cattgtgaat gttttgttgg atctcttctg cagcatataa
     8941 ctactgtatg tgctatggta tggactatgg aatatgatta aagataagga gctccggtga
     9001 cggacccatg gcttcgttga acaacggaaa ctcgacttgc cttccgcaca atacatcatt
     9061 tcttcttagc tttttttctt cttcttcgtt catacagttt ttttttgttt atcagcttac
     9121 attttcttga accgtagctt tcgttttctt ctttttaact ttccattcgg agtttttgta
     9181 tcttgtttca tagtttgtcc caggattaga atgattaggc atcgaacctt caagaatttg
     9241 attgaataaa acatcttcat tcttaagata tgaagataat cttcaaaagg cccctgggaa
     9301 tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt tcttatatag
     9361 gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa gaaaacgaag
     9421 ctgagtttat atacagctag agtcgaagta gtgattagag gcaggtgccg ctacagggcg
     9481 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     9541 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     9601 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     9661 cactataggg cgaattgggt accgggcccc ccctcgaggt cctccagctt ttgttccctt
     9721 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     9781 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     9841 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccac
     9901 ctgcggtcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     9961 gaaaaagtgg caccgagtcg gtgctttttt tcccggtggc actggccgtc gttttacaac
    10021 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
    10081 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
    10141 gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt tcccgccttc
    10201 agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg
    10261 tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg
    10321 tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc
    10381 gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt
    10441 cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg
    10501 tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca
    10561 tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg
    10621 acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga
    10681 agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc
    10741 ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac
    10801 tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt
    10861 gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg
    10921 agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc
    10981 gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg
    11041 agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc
    11101 gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc
    11161 ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga
    11221 atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt
    11281 cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc
    11341 gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc
    11401 ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg
    11461 tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa
    11521 taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg
    11581 tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat
    11641 gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa
    11701 gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc
    11761 atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg
    11821 tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata
    11881 tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg
    11941 ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt
    12001 gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg
    12061 agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac
    12121 gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat
    12181 gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac
    12241 gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca
    12301 actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca
    12361 agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat
    12421 gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa
    12481 ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta
    12541 agcggctggg ttgcctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg
    12601 gcgtgagcgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc
    12661 tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag gcagaagcac
    12721 gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc cggcaaccgc
    12781 cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa ccagattttt
    12841 tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg
    12901 ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac gagcttccag
    12961 acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg gattacgacc
    13021 tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg gaagggaagg
    13081 gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag ttctgccggc
    13141 gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg ttaaacacca
    13201 cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg acggtatccg
    13261 agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg cggccggagt
    13321 acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc aagaacccgg
    13381 acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc cgttttctct
    13441 accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc aagacgatct
    13501 acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg cgcaagctga
    13561 tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag gctggcccga
    13621 tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt tcctaatgta
    13681 cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa ggtctctttc
    13741 ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg aacccgtaca
    13801 ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact gatataaaag
    13861 agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact cttaaaaccc
    13921 gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa aaagcgccta
    13981 cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg
    14041 gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga caagccgcgc
    14101 cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat
    14161 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
    14221 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
    14281 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
    14341 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
    14401 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
    14461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
    14521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
    14581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
    14641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
    14701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
    14761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
    14821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
    14881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
    14941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
//
