LOCUS       sequence-247528-       15357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPR/Cas9 delivery in maize. Expresses Cas9 with rice 
            ubiquitin promoter, BAR with 35S promoter, and sgRNA/PTG with rice 
            snoRNA U3 promoter (pol III). Can produce 1 or more gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15357)
  AUTHORS   Hunter CT
  TITLE     CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize.
  JOURNAL   Plants (Basel). 2021 Apr 8;10(4). pii: plants10040723. doi: 
            10.3390/plants10040723.
  PUBMED    33917747
REFERENCE   2  (bases 1 to 15357)
  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: "Plants 
            (Basel)."; date: "2021-04-8"; volume: "10(4). pii: plants10040723. 
            doi"; pages: " 10.3390/plants10040723"
COMMENT     Sequence Label: pRGEB32-BAR
FEATURES             Location/Qualifiers
     source          1..15357
                     /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(7117..7668)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(7709..8385)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(7752..7772)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    8576..8597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8612..8617,8618..8635,8636..8642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8650..8666
                     /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     8655..8677
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8674..8690
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8674..8690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8708..9088
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     misc_RNA        9105..9180
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        9261..10970
                     /gene="RUBQ2"
                     /label=Ubi promoter
                     /note="promoter for the rice (Oryza sativa) polyubiquitin 
                     gene RUBQ2"
     intron          10009..10970
                     /note="intron upstream of the coding sequence for the rice 
                     polyubiquitin gene RUBQ2"
     protein_bind    10981..11005
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             11050..11115
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             11122..11142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11167..15267
                     /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             11167..15267
                     /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             15268..15315
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    complement(join(15348..15357,1..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 gagcgatcag atctcggtga cgggcaggac
     7141 cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgt catgccagtt
     7201 cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccga gcgcctcgtg
     7261 catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg accacgctct tgaagccctg
     7321 tgcctccagg gacttcagca ggtgggtgta gagcgtggag cccagtcccg tccgctggtg
     7381 gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgc gtgccttcca
     7441 ggggcccgcg taggcgatgc cggcgacctc gccgtccacc tcggcgacga gccagggata
     7501 gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcg gctcggtacg
     7561 gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccg ccggcatgtc
     7621 cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct gggctcatgg ctcgagagag
     7681 atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga aatgaacttc
     7741 cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag
     7801 tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca
     7861 cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttgaa
     7921 cgatagcctt tcctttatcg caatgatggc atttgtaggt gccaccttcc ttttctactg
     7981 tccttttgat gaagtgacag atagctgggc aatggaatcc gaggaggttt cccgatatta
     8041 ccctttgttg aaaagtctca atagcccttt ggtcttctga gactgtatct ttgatattct
     8101 tggagtagac gagagtgtcg tgctccacca tgttcacatc aatccacttg ctttgaagac
     8161 gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga
     8221 ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg
     8281 taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg
     8341 aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct
     8401 tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg
     8461 gcaagctgct ctagccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     8521 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     8581 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     8641 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacgc
     8701 caagcttaag gaatctttaa acatacgaac agatcactta aagttcttct gaagcaactt
     8761 aaagttatca ggcatgcatg gatcttggag gaatcagatg tgcagtcagg gaccatagca
     8821 caagacaggc gtcttctact ggtgctacca gcaaatgctg gaagccggga acactgggta
     8881 cgttggaaac cacgtgatgt gaagaagtaa gataaactgt aggagaaaag catttcgtag
     8941 tgggccatga agcctttcag gacatgtatt gcagtatggg ccggcccatt acgcaattgg
     9001 acgacaacaa agactagtat tagtaccacc tcggctatcc acatagatca aagctgattt
     9061 aaaagagttg tgcagatgat ccgtggcagg agaccgaggt ctcggtttta gagctagaaa
     9121 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     9181 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
     9241 gcatgcctgc aggtccacaa attcgggtca aggcggaagc cagcgcgcca ccccacgtca
     9301 gcaaatacgg aggcgcgggg ttgacggcgt cacccggtcc taacggcgac caacaaacca
     9361 gccagaagaa attacagtaa aaaaaaagta aattgcactt tgatccacct tttattacct
     9421 aagtctcaat ttggatcacc cttaaaccta tcttttcaat ttgggccggg ttgtggtttg
     9481 gactaccatg aacaactttt cgtcatgtct aacttccctt tcagcaaaca tatgaaccat
     9541 atatagagga gatcggccgt atactagagc tgatgtgttt aaggtcgttg attgcacgag
     9601 aaaaaaaaat ccaaatcgca acaatagcaa atttatctgg ttcaaagtga aaagatatgt
     9661 ttaaaggtag tccaaagtaa aacttataga taataaaatg tggtccaaag cgtaattcac
     9721 tcaaaaaaaa tcaacgagac gtgtaccaaa cggagacaaa cggcatcttc tcgaaatttc
     9781 ccaaccgctc gctcgcccgc ctcgtcttcc cggaaaccgc ggtggtttca gcgtggcgga
     9841 ttctccaagc agacggagac gtcacggcac gggactcctc ccaccaccca accgccataa
     9901 ataccagccc cctcatctcc tctcctcgca tcagctccac ccccgaaaaa tttctcccca
     9961 atctcgcgag gctctcgtcg tcgaatcgaa tcctctcgcg tcctcaaggt acgctgcttc
    10021 tcctctcctc gcttcgtttc gattcgattt cggacgggtg aggttgtttt gttgctagat
    10081 ccgattggtg gttagggttg tcgatgtgat tatcgtgaga tgtttagggg ttgtagatct
    10141 gatggttgtg atttgggcac ggttggttcg ataggtggaa tcgtggttag gttttgggat
    10201 tggatgttgg ttctgatgat tggggggaat ttttacggtt agatgaattg ttggatgatt
    10261 cgattgggga aatcggtgta gatctgttgg ggaattgtgg aactagtcat gcctgagtga
    10321 ttggtgcgat ttgtagcgtg ttccatcttg taggccttgt tgcgagcatg ttcagatcta
    10381 ctgttccgct cttgattgag ttattggtgc catgggttgg tgcaaacaca ggctttaata
    10441 tgttatatct gttttgtgtt tgatgtagat ctgtagggta gttcttctta gacatggttc
    10501 aattatgtag cttgtgcgtt tcgatttgat ttcatatgtt cacagattag ataatgatga
    10561 actcttttaa ttaattgtca atggtaaata ggaagtcttg tcgctatatc tgtcataatg
    10621 atctcatgtt actatctgcc agtaatttat gctaagaact atattagaat atcatgttac
    10681 aatctgtagt aatatcatgt tacaatctgt agttcatcta tataatctat tgtggtaatt
    10741 tctttttact atctgtgtga agattattgc cactagttca ttctacttat ttctgaagtt
    10801 caggatacgt gtgctgttac tacctatctg aatacatgtg tgatgtgcct gttactatct
    10861 ttttgaatac atgtatgttc tgttggaata tgtttgctgt ttgatccgtt gttgtgtcct
    10921 taatcttgtg ctagttctta ccctatctgt ttggtgatta tttcttgcag atagttatca
    10981 acaagtttgt acaaaaaagc aggcttcgaa ggagatagaa ccaattctct aaggaaatac
    11041 ttaaccatgg actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa
    11101 gacgatgacg ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca
    11161 gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc
    11221 gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac
    11281 cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc
    11341 gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc
    11401 tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac
    11461 agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc
    11521 ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga
    11581 aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc
    11641 cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc
    11701 gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac
    11761 cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc
    11821 agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga
    11881 aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc
    11941 gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg
    12001 gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc
    12061 atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc
    12121 tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg
    12181 cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac
    12241 gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc
    12301 ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg
    12361 cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg
    12421 cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag
    12481 atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac
    12541 agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag
    12601 gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat
    12661 aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc
    12721 gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc
    12781 ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg
    12841 accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa
    12901 atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa
    12961 attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc
    13021 gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat
    13081 gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg
    13141 ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc
    13201 ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac
    13261 gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc
    13321 ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag
    13381 acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc
    13441 gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag
    13501 agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac
    13561 cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg
    13621 cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac
    13681 catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga
    13741 agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg
    13801 aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat
    13861 ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga
    13921 cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg
    13981 aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag
    14041 tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac
    14101 aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa
    14161 aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg
    14221 aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac
    14281 agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag
    14341 cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat
    14401 tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag
    14461 gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg
    14521 atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg
    14581 gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt
    14641 gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc
    14701 gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct
    14761 aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa
    14821 ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc
    14881 agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg
    14941 gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga
    15001 gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat
    15061 aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga
    15121 gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc
    15181 aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg
    15241 atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag
    15301 gcaaaaaaga aaaagtaaga attcgcggcc gcactcgaga tatctagacc cagcttt
//
