LOCUS       sequence-453880-       16738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPR/Cas-mediated gene editing in model fern species, 
            Ceratopteris richardii; expresses guide RNAs from CrActin promoter 
            and Cas9 from maize ubiquitin (ZmUbi) promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16738)
  AUTHORS   Jiang W, Deng F, Babla M, Chen C, Yang D, Tong T, Qin Y, Chen G, 
            Marchant B, Soltis P, Soltis DE, Zeng F, Chen ZH
  TITLE     Efficient gene editing of a model fern species through 
            gametophyte-based transformation.
  JOURNAL   Plant Physiol. 2024 Sep 12:kiae473. doi: 10.1093/plphys/kiae473.
  PUBMED    39268871
REFERENCE   2  (bases 1 to 16738)
  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 
            Physiol. 2024 Sep 12:kiae473. doi: 10.1093/plphys/kiae473."
COMMENT     Sequence Label: pRGEB32-CrActin-UBI
FEATURES             Location/Qualifiers
     source          1..16738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             26..49
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      71..323
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(332..349)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(332..348)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(341..363)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    551..575
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1876..2505
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2934..4007
                     /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      4073..4267
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4503..4525)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4611..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4666..4685
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4766..4783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4937..5525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5017..5036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5612..6406)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6831..6855
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    6933..7107
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(7148..8173)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(8240..8916)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(8283..8303)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    9107..9128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9143..9148,9149..9166,9167..9173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9181..9197
                     /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     9186..9208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9205..9221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9205..9221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        10233..10308
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      10382..10634
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        10642..12351
                     /gene="RUBQ2"
                     /label=Ubi promoter
                     /note="promoter for the rice (Oryza sativa) polyubiquitin 
                     gene RUBQ2"
     intron          11390..12351
                     /note="intron upstream of the coding sequence for the rice 
                     polyubiquitin gene RUBQ2"
     protein_bind    12362..12386
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             12431..12496
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             12503..12523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12548..16648
                     /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             12548..16648
                     /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             16649..16696
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(join(16729..16738,1..15))
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc
       61 gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc
      121 cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa
      181 catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata
      241 catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
      301 ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacaa cgtcgtgact
      361 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
      421 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
      481 gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac
      541 tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga
      601 ataatcggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat
      661 gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata
      721 gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt
      781 cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt
      841 ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga
      901 gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca
      961 accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg
     1021 gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg
     1081 ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg
     1141 ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca
     1201 ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc
     1261 gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga
     1321 agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg
     1381 accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc
     1441 tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg
     1501 ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc
     1561 aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga
     1621 agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc
     1681 aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc
     1741 ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga
     1801 gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat
     1861 acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag
     1921 acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta
     1981 gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg
     2041 ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg
     2101 cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc
     2161 aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc
     2221 gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg
     2281 gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg
     2341 ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca
     2401 ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc
     2461 gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag
     2521 agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca
     2581 aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt
     2641 tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta
     2701 ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg
     2761 agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc
     2821 gacgccgtgg aatgccccat gtgttgagga acgggcggtt ggccaggcgt aagcggctgg
     2881 gttgcctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgagcg
     2941 gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga
     3001 agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg
     3061 aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg
     3121 gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga
     3181 tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc
     3241 tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg
     3301 tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga
     3361 tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc
     3421 ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg
     3481 gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg
     3541 ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag
     3601 ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga
     3661 tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga
     3721 cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg
     3781 cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca
     3841 gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa
     3901 atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca
     3961 tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga
     4021 tgctagggca aattgcccta gcaggggaaa aaggtcgaaa agcactcttt cctgtgaata
     4081 gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc
     4141 caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag
     4201 gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct
     4261 gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt
     4321 cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa
     4381 aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac
     4441 tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa
     4501 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4561 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4621 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4681 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     4741 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5341 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5581 gttaagggat tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat
     5641 attttatttt ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact
     5701 gttcttcccc gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt
     5761 ccgccctgcc gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga
     5821 tgttgctgtc tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct
     5881 ttaaaaaatc atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc
     5941 aatccacatc ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta
     6001 agctattcgt atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg
     6061 catacagctc gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga
     6121 cgccatcggc ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga
     6181 cctttggaac aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat
     6241 cataggtggt ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc
     6301 ccaccagctt atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt
     6361 tttcgatcag ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc
     6421 ctcttttcta cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa
     6481 ttcactgttc cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt
     6541 ttcaaagttg gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca
     6601 ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt
     6661 ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc
     6721 tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg
     6781 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata
     6841 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta
     6901 atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt
     6961 tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg
     7021 tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg
     7081 aactactcac acattattat ggagaaactc gagcttgtcg atcgacagat cccggtcggc
     7141 atctactcta tttctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga
     7201 gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc
     7261 cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat
     7321 catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat
     7381 acgcccggag tcgtggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct
     7441 gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg
     7501 aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca
     7561 ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg
     7621 cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca
     7681 cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag
     7741 tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg
     7801 ccgcagcgat cgcatccata gcctccgcga ccggttgtag aacagcgggc agttcggttt
     7861 caggcaggtc ttgcaacgtg acaccctgtg aacggcggga gatgcaatag gtcaggctct
     7921 cgctaaactc cccaatgtca agcacttccg gaatcgggag cgcggccgat gcaaagtgcc
     7981 gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc aggacatatc
     8041 cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag agctgcatca
     8101 ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc gcggtgagtt
     8161 caggcttttt catatctcat tgccccccgg atctgcgaaa gctcgagaga gatagatttg
     8221 tagagagaga ctggtgattt cagcgtgtcc tctccaaatg aaatgaactt ccttatatag
     8281 aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat
     8341 cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc
     8401 tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct
     8461 ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga
     8521 tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt
     8581 gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga
     8641 cgagagtgtc gtgctccacc atgttcacat caatccactt gctttgaaga cgtggttgga
     8701 acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg
     8761 gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt gtaggtgcca
     8821 ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg gaatccgagg
     8881 aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc ttctgagact
     8941 gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt ggcaagctgc
     9001 tctagccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     9061 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     9121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     9181 ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg ccaagctttc
     9241 tcatattgct gcttattcta taaaataaat gaaaattgtg tagaaatctg ctaatatggt
     9301 ctagatctct ttaattctgc ctttacatac cttcactatt tagcttagat tgtgttttct
     9361 gtgtttcgta taagggatct tttttatttt cgtgctgctt agcgttcaac tcaggtttta
     9421 aatttgtttt atcaactagg tccattatct aacaaagcta agggaaaatg gctgatgtgg
     9481 acgaagttca accgcttgtt tgtgataatg gttctgggat ggtcaaggta atgccaaagt
     9541 tttgtctggt ttcttgtcta ttgcttcatg tacaacaaca acacatccat tttcttcaat
     9601 tcaggctgga tttgctggcg atgatgcccc tcgggctgta tttcctagca ttgtgggtcg
     9661 tcctagacat actggggtca tggttggtat gggccaaaaa gatgcatatg ttggtgatga
     9721 ggctcaatca aagcgtggga ttcttacatt gaagtaccct atcgagcatg gtattgtgac
     9781 gaattgggat gatatggaga agatatggca ccatactttc tataatgagc tacgtgttgc
     9841 tccagaggag catccagttc tgctcactga agctccattg aatccgaagg ccaatcgaga
     9901 aaaaatgaca cagatcatgt ttgacacgtt caatgctcca gctatgtatg ttgcaattca
     9961 agctgtgtta tcactttatg caagtggaag gaccacaggt atgtatttta tgtctttcct
    10021 tgaatgctat ttgtatttca acaaatacat atataatttc taagtccaaa aaaatcatgg
    10081 gttgatgagc ttctcaggta tcgtgctgga ttctggtgat ggagtcacgc acacagtgcc
    10141 catttatgag ggttatgcat tgccccatgc tatcctgcga ctggatcttg caggtcggga
    10201 tttaactgat gctcttggag accgaggtct cggttttaga gctagaaata gcaagttaaa
    10261 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta
    10321 gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc atgcctgcag
    10381 ggatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
    10441 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
    10501 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
    10561 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
    10621 tatgttacta gatctccaca aattcgggtc aaggcggaag ccagcgcgcc accccacgtc
    10681 agcaaatacg gaggcgcggg gttgacggcg tcacccggtc ctaacggcga ccaacaaacc
    10741 agccagaaga aattacagta aaaaaaaagt aaattgcact ttgatccacc ttttattacc
    10801 taagtctcaa tttggatcac ccttaaacct atcttttcaa tttgggccgg gttgtggttt
    10861 ggactaccat gaacaacttt tcgtcatgtc taacttccct ttcagcaaac atatgaacca
    10921 tatatagagg agatcggccg tatactagag ctgatgtgtt taaggtcgtt gattgcacga
    10981 gaaaaaaaaa tccaaatcgc aacaatagca aatttatctg gttcaaagtg aaaagatatg
    11041 tttaaaggta gtccaaagta aaacttatag ataataaaat gtggtccaaa gcgtaattca
    11101 ctcaaaaaaa atcaacgaga cgtgtaccaa acggagacaa acggcatctt ctcgaaattt
    11161 cccaaccgct cgctcgcccg cctcgtcttc ccggaaaccg cggtggtttc agcgtggcgg
    11221 attctccaag cagacggaga cgtcacggca cgggactcct cccaccaccc aaccgccata
    11281 aataccagcc ccctcatctc ctctcctcgc atcagctcca cccccgaaaa atttctcccc
    11341 aatctcgcga ggctctcgtc gtcgaatcga atcctctcgc gtcctcaagg tacgctgctt
    11401 ctcctctcct cgcttcgttt cgattcgatt tcggacgggt gaggttgttt tgttgctaga
    11461 tccgattggt ggttagggtt gtcgatgtga ttatcgtgag atgtttaggg gttgtagatc
    11521 tgatggttgt gatttgggca cggttggttc gataggtgga atcgtggtta ggttttggga
    11581 ttggatgttg gttctgatga ttggggggaa tttttacggt tagatgaatt gttggatgat
    11641 tcgattgggg aaatcggtgt agatctgttg gggaattgtg gaactagtca tgcctgagtg
    11701 attggtgcga tttgtagcgt gttccatctt gtaggccttg ttgcgagcat gttcagatct
    11761 actgttccgc tcttgattga gttattggtg ccatgggttg gtgcaaacac aggctttaat
    11821 atgttatatc tgttttgtgt ttgatgtaga tctgtagggt agttcttctt agacatggtt
    11881 caattatgta gcttgtgcgt ttcgatttga tttcatatgt tcacagatta gataatgatg
    11941 aactctttta attaattgtc aatggtaaat aggaagtctt gtcgctatat ctgtcataat
    12001 gatctcatgt tactatctgc cagtaattta tgctaagaac tatattagaa tatcatgtta
    12061 caatctgtag taatatcatg ttacaatctg tagttcatct atataatcta ttgtggtaat
    12121 ttctttttac tatctgtgtg aagattattg ccactagttc attctactta tttctgaagt
    12181 tcaggatacg tgtgctgtta ctacctatct gaatacatgt gtgatgtgcc tgttactatc
    12241 tttttgaata catgtatgtt ctgttggaat atgtttgctg tttgatccgt tgttgtgtcc
    12301 ttaatcttgt gctagttctt accctatctg tttggtgatt atttcttgca gatagttatc
    12361 aacaagtttg tacaaaaaag caggcttcga aggagataga accaattctc taaggaaata
    12421 cttaaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
    12481 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
    12541 agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
    12601 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
    12661 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
    12721 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
    12781 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
    12841 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
    12901 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
    12961 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
    13021 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
    13081 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
    13141 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
    13201 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
    13261 aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
    13321 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
    13381 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
    13441 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
    13501 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
    13561 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
    13621 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
    13681 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
    13741 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
    13801 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
    13861 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
    13921 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
    13981 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
    14041 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
    14101 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
    14161 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
    14221 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
    14281 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
    14341 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
    14401 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
    14461 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
    14521 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
    14581 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
    14641 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
    14701 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
    14761 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
    14821 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
    14881 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
    14941 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
    15001 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
    15061 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
    15121 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
    15181 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
    15241 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
    15301 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
    15361 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
    15421 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
    15481 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
    15541 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
    15601 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
    15661 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
    15721 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
    15781 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
    15841 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
    15901 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
    15961 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
    16021 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
    16081 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
    16141 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
    16201 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
    16261 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
    16321 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
    16381 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
    16441 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
    16501 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
    16561 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
    16621 gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
    16681 ggcaaaaaag aaaaagtaag aattcgcggc cgcactcgag atatctagac ccagcttt
//
