LOCUS       sequence-453879-       16023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPR/Cas-mediated gene editing in model fern species, 
            Ceratopteris richardii; expresses guide RNAs from CrU6-2 promoter 
            and Cas9 from maize ubiquitin (ZmUbi) promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16023)
  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 16023)
  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-CrU6-2-UBI
FEATURES             Location/Qualifiers
     source          1..16023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1097..1726
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2155..3228
                     /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      3294..3488
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3724..3746)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3832..3972
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3887..3906
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3987..4004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4158..4746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4238..4257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4833..5627)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6052..6076
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    6154..6328
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(6369..7394)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(7461..8137)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(7504..7524)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    8328..8349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8364..8369,8370..8387,8388..8394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8402..8418
                     /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     8407..8429
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8426..8442
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8426..8442
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        8992..9067
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        9148..10857
                     /gene="RUBQ2"
                     /label=Ubi promoter
                     /note="promoter for the rice (Oryza sativa) polyubiquitin 
                     gene RUBQ2"
     intron          9896..10857
                     /note="intron upstream of the coding sequence for the rice 
                     polyubiquitin gene RUBQ2"
     protein_bind    10868..10892
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             10937..11002
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             11009..11029
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11054..15154
                     /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             11054..15154
                     /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             15155..15202
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(15235..15259)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             15270..15293
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      15315..15567
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(15576..15593)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(15576..15592)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(15585..15607)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    15795..15819
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt
       61 tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag
      121 agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc
      181 aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc
      241 ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac
      301 gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt
      361 gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac
      421 accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag
      481 cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg
      541 aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc
      601 gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc
      661 ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt
      721 gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc
      781 caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg
      841 aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct
      901 caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc
      961 cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg
     1021 agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa
     1081 tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa
     1141 gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt
     1201 agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc
     1261 gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg
     1321 gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat
     1381 caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac
     1441 cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc
     1501 ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc
     1561 gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc
     1621 aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg
     1681 cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa
     1741 gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc
     1801 aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag
     1861 ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt
     1921 accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat
     1981 gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac
     2041 cgacgccgtg gaatgcccca tgtgttgagg aacgggcggt tggccaggcg taagcggctg
     2101 ggttgcctgc cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgagc
     2161 ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag
     2221 aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt
     2281 gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc
     2341 ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg
     2401 atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt
     2461 ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac
     2521 gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg
     2581 atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag
     2641 cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat
     2701 ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt
     2761 gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa
     2821 gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag
     2881 atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg
     2941 acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg
     3001 gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc
     3061 agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca
     3121 aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc
     3181 atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag
     3241 atgctagggc aaattgccct agcaggggaa aaaggtcgaa aagcactctt tcctgtgaat
     3301 agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac
     3361 ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa
     3421 ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc
     3481 tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg
     3541 tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca
     3601 aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca
     3661 ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga
     3721 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3781 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     3841 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     3901 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     3961 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4021 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4081 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4561 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4801 cgttaaggga ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa
     4861 tattttattt tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac
     4921 tgttcttccc cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg
     4981 tccgccctgc cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag
     5041 atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc
     5101 tttaaaaaat catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg
     5161 caatccacat cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct
     5221 aagctattcg tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc
     5281 gcatacagct cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg
     5341 acgccatcgg cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg
     5401 acctttggaa caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca
     5461 tcataggtgg tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct
     5521 cccaccagct tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat
     5581 ttttcgatca gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt
     5641 cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca
     5701 attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt
     5761 tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac
     5821 aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg
     5881 tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt
     5941 ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc
     6001 gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat
     6061 atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt
     6121 aatgtactga attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg
     6181 ttttaggaat tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg
     6241 gtttcttata tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg
     6301 gaactactca cacattatta tggagaaact cgagcttgtc gatcgacaga tcccggtcgg
     6361 catctactct atttctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg
     6421 agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc
     6481 ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca
     6541 tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata
     6601 tacgcccgga gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc
     6661 tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg
     6721 gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc
     6781 aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg
     6841 gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc
     6901 acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta
     6961 gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg
     7021 gccgcagcga tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt
     7081 tcaggcaggt cttgcaacgt gacaccctgt gaacggcggg agatgcaata ggtcaggctc
     7141 tcgctaaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc
     7201 cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat
     7261 ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc
     7321 aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt
     7381 tcaggctttt tcatatctca ttgccccccg gatctgcgaa agctcgagag agatagattt
     7441 gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata
     7501 gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata
     7561 tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc
     7621 ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc
     7681 tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg
     7741 atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt
     7801 tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag
     7861 acgagagtgt cgtgctccac catgttcaca tcaatccact tgctttgaag acgtggttgg
     7921 aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc
     7981 ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc
     8041 accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag
     8101 gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac
     8161 tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tggcaagctg
     8221 ctctagccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     8281 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     8341 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     8401 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gccaagcttt
     8461 tagggttata ggtgtatgta tatacgtgcg gagcggacaa aaagagctcc taaagaggga
     8521 agaacgtgag ggggtggtgc tcagtgttga actttctatg aatgtagttt gttgtaaaga
     8581 tgctttctag aattggaatt ggagcttttt ctaggattcg agggtttgtg gaattggaat
     8641 tggagctatt ttttaagata taatcttgtt tctggggcat ttgactccca tagctccata
     8701 tgttttgagg ttctcggtgc gtttccctct tcgtgatggt gtttgtcgta ctacctttga
     8761 tctcactgtt ttgttcgtcc gtgtattgca gaggggtttt gtgccgattg aaaagctgtg
     8821 gcattttagt gtgttggtgt gtcgactgag ggtagttcaa cggaagaggc gcgcgtggat
     8881 aggaattatt aataatgtgg ggaggagaaa acccacatgt gaatcacaac cctgaaagat
     8941 gagcctacat attggccggc cagcgattgg ctgctggaga ccgaggtctc ggttttagag
     9001 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     9061 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt
     9121 agcgcgtgca tgcctgcagg tccacaaatt cgggtcaagg cggaagccag cgcgccaccc
     9181 cacgtcagca aatacggagg cgcggggttg acggcgtcac ccggtcctaa cggcgaccaa
     9241 caaaccagcc agaagaaatt acagtaaaaa aaaagtaaat tgcactttga tccacctttt
     9301 attacctaag tctcaatttg gatcaccctt aaacctatct tttcaatttg ggccgggttg
     9361 tggtttggac taccatgaac aacttttcgt catgtctaac ttccctttca gcaaacatat
     9421 gaaccatata tagaggagat cggccgtata ctagagctga tgtgtttaag gtcgttgatt
     9481 gcacgagaaa aaaaaatcca aatcgcaaca atagcaaatt tatctggttc aaagtgaaaa
     9541 gatatgttta aaggtagtcc aaagtaaaac ttatagataa taaaatgtgg tccaaagcgt
     9601 aattcactca aaaaaaatca acgagacgtg taccaaacgg agacaaacgg catcttctcg
     9661 aaatttccca accgctcgct cgcccgcctc gtcttcccgg aaaccgcggt ggtttcagcg
     9721 tggcggattc tccaagcaga cggagacgtc acggcacggg actcctccca ccacccaacc
     9781 gccataaata ccagccccct catctcctct cctcgcatca gctccacccc cgaaaaattt
     9841 ctccccaatc tcgcgaggct ctcgtcgtcg aatcgaatcc tctcgcgtcc tcaaggtacg
     9901 ctgcttctcc tctcctcgct tcgtttcgat tcgatttcgg acgggtgagg ttgttttgtt
     9961 gctagatccg attggtggtt agggttgtcg atgtgattat cgtgagatgt ttaggggttg
    10021 tagatctgat ggttgtgatt tgggcacggt tggttcgata ggtggaatcg tggttaggtt
    10081 ttgggattgg atgttggttc tgatgattgg ggggaatttt tacggttaga tgaattgttg
    10141 gatgattcga ttggggaaat cggtgtagat ctgttgggga attgtggaac tagtcatgcc
    10201 tgagtgattg gtgcgatttg tagcgtgttc catcttgtag gccttgttgc gagcatgttc
    10261 agatctactg ttccgctctt gattgagtta ttggtgccat gggttggtgc aaacacaggc
    10321 tttaatatgt tatatctgtt ttgtgtttga tgtagatctg tagggtagtt cttcttagac
    10381 atggttcaat tatgtagctt gtgcgtttcg atttgatttc atatgttcac agattagata
    10441 atgatgaact cttttaatta attgtcaatg gtaaatagga agtcttgtcg ctatatctgt
    10501 cataatgatc tcatgttact atctgccagt aatttatgct aagaactata ttagaatatc
    10561 atgttacaat ctgtagtaat atcatgttac aatctgtagt tcatctatat aatctattgt
    10621 ggtaatttct ttttactatc tgtgtgaaga ttattgccac tagttcattc tacttatttc
    10681 tgaagttcag gatacgtgtg ctgttactac ctatctgaat acatgtgtga tgtgcctgtt
    10741 actatctttt tgaatacatg tatgttctgt tggaatatgt ttgctgtttg atccgttgtt
    10801 gtgtccttaa tcttgtgcta gttcttaccc tatctgtttg gtgattattt cttgcagata
    10861 gttatcaaca agtttgtaca aaaaagcagg cttcgaagga gatagaacca attctctaag
    10921 gaaatactta accatggact ataaggacca cgacggagac tacaaggatc atgatattga
    10981 ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg
    11041 agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg
    11101 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
    11161 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
    11221 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
    11281 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
    11341 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
    11401 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
    11461 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
    11521 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
    11581 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
    11641 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
    11701 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
    11761 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
    11821 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
    11881 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
    11941 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
    12001 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
    12061 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
    12121 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
    12181 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
    12241 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
    12301 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
    12361 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
    12421 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
    12481 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
    12541 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
    12601 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
    12661 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
    12721 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
    12781 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
    12841 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
    12901 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
    12961 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
    13021 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
    13081 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
    13141 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
    13201 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
    13261 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
    13321 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
    13381 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
    13441 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
    13501 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
    13561 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
    13621 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
    13681 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
    13741 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
    13801 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
    13861 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
    13921 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
    13981 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
    14041 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
    14101 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
    14161 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
    14221 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
    14281 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
    14341 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
    14401 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
    14461 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
    14521 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
    14581 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
    14641 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
    14701 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
    14761 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
    14821 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
    14881 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
    14941 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
    15001 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
    15061 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
    15121 gacacggatc gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc
    15181 cggccaggca aaaaagaaaa agtaagaatt cgcggccgca ctcgagatat ctagacccag
    15241 ctttcttgta caaagtggtt gataacagcg actacaagga tgacgatgac aaggcttaga
    15301 gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
    15361 ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa
    15421 ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat
    15481 tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc
    15541 gcgcggtgtc atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt
    15601 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
    15661 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
    15721 aatggcgaat gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt
    15781 aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat
    15841 tagaataatc ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt
    15901 gcatgccaac cacagggttc ccctcgggat caaagtactt tgatccaacc cctccgctgc
    15961 tatagtgcag tcggcttctg acgttcagtg cagccgtctt ctgaaaacga catgtcgcac
    16021 aag
//
