LOCUS       sequence-211913-       16427 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of Cas9 in plants, cauliflower mosaic virus 35S promoter 
            (P35S), hygro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16427)
  AUTHORS   Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, 
            Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X,
            Dong Z, Liu YG
  TITLE     A robust CRISPR/Cas9 system for convenient, high-efficiency 
            multiplex genome editing in monocot and dicot plants.
  JOURNAL   Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007.
  PUBMED    25917172
REFERENCE   2  (bases 1 to 16427)
  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: "Mol Plant. 
            2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 
            10.1016/j.molp.2015.04.007."
COMMENT     Sequence Label: pYLCRISPR/Cas9P35S-H
FEATURES             Location/Qualifiers
     source          1..16427
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    349..370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(385..390,391..408,409..415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    423..439
                     /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     447..463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(804..851)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(852..4952)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(852..4952)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             3758..3766
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             complement(4977..4997)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(5146..5490)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     protein_bind    6003..6024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6039..6044,6045..6062,6063..6069)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6077..6093
                     /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        6135..6153
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6135..6152
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             6227..6529
                     /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRTMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    6739..6772
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6802..6819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6802..6818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6811..6833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    7021..7045
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             8346..8975
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             9404..10477
                     /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      10543..10737
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(10973..10995)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    11081..11221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     11136..11155
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(11236..11253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11407..11995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11487..11506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12082..12876)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    14500..14524
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    14602..14776
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(14817..15842)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(join(15909..16427,1..158))
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
ORIGIN
        1 cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc
       61 caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga
      121 ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga
      181 ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct
      241 gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      301 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      361 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      421 aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta cgaattcgag
      481 ctcggtaccg ctcgctacct taagagagga tatccctcca tcctataatg taggctatag
      541 gaactagggc aaggccggcc ttatgcggcc gcgtcactgg attttggttt taggaattag
      601 aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc
      661 tcaacacatg agcgaaaccc tataagaacc ctaattccct tatctgggaa ctactcacac
      721 attattctgg agaaaaatag agagagatag atttgtagag agagactggt gatttttgcg
      781 gactctagat caggtggatc ctacttcttt ttcttagcct gtccggcctt tttggtggca
      841 gcaggacgct tatcaccacc aagctgggaa aggtcgatac gagtctcgta aagaccggtg
      901 atggactggt ggatgagagt agcgtcgaga acctccttgg tggacgtgta acgcttcctg
      961 tcgatggtgg tgtcgaagta cttgaaagca gcaggggcgc cgaggttcgt gagcgtgaag
     1021 aggtggatga tgttctcggc ctgctcgcgg atgggcttgt cgcggtgctt gttgtaggcg
     1081 gagaggacct tgtcgaggtt agcgtcagcg aggatgacgc gcttggagaa ctcggagatc
     1141 tgctcgatga tctcgtcgag gtagtgcttg tgctgctcca cgaagagctg cttctgctcg
     1201 ttatcctcag gggaaccctt gagcttctcg tagtgggagg cgaggtagag gaagttcacg
     1261 tacttggaag gaagagcaag ctcgttaccc ttctggagct caccagcgga agccagcatc
     1321 ctcttacgac cgttctcgag ctcgaaaaga gagtacttgg ggagcttgat gatgaggtcc
     1381 ttcttgacct ccttgtagcc cttggcctcg aggaagtcga tcgggttctt ctcgaaggag
     1441 gagcgctcca tgatggtgat gccgaggagc tccttgacgg acttgagctt cttcgactta
     1501 cccttctcca ccttggcgac cacgaggacg gagtaggcga cagtagggga gtcgaaacca
     1561 ccgtacttct tagggtccca atccttcttc ctagcgatga gcttgtccga gtttctcttt
     1621 ggaaggatag actccttgga gaagccaccg gtctggacct cggtcttctt gacgatgtta
     1681 acctgaggca tagaaaggac cttgcgaaca gtagcgaagt cgcgaccctt gtcccaaacg
     1741 atctcaccag tctcaccgtt cgtctcgata agagggcgct tgcggatctc gccgttggcg
     1801 agggtgatct cggtcttgaa gaagttcatg atgttggagt agaagaagta cttggcggtg
     1861 gccttgccga tctcctgctc ggacttggcg atcatcttac gaacgtcgta gaccttgtag
     1921 tcaccgtaga cgaactcgga ctcaagctta gggtacttct tgataagagc ggtaccaacg
     1981 acagcgttaa ggtaagcatc gtgagcgtgg tggtagttgt tgatctcgcg gaccttgtag
     2041 aactggaagt ccttgcggaa gtcggagacg agcttggact tgagggtgat caccttgacc
     2101 tcgcggatga gcttgtcgtt ctcgtcgtac ttggtgttca tcctagaatc gaggatctga
     2161 gcaacgtgct tggtaatctg cctcgtctca acaagctgcc tcttgatgaa accagccttg
     2221 tcaagctcgg aaaggccacc cctctcagcc ttcgtgaggt tgtcgaactt cctctgggta
     2281 atgagcttag cgttgagaag ctgcctccag tagttcttca tcttcttgac aacctcctcg
     2341 gaagggacgt tgtccgactt acccctgttc ttgtcggacc tcgtgaggac cttgttgtcg
     2401 atggagtcat ccttaaggaa agactgagga acaatgtggt cgacgtcgta gtcagaaagc
     2461 ctgttgatgt cgagctcctg gtcaacgtac atatccctac cgttctggag gtagtagagg
     2521 tagagcttct cgttctggag ctgggtgttc tcgacagggt gctccttaag gatctgagaa
     2581 ccaagctcct tgataccctc ctcaatcctc ttcatgcgct ccctcgagtt cttctgaccc
     2641 ttctgggtag tctggttctc acgagccatc tcgatgacga tgttctcagg cttgtgacga
     2701 cccataacct tgacaagctc atcgacaacc ttaacagtct gaaggatgcc cttcttgata
     2761 gcaggggaac cagcaaggtt agcaatgtgc tcgtggagag agtcgccctg accggacacc
     2821 tgagccttct gaatatcctc cttgaaggta agagagtcat cgtggatgag ctgcatgaag
     2881 ttcctgttag cgaaaccatc agacttgagg aagtcgagga tagtcttgcc gctctgctta
     2941 tccctgatac cgttgatgag cttgcgggag agcctacccc aaccggtgta acggcgacgc
     3001 ttgagctgct tcataacctt gtcatcgaag agatgagcgt aagtcttgag cctctcctcg
     3061 atcatctccc tatcctcgaa gagagtaaga gtgaggacga tgtcctcgag gatgtcctcg
     3121 ttctcctcgt tgtcgaggaa gtccttgtcc ttgatgatct tgaggagatc gtggtaggta
     3181 ccgagagaag cgttgaaacg gtcctcaacg ccgctgatct cgacggagtc gaagcactcg
     3241 atcttcttga agtagtcctc cttgagctgc ttgacggtga ccttgcggtt ggtcttgaag
     3301 aggaggtcaa cgatagcctt cttctgctcg ccggagagga aggcaggctt gcgcataccc
     3361 tcggtgacgt acttgacctt ggtgagctcg ttgtagaccg tgaagtactc gtagaggagg
     3421 gagtgcttgg ggaggacctt ctcgttgggg aggttcttgt cgaagttggt catgcgctcg
     3481 atgaaggact gggcggaagc acccttgtca acaacctcct cgaagttcca aggggtgata
     3541 gtctcctcgg acttcctagt catccaagcg aaacgggagt taccacgagc aagaggacca
     3601 acgtagtaag gaatacggaa agtaaggatc ttctcgatct tctcacggtt gtccttgagg
     3661 aaagggtaga agtcctcctg cctacgaagg atggcgtgaa gctcaccaag gtggatctgg
     3721 tgagggatag agccgttatc gaaagtcctc tgcttcctaa ggaggtcctc acggttaagc
     3781 ttaacgagaa gctcctcagt accatccatc ttctcaagga ttggcttgat gaacttgtag
     3841 aactcctcct ggctagctcc accgtcaatg taaccggcgt agccgttctt ggactggtcg
     3901 aagaagatct ccttgtactt ctcggggagc tgctgacgaa caagagcctt gaggagggtg
     3961 aggtcctggt ggtgctcgtc gtagcgcttg atcatggaag cagaaagagg agccttagtg
     4021 atctcagtgt taaccctaag gatatccgaa aggaggatag catcggagag gttcttagca
     4081 gcaaggaaga gatcagcgta ctgatctcca atctgagcga ggaggttgtc gagatcatcg
     4141 tcgtaggtat ccttggagag ctgaagctta gcgtcctcag cgaggtcgaa gttggacttg
     4201 aagttagggg tcagaccgag ggagagagcg atgaggttac cgaaaagacc gttcttcttc
     4261 tcaccaggga gctgggcgat gaggttctcg agacgcctgg acttggagag cctagcggaa
     4321 aggatcgcct tagcgtcgac accggaagcg ttgatagggt tctcctcgaa gagctggttg
     4381 taggtctgga cgagctggat gaagagcttg tccacgtcgg agttatcagg gttaaggtca
     4441 ccctcgataa ggaagtgacc acggaacttg atcatgtgag cgagagcaag gtagatgaga
     4501 cgaagatcag ccttatcagt agagtcaaca agcttcttac gaaggtggta gatagtgggg
     4561 tacttctcgt ggtaggcgac ctcgtcgacg atgttgccga agatggggtg gcgctcgtgc
     4621 ttcttgtcct cctccacgag gaaggactcc tcgaggcggt ggaagaagga gtcgtcgacc
     4681 ttcgccatct cgttggagaa gatctcctgg aggtagcaga tgcggttctt gcggcgggtg
     4741 tagcggcggc gggcggtgcg cttgaggcgg gtcgcctccg ccgtctcgcc ggagtcgaag
     4801 aggagggcgc cgatgaggtt cttcttgatg gagtggcggt cggtgttgcc caggaccttg
     4861 aacttcttgg acgggacctt gtactcgtcg gtgatcaccg cccagccgac gctgttggtg
     4921 ccgatgtcga ggccgatgga gtacttcttg tcagccgcag gcaccccgtg aataccaacc
     4981 ttccgcttct tcttaggagc catggctatc gttcgtaaat ggtgaaaatt ttcagaaaat
     5041 tgcttttgct ttaaaagaaa tgatttaaat tgctgcaata gaagtagaat gcttgattgc
     5101 ttgagattcg tttgttttgt atatgttgtg ttgagaactc tcgacgtcct ctccaaatga
     5161 aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc
     5221 cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct
     5281 tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag
     5341 gcatcttcaa cgatggcctt tcctttatcg caatgatggc atttgtagga gccaccttcc
     5401 ttttccacta tcttcacaat aaagtgacag atagctgggc aatggaatcc gaggaggttt
     5461 ccggatatta ccctttgttg aaaagtctca attgcccttt ggtcttctga gactgtatct
     5521 ttgatatttt tggagtagac aagtgtgtcg tgctccacca tgttatcaca tcaatccact
     5581 tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt gggtgggggt
     5641 ccatctttgg gaccactgtc ggcagaggca tcttcaacga tggcctttcc tttatcgcaa
     5701 tgatggcatt tgtaggagcc accttccttt tccactatct tcacaataaa gtgacagata
     5761 gctgggcaat ggaatccgag gaggtttccg gatattaccc tttgttgaaa agtctcaatt
     5821 gccctttggt cttctgagac tgtatctttg atatttttgg agtagacaag tgtgtcgtgc
     5881 tccacatgtt gaccggtaag gcgcgccgta gtgctcgaga gacctctgaa gtggccgatt
     5941 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     6001 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     6061 cgtatgttgt gtggaattgt gagcggataa caatttcaca caagaaacag ctatgaccat
     6121 gattacgcca agctatttag gtgacactat agaatactca agctatgcat caagctcaat
     6181 gggtctagtc tgtagatacc catcacactg gcgaccgctc gaacatcagt ttaaggttta
     6241 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
     6301 cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
     6361 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca cgatgaccac
     6421 cgatatggcc agtgtgcctg tctccgttat cggggaagaa gtggctgatc tcagccaccg
     6481 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcct
     6541 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtga gcgtgggtct
     6601 cgcggtatca ttggcgcgcc tctcgagcta gcggccgcat gcatcgatct cctacatcgt
     6661 ataaattagc ctatacgaag ttattgcatc tatgtcgggt gcggagaaag aggtaatgaa
     6721 atggcagtat tagatctgat aacttcgtat aatgtatgct atacgaagtt atgactgcaa
     6781 gtcgacaccc ataagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6841 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6901 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcta
     6961 gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt
     7021 tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat
     7081 atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca
     7141 gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg
     7201 cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt cctaagttac
     7261 gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat
     7321 aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc
     7381 tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca accaacgggc
     7441 cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg
     7501 cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt
     7561 gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat
     7621 ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc
     7681 ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat
     7741 catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc
     7801 ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg
     7861 ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc
     7921 acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga
     7981 ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca
     8041 agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag
     8101 gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg
     8161 ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg
     8221 gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc
     8281 ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg
     8341 aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg
     8401 caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg
     8461 atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg
     8521 tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg
     8581 tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg
     8641 acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg
     8701 tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg
     8761 tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt
     8821 acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg
     8881 ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg
     8941 cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag
     9001 caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac
     9061 gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga
     9121 ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct
     9181 atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa
     9241 ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg
     9301 aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgcctgcc
     9361 ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc
     9421 atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc
     9481 cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca
     9541 agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc
     9601 gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga
     9661 cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg
     9721 tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc
     9781 cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc
     9841 ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt
     9901 gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca
     9961 gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg
    10021 tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag
    10081 ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc
    10141 tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc
    10201 cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc
    10261 cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc
    10321 cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc
    10381 ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg
    10441 caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca
    10501 aattgcccta gcaggggaaa aaggtcgaaa agttctcttt cctgtggata gcacgtacat
    10561 tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta
    10621 cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc
    10681 cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact
    10741 gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc
    10801 cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg
    10861 cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg
    10921 gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
    10981 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
    11041 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
    11101 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
    11161 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
    11221 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
    11281 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
    11341 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
    11401 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
    11461 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
    11521 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
    11581 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
    11641 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
    11701 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
    11761 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
    11821 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
    11881 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
    11941 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
    12001 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
    12061 tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt
    12121 ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc
    12181 gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc
    12241 gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc
    12301 tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc
    12361 atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc
    12421 ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt
    12481 atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc
    12541 gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc
    12601 ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac
    12661 aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt
    12721 ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt
    12781 atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag
    12841 ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta
    12901 cagtatttaa agatacccca agaagctaag gaaggtgcga acaagtccct gatatgagat
    12961 catgtttgtc atctggagcc atagaacagg gttcatcatg agtcatcaac ttaccttcgc
    13021 cgacagtgaa ttcagcagta agcgccgtca gaccagaaaa gagattttct tgtcccgcat
    13081 ggagcagatt ctgccatggc aaaacatggt ggaagtcatc gagccgtttt accccaaggc
    13141 tggtaatggc cggcgacctt atccgctgga aaccatgcta cgcattcact gcatgcagca
    13201 ttggtacaac ctgagcgatg gcgcgatgga agatgctctg tacgaaatcg cctccatgcg
    13261 tctgtttgcc cggttatccc tggatagcgc cttgccggac cgcaccacca tcatgaattt
    13321 ccgccacctg ctggagcagc atcaactggc ccgccaattg ttcaagacca tcaatcgctg
    13381 gctggccgaa gcaggcgtca tgatgactca aggcaccttg gtcgatgcca ccatcattga
    13441 ggcacccagc tcgaccaaga acaaagagca gcaacgcgat ccggagatgc atcagaccaa
    13501 gaaaggcaat cagtggcact ttggcatgaa ggcccacatt ggtgtcgatg ccaagagtgg
    13561 cctgacccac agcctggtca ccaccgcggc caacgagcat gacctcaatc agctgggtaa
    13621 tctgctgcat ggagaggagc aatttgtctc agccgatgcc ggctaccaag gggcgccaca
    13681 gcgcgaggag ctggccgagg tggatgtgga ctggctgatc gccgagcgcc ccggcaaggt
    13741 aagaaccttg aaacagcatc cacgcaagaa caaaacggcc atcaacatcg aatacatgaa
    13801 agccagcatc cgggccaggg tggagcaccc atttcgcatc atcaagcgac agttcggctt
    13861 cgtgaaagcc agatacaagg ggttgctgaa aaacgataac caactggcga tgttattcac
    13921 gctggccaac ctgtttcggg cggaccaaat gatacgtcag tgggagagat ctcactaaaa
    13981 actggggata acgccttaaa tggcgaagaa acggtctaaa taggctgatt caaggcattt
    14041 acgggagaaa aaatcggctc aaacatgaag aaatgaaatg actgagtcag ccgagaagaa
    14101 tttccccgct tattcgcacc ttccctaatt ataacaagac gaactccaat tcactgttcc
    14161 ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg
    14221 cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc
    14281 tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg
    14341 cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac
    14401 aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt
    14461 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
    14521 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgaat
    14581 taacgccgaa ttaattcggg ggatctggat tttagtactg gattttggtt ttaggaatta
    14641 gaaattttat tgatagaagt attttacaaa tacaaataca tactaagggt ttcttatatg
    14701 ctcaacacat gagcgaaacc ctataggaac cctaattccc ttatctggga actactcaca
    14761 cattattatg gagaaactcg agcttgtcga tcgacagatc ccggtcggca tctactctat
    14821 ttctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca
    14881 cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg
    14941 gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
    15001 ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagt
    15061 cgtggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
    15121 caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac
    15181 atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg
    15241 gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
    15301 agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag
    15361 tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
    15421 attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc
    15481 gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct
    15541 tgcaacgtga caccctgtga acggcgggag atgcaatagg tcaggctctc gctaaactcc
    15601 ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa
    15661 cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct
    15721 acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg
    15781 ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc
    15841 atatctcatt gccccccgga tctgcgaaag ctcgagagag atagatttgt agagagagac
    15901 tggtgatttc agcgtgtcct ctccaaatga aatgaacttc cttatataga ggaagggtct
    15961 tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc
    16021 acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctctt cgtgggtggg
    16081 ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg
    16141 caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag
    16201 atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca
    16261 atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg
    16321 tgctccacca tgttcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt
    16381 ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgt
//
