LOCUS       sequence-379799-       15578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for genome editing in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15578)
  AUTHORS   Yan P, Tuo D, Shen W, Deng H, Zhou P, Gao X
  TITLE     A Nimble Cloning-compatible vector system for high-throughput gene 
            functional analysis in plants.
  JOURNAL   Plant Commun. 2023 Mar 13;4(2):100471. doi: 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9.
  PUBMED    36352791
REFERENCE   2  (bases 1 to 15578)
  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 
            Commun."; date: "2023-03-13"; volume: "4(2):100471. doi"; pages: " 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9"
COMMENT     Sequence Label: pNC-BSE401
FEATURES             Location/Qualifiers
     source          1..15578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    21..45
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     233..255
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     247..264
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     248..264
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        879..981
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1012..1317
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1196..1215
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1374..1449
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2063..2407
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             2560..2625
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2632..2652
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2677..6777
                     /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             2677..6777
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             6690..6698
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6778..6825
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7483..7499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7483..7499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7496..7518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7507..7523
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7531..7537,7538..7555,7556..7561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7576..7597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7788..8464
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             8509..9060
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     polyA_signal    9067..9241
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    9319..9343
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             9768..10562
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      10649..11237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11138..11157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11391..11408
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    11423..11563
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(11489..11508)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11649..11671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      11907..12101
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(12167..13240)
                     /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"
     CDS             complement(13669..14298)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
        1 taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta
       61 aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc
      121 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      181 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      241 cgacgttgta aaacgacggc cagtgccaag cttcgacttg ccttccgcac aatacatcat
      301 ttcttcttag ctttttttct tcttcttcgt tcatacagtt tttttttgtt tatcagctta
      361 cattttcttg aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt
      421 atcttgtttc atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt
      481 gattgaataa aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga
      541 atctgaaaga agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata
      601 ggcccattta agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa
      661 gctgagttta tatacagcta gagtcgaagt agtgattgag tggtctctgt ccagtcctgg
      721 cctcgtcggc cattctcgac taagttggca gcatcacccg acgcactttg cgccgaataa
      781 atacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc
      841 gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc
      901 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt
      961 tcaggagcta aggaagctaa acttttgctg acgagaacag ggactggtga aatgcagttt
     1021 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     1081 attattgaca cgcctgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     1141 gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     1201 atgaccaccg atatggccag tgtgccggta tccgttatcg gggaagaagt ggctgatctc
     1261 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     1321 tcaggctccc ttatacacag ggccagtctg gccacttgtg gtctgctgag accgttttag
     1381 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     1441 agtcggtgct tttttttgca aaattttcca gatcgatttc ttcttcctct gttcttcggc
     1501 gttcaatttc tggggttttc tcttcgtttt ctgtaactga aacctaaaat ttgacctaaa
     1561 aaaaatctca aataatatga ttcagtggtt ttgtactttt cagttagttg agttttgcag
     1621 ttccgatgag ataaaccaat aagcttgcat gcctgcaggt caacatggtg gagcacgaca
     1681 cacttgtcta ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga
     1741 cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc
     1801 actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata
     1861 aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac
     1921 ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt
     1981 gatgtgataa catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag
     2041 tctcagaaga ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc
     2101 tcggattcca ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg
     2161 gctcctacaa atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg
     2221 acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc
     2281 caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg
     2341 cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg
     2401 agaggacctc gacctcaaca caacatatac aaaacaaacg aatctcaagc aatcaagcat
     2461 tctacttcta ttgcagcaat ttaaatcatt tcttttaaag caaaagcaat tttctgaaaa
     2521 ttttcaccat ttacgaacga tactcgagta atctagatgg attacaagga ccacgacggg
     2581 gattacaagg accacgacat tgattacaag gatgatgatg acaagatggc tccgaagaag
     2641 aagaggaagg ttggcatcca cggggtgcca gctgctgaca agaagtactc gatcggcctc
     2701 gatattggga ctaactctgt tggctgggcc gtgatcaccg acgagtacaa ggtgccctca
     2761 aagaagttca aggtcctggg caacaccgat cggcattcca tcaagaagaa tctcattggc
     2821 gctctcctgt tcgacagcgg cgagacggct gaggctacgc ggctcaagcg caccgcccgc
     2881 aggcggtaca cgcgcaggaa gaatcgcatc tgctacctgc aggagatttt ctccaacgag
     2941 atggcgaagg ttgacgattc tttcttccac aggctggagg agtcattcct cgtggaggag
     3001 gataagaagc acgagcggca tccaatcttc ggcaacattg tcgacgaggt tgcctaccac
     3061 gagaagtacc ctacgatcta ccatctgcgg aagaagctcg tggactccac agataaggcg
     3121 gacctccgcc tgatctacct cgctctggcc cacatgatta agttcagggg ccatttcctg
     3181 atcgaggggg atctcaaccc ggacaatagc gatgttgaca agctgttcat ccagctcgtg
     3241 cagacgtaca accagctctt cgaggagaac cccattaatg cgtcaggcgt cgacgcgaag
     3301 gctatcctgt ccgctaggct ctcgaagtct cggcgcctcg agaacctgat cgcccagctg
     3361 ccgggcgaga agaagaacgg cctgttcggg aatctcattg cgctcagcct ggggctcacg
     3421 cccaacttca agtcgaattt cgatctcgct gaggacgcca agctgcagct ctccaaggac
     3481 acatacgacg atgacctgga taacctcctg gcccagatcg gcgatcagta cgcggacctg
     3541 ttcctcgctg ccaagaatct gtcggacgcc atcctcctgt ctgatattct cagggtgaac
     3601 accgagatta cgaaggctcc gctctcagcc tccatgatca agcgctacga cgagcaccat
     3661 caggatctga ccctcctgaa ggcgctggtc aggcagcagc tccccgagaa gtacaaggag
     3721 atcttcttcg atcagtcgaa gaacggctac gctgggtaca ttgacggcgg ggcctctcag
     3781 gaggagttct acaagttcat caagccgatt ctggagaaga tggacggcac ggaggagctg
     3841 ctggtgaagc tcaatcgcga ggacctcctg aggaagcagc ggacattcga taacggcagc
     3901 atcccacacc agattcatct cggggagctg cacgctatcc tgaggaggca ggaggacttc
     3961 taccctttcc tcaaggataa ccgcgagaag atcgagaaga ttctgacttt caggatcccg
     4021 tactacgtcg gcccactcgc taggggcaac tcccgcttcg cttggatgac ccgcaagtca
     4081 gaggagacga tcacgccgtg gaacttcgag gaggtggtcg acaagggcgc tagcgctcag
     4141 tcgttcatcg agaggatgac gaatttcgac aagaacctgc caaatgagaa ggtgctccct
     4201 aagcactcgc tcctgtacga gtacttcaca gtctacaacg agctgactaa ggtgaagtat
     4261 gtgaccgagg gcatgaggaa gccggctttc ctgtctgggg agcagaagaa ggccatcgtg
     4321 gacctcctgt tcaagaccaa ccggaaggtc acggttaagc agctcaagga ggactacttc
     4381 aagaagattg agtgcttcga ttcggtcgag atctctggcg ttgaggaccg cttcaacgcc
     4441 tccctgggga cctaccacga tctcctgaag atcattaagg ataaggactt cctggacaac
     4501 gaggagaatg aggatatcct cgaggacatt gtgctgacac tcactctgtt cgaggaccgg
     4561 gagatgatcg aggagcgcct gaagacttac gcccatctct tcgatgacaa ggtcatgaag
     4621 cagctcaaga ggaggaggta caccggctgg gggaggctga gcaggaagct catcaacggc
     4681 attcgggaca agcagtccgg gaagacgatc ctcgacttcc tgaagagcga tggcttcgcg
     4741 aaccgcaatt tcatgcagct gattcacgat gacagcctca cattcaagga ggatatccag
     4801 aaggctcagg tgagcggcca gggggactcg ctgcacgagc atatcgcgaa cctcgctggc
     4861 tcgccagcta tcaagaaggg gattctgcag accgtgaagg ttgtggacga gctggtgaag
     4921 gtcatgggca ggcacaagcc tgagaacatc gtcattgaga tggcccggga gaatcagacc
     4981 acgcagaagg gccagaagaa ctcacgcgag aggatgaaga ggatcgagga gggcattaag
     5041 gagctggggt cccagatcct caaggagcac ccggtggaga acacgcagct gcagaatgag
     5101 aagctctacc tgtactacct ccagaatggc cgcgatatgt atgtggacca ggagctggat
     5161 attaacaggc tcagcgatta cgacgtcgat catatcgttc cacagtcatt cctgaaggat
     5221 gactccattg acaacaaggt cctcaccagg tcggacaaga accggggcaa gtctgataat
     5281 gttccttcag aggaggtcgt taagaagatg aagaactact ggcgccagct cctgaatgcc
     5341 aagctgatca cgcagcggaa gttcgataac ctcacaaagg ctgagagggg cgggctctct
     5401 gagctggaca aggcgggctt catcaagagg cagctggtcg agacacggca gatcactaag
     5461 cacgttgcgc agattctcga ctcacggatg aacactaagt acgatgagaa tgacaagctg
     5521 atccgcgagg tgaaggtcat caccctgaag tcaaagctcg tctccgactt caggaaggat
     5581 ttccagttct acaaggttcg ggagatcaac aattaccacc atgcccatga cgcgtacctg
     5641 aacgcggtgg tcggcacagc tctgatcaag aagtacccaa agctcgagag cgagttcgtg
     5701 tacggggact acaaggttta cgatgtgagg aagatgatcg ccaagtcgga gcaggagatt
     5761 ggcaaggcta ccgccaagta cttcttctac tctaacatta tgaatttctt caagacagag
     5821 atcactctgg ccaatggcga gatccggaag cgccccctca tcgagacgaa cggcgagacg
     5881 ggggagatcg tgtgggacaa gggcagggat ttcgcgaccg tcaggaaggt tctctccatg
     5941 ccacaagtga atatcgtcaa gaagacagag gtccagactg gcgggttctc taaggagtca
     6001 attctgccta agcggaacag cgacaagctc atcgcccgca agaaggactg ggatccgaag
     6061 aagtacggcg ggttcgacag ccccactgtg gcctactcgg tcctggttgt ggcgaaggtt
     6121 gagaagggca agtccaagaa gctcaagagc gtgaaggagc tgctggggat cacgattatg
     6181 gagcgctcca gcttcgagaa gaacccgatc gatttcctgg aggcgaaggg ctacaaggag
     6241 gtgaagaagg acctgatcat taagctcccc aagtactcac tcttcgagct ggagaacggc
     6301 aggaagcgga tgctggcttc cgctggcgag ctgcagaagg ggaacgagct ggctctgccg
     6361 tccaagtatg tgaacttcct ctacctggcc tcccactacg agaagctcaa gggcagcccc
     6421 gaggacaacg agcagaagca gctgttcgtc gagcagcaca agcattacct cgacgagatc
     6481 attgagcaga tttccgagtt ctccaagcgc gtgatcctgg ccgacgcgaa tctggataag
     6541 gtcctctccg cgtacaacaa gcaccgcgac aagccaatca gggagcaggc tgagaatatc
     6601 attcatctct tcaccctgac gaacctcggc gcccctgctg ctttcaagta cttcgacaca
     6661 actatcgatc gcaagaggta cacaagcact aaggaggtcc tggacgcgac cctcatccac
     6721 cagtcgatta ccggcctcta cgagacgcgc atcgacctgt ctcagctcgg gggcgacaag
     6781 cggccagcgg cgacgaagaa ggcggggcag gcgaagaaga agaagtgagc tcagagcttt
     6841 cgttcgtatc atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt tcattgcgca
     6901 cacaccagaa tcctactgag tttgagtatt atggcattgg gaaaactgtt tttcttgtac
     6961 catttgttgt gcttgtaatt tactgtgttt tttattcggt tttcgctatc gaactgtgaa
     7021 atggaaatgg atggagaaga gttaatgaat gatatggtcc ttttgttcat tctcaaatta
     7081 atattatttg ttttttctct tatttgttgt gtgttgaatt tgaaattata agagatatgc
     7141 aaacattttg ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc gaagttaata
     7201 tgaggagtaa aacacttgta gttgtaccat tatgcttatt cactaggcaa caaatatatt
     7261 ttcagaccta gaaaagctgc aaatgttact gaatacaagt atgtcctctt gtgttttaga
     7321 catttatgaa ctttccttta tgtaattttc cagaatcctt gtcagattct aatcattgct
     7381 ttataattat agttatactc atggatttgt agttgagtat gaaaatattt tttaatgcat
     7441 tttatgactt gccaattgat tgacaacgaa ttcgtaatca tggtcatagc tgtttcctgt
     7501 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     7561 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     7621 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     7681 aggcggtttg cgtattggct agagcagctt gccaacatgg tggagcacga cactctcgtc
     7741 tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga gacttttcaa
     7801 caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg tcacttcatc
     7861 aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga taaaggaaag
     7921 gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg
     7981 agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgaa
     8041 catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga
     8101 ccaaagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca
     8161 ttgcccagct atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa
     8221 atgccatcat tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc
     8281 caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc
     8341 ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca
     8401 ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agaggacacg
     8461 ctgaaatcac cagtctctct ctacaaatct atctctctcg agtctaccat gagcccagaa
     8521 cgacgcccgg ccgacatccg ccgtgccacc gaggcggaca tgccggcggt ctgcaccatc
     8581 gtcaaccact acatcgagac aagcacggtc aacttccgta ccgagccgca ggaaccgcag
     8641 gagtggacgg acgacctcgt ccgtctgcgg gagcgctatc cctggctcgt cgccgaggtg
     8701 gacggcgagg tcgccggcat cgcctacgcg ggcccctgga aggcacgcaa cgcctacgac
     8761 tggacggccg agtcgaccgt gtacgtctcc ccccgccacc agcggacggg actgggctcc
     8821 acgctctaca cccacctgct gaagtccctg gaggcacagg gcttcaagag cgtggtcgct
     8881 gtcatcgggc tgcccaacga cccgagcgtg cgcatgcacg aggcgctcgg atatgccccc
     8941 cgcggcatgc tgcgggcggc cggcttcaag cacgggaact ggcatgacgt gggtttctgg
     9001 cagctggact tcagcctgcc ggtaccgccc cgtccggtcc tgcccgtcac cgagatttga
     9061 ctcgagtttc tccataataa tgtgtgagta gttcccagat aagggaatta gggttcctat
     9121 agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa
     9181 atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat
     9241 cccccgaatt aattcggcgt taattcagta cattaaaaac gtccgcaatg tgttattaag
     9301 ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca gccaacagct
     9361 ccccgaccgg cagctcggca caaaatcacc actcgataca ggcagcccat cagtccggga
     9421 cggcgtcagc gggagagccg ttgtaaggcg gcagactttg ctcatgttac cgatgctatt
     9481 cggaagaacg gcaactaagc tgccgggttt gaaacacgga tgatctcgcg gagggtagca
     9541 tgttgattgt aacgatgaca gagcgttgct gcctgtgatc accgcggttt caaaatcggc
     9601 tccgtcgata ctatgttata cgccaacttt gaaaacaact ttgaaaaagc tgttttctgg
     9661 tatttaaggt tttagaatgc aaggaacagt gaattggagt tcgtcttgtt ataattagct
     9721 tcttggggta tctttaaata ctgtagaaaa gaggaaggaa ataataaatg gctaaaatga
     9781 gaatatcacc ggaattgaaa aaactgatcg aaaaataccg ctgcgtaaaa gatacggaag
     9841 gaatgtctcc tgctaaggta tataagctgg tgggagaaaa tgaaaaccta tatttaaaaa
     9901 tgacggacag ccggtataaa gggaccacct atgatgtgga acgggaaaag gacatgatgc
     9961 tatggctgga aggaaagctg cctgttccaa aggtcctgca ctttgaacgg catgatggct
    10021 ggagcaatct gctcatgagt gaggccgatg gcgtcctttg ctcggaagag tatgaagatg
    10081 aacaaagccc tgaaaagatt atcgagctgt atgcggagtg catcaggctc tttcactcca
    10141 tcgacatatc ggattgtccc tatacgaata gcttagacag ccgcttagcc gaattggatt
    10201 acttactgaa taacgatctg gccgatgtgg attgcgaaaa ctgggaagaa gacactccat
    10261 ttaaagatcc gcgcgagctg tatgattttt taaagacgga aaagcccgaa gaggaacttg
    10321 tcttttccca cggcgacctg ggagacagca acatctttgt gaaagatggc aaagtaagtg
    10381 gctttattga tcttgggaga agcggcaggg cggacaagtg gtatgacatt gccttctgcg
    10441 tccggtcgat cagggaggat atcggggaag aacagtatgt cgagctattt tttgacttac
    10501 tggggatcaa gcctgattgg gagaaaataa aatattatat tttactggat gaattgtttt
    10561 agtacctaga atgcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    10621 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    10681 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    10741 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
    10801 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    10861 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    10921 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    10981 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    11041 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    11101 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    11161 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    11221 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
    11281 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
    11341 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
    11401 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
    11461 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
    11521 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
    11581 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
    11641 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
    11701 gcagggtgcc ttgatgtggg cgccggcggt cgagtggcga cggcgcggct tgtccgcgcc
    11761 ctggtagatt gcctggccgt aggccagcca tttttgagcg gccagcggcc gcgataggcc
    11821 gacgcgaagc ggcggggcgt agggagcgca gcgaccgaag ggtaggcgct ttttgcagct
    11881 cttcggctgt gcgctggcca gacagttatg cacaggccag gcgggtttta agagttttaa
    11941 taagttttaa agagttttag gcggaaaaat cgcctttttt ctcttttata tcagtcactt
    12001 acatgtgtga ccggttccca atgtacggct ttgggttccc aatgtacggg ttccggttcc
    12061 caatgtacgg ctttgggttc ccaatgtacg tgctatccac aggaaacaga ccttttcgac
    12121 ctttttcccc tgctagggca atttgcccta gcatctgctc cgtacattag gaaccggcgg
    12181 atgcttcgcc ctcgatcagg ttgcggtagc gcatgactag gatcgggcca gcctgccccg
    12241 cctcctcctt caaatcgtac tccggcaggt catttgaccc gatcagcttg cgcacggtga
    12301 aacagaactt cttgaactct ccggcgctgc cactgcgttc gtagatcgtc ttgaacaacc
    12361 atctggcttc tgccttgcct gcggcgcggc gtgccaggcg gtagagaaaa cggccgatgc
    12421 cgggatcgat caaaaagtaa tcggggtgaa ccgtcagcac gtccgggttc ttgccttctg
    12481 tgatctcgcg gtacatccaa tcagctagct cgatctcgat gtactccggc cgcccggttt
    12541 cgctctttac gatcttgtag cggctaatca aggcttcacc ctcggatacc gtcaccaggc
    12601 ggccgttctt ggccttcttc gtacgctgca tggcaacgtg cgtggtgttt aaccgaatgc
    12661 aggtttctac caggtcgtct ttctgctttc cgccatcggc tcgccggcag aacttgagta
    12721 cgtccgcaac gtgtggacgg aacacgcggc cgggcttgtc tcccttccct tcccggtatc
    12781 ggttcatgga ttcggttaga tgggaaaccg ccatcagtac caggtcgtaa tcccacacac
    12841 tggccatgcc cgccggccct gcggaaacct ctacgtgccc gtctggaagc tcgtagcgga
    12901 tcacctcgcc agctcgtcgg tcacgcttcg acagacggaa aacggccacg tccatgatgc
    12961 tgcgactatc gcgggtgccc acgtcataga gcatcggaac gaaaaaatct ggttgctcgt
    13021 cgcccttggg cggcttccta atcgacggcg caccggctgc cggcggttgc cgggattctt
    13081 tgcggattcg atcagcggcc gcttgccacg attcaccggg gcgtgcttct gcctcgatgc
    13141 gttgccgctg ggcggcctgc gccgccttca acttctccac caggtcatca cccagcgccg
    13201 cgccgatttg taccgggccg gatggtttgc gaccgctcac gccgattcct cgggcttggg
    13261 ggttccagtg ccattgcagg gccggcaggc aacccagccg cttacgcctg gccaaccgcc
    13321 cgttcctcca cacatggggc attccacggc gtcggtgcct ggttgttctt gattttccat
    13381 gccgcctcct ttagccgcta aaattcatct actcatttat tcatttgctc atttactctg
    13441 gtagctgcgc gatgtattca gatagcagct cggtaatggt cttgccttgg cgtaccgcgt
    13501 acatcttcag cttggtgtga tcctccgccg gcaactgaaa gttgacccgc ttcatggctg
    13561 gcgtgtctgc caggctggcc aacgttgcag ccttgctgct gcgtgcgctc ggacggccgg
    13621 cacttagcgt gtttgtgctt ttgctcattt tctctttacc tcattaactc aaatgagttt
    13681 tgatttaatt tcagcggcca gcgcctggac ctcgcgggca gcgtcgccct cgggttctga
    13741 ttcaagaacg gttgtgccgg cggcggcagt gcctgggtag ctcacgcgct gcgtgatacg
    13801 ggactcaaga atgggcagct cgtacccggc cagcgcctcg gcaacctcac cgccgatgcg
    13861 cgtgcctttg atcgcccgcg acacgacaaa ggccgcttgt agccttccat ccgtgacctc
    13921 aatgcgctgc ttaaccagct ccaccaggtc ggcggtggcc catatgtcgt aagggcttgg
    13981 ctgcaccgga atcagcacga agtcggctgc cttgatcgcg gacacagcca agtccgccgc
    14041 ctggggcgct ccgtcgatca ctacgaagtc gcgccggccg atggccttca cgtcgcggtc
    14101 aatcgtcggg cggtcgatgc cgacaacggt tagcggttga tcttcccgca cggccgccca
    14161 atcgcgggca ctgccctggg gatcggaatc gactaacaga acatcggccc cggcgagttg
    14221 cagggcgcgg gctagatggg ttgcgatggt cgtcttgcct gacccgcctt tctggttaag
    14281 tacagcgata accttcatgc gttccccttg cgtatttgtt tatttactca tcgcatcata
    14341 tacgcagcga ccgcatgacg caagctgttt tactcaaata cacatcacct ttttagacgg
    14401 cggcgctcgg tttcttcagc ggccaagctc gccggccagg ccgccagctt ggcatcagac
    14461 aaaccggcca ggatttcatg cagccgcacg gttgagacgt gcgcgggcgg ctcgaacacg
    14521 tacccggccg cgatcatctc cgcctcgatc tcttcggtaa tgaaaaacgg ttcgtcctgg
    14581 ccgtcctggt gcggtttcat gcttgttcct cttggcgttc attctcggcg gccgccaggg
    14641 cgtcggcctc ggtcaatgcg tcctcacgga aggcaccgcg ccgcctggcc tcggtgggcg
    14701 tcacttcctc gctgcgctca agtgcgcggt acagggtcga gcgatgcacg ccaagcagtg
    14761 cagccgcctc tttcacggtg cggccttcct ggtcgatcag ctcgcgggcg tgcgcgatct
    14821 gtgccggggt gagggtaggg cgggggccaa acttcacgcc tcgcgccttg gcggcctcgc
    14881 gcccgctccg ggtgcggtcg atgattaggg aacgctcgaa ctcggcaatg ccggcgaaca
    14941 cggtcaacac catgcggccg gccggcgtgg tggtgtcggc ccacggctct gccaggctac
    15001 gcaggcccgc gccggcctcc tggatgcgct cggcaatgtc cagtaggtcg cgggtgctgc
    15061 gggccaggcg gtctagcctg gtcactgtca caacgtcgcc agggcgtagg tggtcaagca
    15121 tcctggccag ctccgggcgg tcgcgcctgg tgccggtgat cttctcggaa aacagcttgg
    15181 tgcagccggc cgcgtgcagt tcggcccgtt ggttggtcaa gtcctggtcg tcggtgctga
    15241 cgcgggcata gcccagcagg ccagcggcgg cgctcttgtt catggcgtaa tgtctccggt
    15301 tctagtcgca agtattctac tttatgcgac taaaacacgc gacaagaaaa cgccaggaaa
    15361 agggcagggc ggcagcctgt cgcgtaactt aggacttgtg cgacatgtcg ttttcagaag
    15421 acggctgcac tgaacgtcag aagccgactg cactatagca gcggaggggt tggatcaaag
    15481 tactttgatc ccgaggggaa ccctgtggtt ggcatgcaca tacaaatgga cgaacggata
    15541 aaccttttca cgccctttta aatatccgat tattctaa
//
