pKSE401

LOCUS       pKSE401               16437 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR/Cas based plant genome editing and gene regulation; expresses
            3×FLAG-NLS-zCas9-NLS, gRNA scaffold for insertion of target sequence
            (AtU6-26 promoter), Kan resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pKSE401
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16437)
  AUTHORS   Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
  TITLE     A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
  JOURNAL   BMC Plant Biol. 2014 Nov 29;14(1):327.
  PUBMED    25432517
REFERENCE   2  (bases 1 to 16437)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..16437
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     CDS             977..1768
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        1920..1995
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2609..2953
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3106..3171
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3178..3198
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3223..7323
                     /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             7236..7244
                     /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             7324..7371
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8028..8044)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8028..8044)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8041..8063)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8052..8068
                     /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(8076..8106)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8121..8142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8333..9009
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     CDS             9072..9869
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(9129..9148)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9739..9758
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    9926..10100
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    10178..10202
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             10627..11421
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      11508..12096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11997..12016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12250..12267
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    12282..12422
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(12348..12367)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12508..12530
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      12766..12960
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13026..14099)
                     /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(14528..15157)
                     /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 agtgattggg agaccaaccc agtggacata
      721 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
      781 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
      841 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
      901 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
      961 aacaaagtta aacatcatgg gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     1021 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     1081 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     1141 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     1201 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     1261 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     1321 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     1381 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     1441 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     1501 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     1561 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     1621 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     1681 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     1741 cgagatcacc aaggtagtcg gcaaataatg tctagctaga aattcgttca agccgacgcc
     1801 gcttcgcggc gcggcttaac tcaagcgtta gatgcactaa gcacataatt gctcacagcc
     1861 aaactatcag gtcaagtctg cttttattat ttttaagcgt gcataataag ccggtctcgg
     1921 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1981 gcaccgagtc ggtgcttttt tttgcaaaat tttccagatc gatttcttct tcctctgttc
     2041 ttcggcgttc aatttctggg gttttctctt cgttttctgt aactgaaacc taaaatttga
     2101 cctaaaaaaa atctcaaata atatgattca gtggttttgt acttttcagt tagttgagtt
     2161 ttgcagttcc gatgagataa accaataagc ttgcatgcct gcaggtcaac atggtggagc
     2221 acgacacact tgtctactcc aaaaatatca aagatacagt ctcagaagac caaagggcaa
     2281 ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta
     2341 tctgtcactt tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt
     2401 gcgataaagg aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac
     2461 ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag
     2521 tggattgatg tgataacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag
     2581 atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa
     2641 acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg
     2701 aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct
     2761 ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag
     2821 acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg
     2881 atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc
     2941 atttggagag gacctcgacc tcaacacaac atatacaaaa caaacgaatc tcaagcaatc
     3001 aagcattcta cttctattgc agcaatttaa atcatttctt ttaaagcaaa agcaattttc
     3061 tgaaaatttt caccatttac gaacgatact cgagtaatct agatggatta caaggaccac
     3121 gacggggatt acaaggacca cgacattgat tacaaggatg atgatgacaa gatggctccg
     3181 aagaagaaga ggaaggttgg catccacggg gtgccagctg ctgacaagaa gtactcgatc
     3241 ggcctcgata ttgggactaa ctctgttggc tgggccgtga tcaccgacga gtacaaggtg
     3301 ccctcaaaga agttcaaggt cctgggcaac accgatcggc attccatcaa gaagaatctc
     3361 attggcgctc tcctgttcga cagcggcgag acggctgagg ctacgcggct caagcgcacc
     3421 gcccgcaggc ggtacacgcg caggaagaat cgcatctgct acctgcagga gattttctcc
     3481 aacgagatgg cgaaggttga cgattctttc ttccacaggc tggaggagtc attcctcgtg
     3541 gaggaggata agaagcacga gcggcatcca atcttcggca acattgtcga cgaggttgcc
     3601 taccacgaga agtaccctac gatctaccat ctgcggaaga agctcgtgga ctccacagat
     3661 aaggcggacc tccgcctgat ctacctcgct ctggcccaca tgattaagtt caggggccat
     3721 ttcctgatcg agggggatct caacccggac aatagcgatg ttgacaagct gttcatccag
     3781 ctcgtgcaga cgtacaacca gctcttcgag gagaacccca ttaatgcgtc aggcgtcgac
     3841 gcgaaggcta tcctgtccgc taggctctcg aagtctcggc gcctcgagaa cctgatcgcc
     3901 cagctgccgg gcgagaagaa gaacggcctg ttcgggaatc tcattgcgct cagcctgggg
     3961 ctcacgccca acttcaagtc gaatttcgat ctcgctgagg acgccaagct gcagctctcc
     4021 aaggacacat acgacgatga cctggataac ctcctggccc agatcggcga tcagtacgcg
     4081 gacctgttcc tcgctgccaa gaatctgtcg gacgccatcc tcctgtctga tattctcagg
     4141 gtgaacaccg agattacgaa ggctccgctc tcagcctcca tgatcaagcg ctacgacgag
     4201 caccatcagg atctgaccct cctgaaggcg ctggtcaggc agcagctccc cgagaagtac
     4261 aaggagatct tcttcgatca gtcgaagaac ggctacgctg ggtacattga cggcggggcc
     4321 tctcaggagg agttctacaa gttcatcaag ccgattctgg agaagatgga cggcacggag
     4381 gagctgctgg tgaagctcaa tcgcgaggac ctcctgagga agcagcggac attcgataac
     4441 ggcagcatcc cacaccagat tcatctcggg gagctgcacg ctatcctgag gaggcaggag
     4501 gacttctacc ctttcctcaa ggataaccgc gagaagatcg agaagattct gactttcagg
     4561 atcccgtact acgtcggccc actcgctagg ggcaactccc gcttcgcttg gatgacccgc
     4621 aagtcagagg agacgatcac gccgtggaac ttcgaggagg tggtcgacaa gggcgctagc
     4681 gctcagtcgt tcatcgagag gatgacgaat ttcgacaaga acctgccaaa tgagaaggtg
     4741 ctccctaagc actcgctcct gtacgagtac ttcacagtct acaacgagct gactaaggtg
     4801 aagtatgtga ccgagggcat gaggaagccg gctttcctgt ctggggagca gaagaaggcc
     4861 atcgtggacc tcctgttcaa gaccaaccgg aaggtcacgg ttaagcagct caaggaggac
     4921 tacttcaaga agattgagtg cttcgattcg gtcgagatct ctggcgttga ggaccgcttc
     4981 aacgcctccc tggggaccta ccacgatctc ctgaagatca ttaaggataa ggacttcctg
     5041 gacaacgagg agaatgagga tatcctcgag gacattgtgc tgacactcac tctgttcgag
     5101 gaccgggaga tgatcgagga gcgcctgaag acttacgccc atctcttcga tgacaaggtc
     5161 atgaagcagc tcaagaggag gaggtacacc ggctggggga ggctgagcag gaagctcatc
     5221 aacggcattc gggacaagca gtccgggaag acgatcctcg acttcctgaa gagcgatggc
     5281 ttcgcgaacc gcaatttcat gcagctgatt cacgatgaca gcctcacatt caaggaggat
     5341 atccagaagg ctcaggtgag cggccagggg gactcgctgc acgagcatat cgcgaacctc
     5401 gctggctcgc cagctatcaa gaaggggatt ctgcagaccg tgaaggttgt ggacgagctg
     5461 gtgaaggtca tgggcaggca caagcctgag aacatcgtca ttgagatggc ccgggagaat
     5521 cagaccacgc agaagggcca gaagaactca cgcgagagga tgaagaggat cgaggagggc
     5581 attaaggagc tggggtccca gatcctcaag gagcacccgg tggagaacac gcagctgcag
     5641 aatgagaagc tctacctgta ctacctccag aatggccgcg atatgtatgt ggaccaggag
     5701 ctggatatta acaggctcag cgattacgac gtcgatcata tcgttccaca gtcattcctg
     5761 aaggatgact ccattgacaa caaggtcctc accaggtcgg acaagaaccg gggcaagtct
     5821 gataatgttc cttcagagga ggtcgttaag aagatgaaga actactggcg ccagctcctg
     5881 aatgccaagc tgatcacgca gcggaagttc gataacctca caaaggctga gaggggcggg
     5941 ctctctgagc tggacaaggc gggcttcatc aagaggcagc tggtcgagac acggcagatc
     6001 actaagcacg ttgcgcagat tctcgactca cggatgaaca ctaagtacga tgagaatgac
     6061 aagctgatcc gcgaggtgaa ggtcatcacc ctgaagtcaa agctcgtctc cgacttcagg
     6121 aaggatttcc agttctacaa ggttcgggag atcaacaatt accaccatgc ccatgacgcg
     6181 tacctgaacg cggtggtcgg cacagctctg atcaagaagt acccaaagct cgagagcgag
     6241 ttcgtgtacg gggactacaa ggtttacgat gtgaggaaga tgatcgccaa gtcggagcag
     6301 gagattggca aggctaccgc caagtacttc ttctactcta acattatgaa tttcttcaag
     6361 acagagatca ctctggccaa tggcgagatc cggaagcgcc ccctcatcga gacgaacggc
     6421 gagacggggg agatcgtgtg ggacaagggc agggatttcg cgaccgtcag gaaggttctc
     6481 tccatgccac aagtgaatat cgtcaagaag acagaggtcc agactggcgg gttctctaag
     6541 gagtcaattc tgcctaagcg gaacagcgac aagctcatcg cccgcaagaa ggactgggat
     6601 ccgaagaagt acggcgggtt cgacagcccc actgtggcct actcggtcct ggttgtggcg
     6661 aaggttgaga agggcaagtc caagaagctc aagagcgtga aggagctgct ggggatcacg
     6721 attatggagc gctccagctt cgagaagaac ccgatcgatt tcctggaggc gaagggctac
     6781 aaggaggtga agaaggacct gatcattaag ctccccaagt actcactctt cgagctggag
     6841 aacggcagga agcggatgct ggcttccgct ggcgagctgc agaaggggaa cgagctggct
     6901 ctgccgtcca agtatgtgaa cttcctctac ctggcctccc actacgagaa gctcaagggc
     6961 agccccgagg acaacgagca gaagcagctg ttcgtcgagc agcacaagca ttacctcgac
     7021 gagatcattg agcagatttc cgagttctcc aagcgcgtga tcctggccga cgcgaatctg
     7081 gataaggtcc tctccgcgta caacaagcac cgcgacaagc caatcaggga gcaggctgag
     7141 aatatcattc atctcttcac cctgacgaac ctcggcgccc ctgctgcttt caagtacttc
     7201 gacacaacta tcgatcgcaa gaggtacaca agcactaagg aggtcctgga cgcgaccctc
     7261 atccaccagt cgattaccgg cctctacgag acgcgcatcg acctgtctca gctcgggggc
     7321 gacaagcggc cagcggcgac gaagaaggcg gggcaggcga agaagaagaa gtgagctcag
     7381 agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat
     7441 tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc
     7501 ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac
     7561 tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc
     7621 aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag
     7681 atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag
     7741 ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa
     7801 tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt
     7861 tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc
     7921 attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta
     7981 atgcatttta tgacttgcca attgattgac aacgaattcg taatcatgtc atagctgttt
     8041 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     8101 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     8161 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     8221 gggagaggcg gtttgcgtat tggctagagc agcttgccaa catggtggag cacgacactc
     8281 tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt
     8341 ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact
     8401 tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag
     8461 gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca
     8521 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat
     8581 gtgaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca
     8641 gaagaccaaa gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga
     8701 ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc
     8761 tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt
     8821 ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc
     8881 acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa
     8941 tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg
     9001 acacgctgaa atcaccagtc tctctctaca aatctatctc tctcgagctt tcgcagatct
     9061 gtcgatcgac catggggatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     9121 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     9181 tgtttcggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     9241 ccctgaatga actccaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     9301 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     9361 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     9421 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     9481 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     9541 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     9601 cgcgcatgcc cgacggcgag gatctcgtcg tgacacatgg cgatgcctgc ttgccgaata
     9661 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     9721 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     9781 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     9841 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcggat cgatcctcta
     9901 gctagagtcg atcgacaagc tcgagtttct ccataataat gtgtgagtag ttcccagata
     9961 agggaattag ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt
    10021 atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat
    10081 ccagtactaa aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg
    10141 tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc
    10201 caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag
    10261 gcagcccatc agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc
    10321 tcatgttacc gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat
    10381 gatctcgcgg agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca
    10441 ccgcggtttc aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt
    10501 tgaaaaagct gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt
    10561 cgtcttgtta taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa
    10621 taataaatgg ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc
    10681 tgcgtaaaag atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat
    10741 gaaaacctat atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa
    10801 cgggaaaagg acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac
    10861 tttgaacggc atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc
    10921 tcggaagagt atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc
    10981 atcaggctct ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc
    11041 cgcttagccg aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac
    11101 tgggaagaag acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa
    11161 aagcccgaag aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg
    11221 aaagatggca aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg
    11281 tatgacattg ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc
    11341 gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt
    11401 ttactggatg aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt
    11461 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
    11521 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
    11581 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
    11641 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
    11701 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
    11761 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
    11821 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
    11881 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
    11941 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
    12001 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
    12061 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
    12121 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
    12181 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
    12241 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
    12301 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
    12361 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
    12421 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
    12481 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
    12541 catcaccgaa acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac
    12601 ggcgcggctt gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg
    12661 ccagcggccg cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg
    12721 gtaggcgctt tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg
    12781 cgggttttaa gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc
    12841 tcttttatat cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca
    12901 atgtacgggt tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca
    12961 ggaaacagac cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc
    13021 gtacattagg aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg
    13081 atcgggccag cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg
    13141 atcagcttgc gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg
    13201 tagatcgtct tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg
    13261 tagagaaaac ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg
    13321 tccgggttct tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg
    13381 tactccggcc gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc
    13441 tcggataccg tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc
    13501 gtggtgttta accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct
    13561 cgccggcaga acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct
    13621 cccttccctt cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc
    13681 aggtcgtaat cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg
    13741 tctggaagct cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa
    13801 acggccacgt ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg
    13861 aaaaaatctg gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc
    13921 ggcggttgcc gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg
    13981 cgtgcttctg cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc
    14041 aggtcatcac ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgctcacg
    14101 ccgattcctc gggcttgggg gttccagtgc cattgcaggg ccggcaggca acccagccgc
    14161 ttacgcctgg ccaaccgccc gttcctccac acatggggca ttccacggcg tcggtgcctg
    14221 gttgttcttg attttccatg ccgcctcctt tagccgctaa aattcatcta ctcatttatt
    14281 catttgctca tttactctgg tagctgcgcg atgtattcag atagcagctc ggtaatggtc
    14341 ttgccttggc gtaccgcgta catcttcagc ttggtgtgat cctccgccgg caactgaaag
    14401 ttgacccgct tcatggctgg cgtgtctgcc aggctggcca acgttgcagc cttgctgctg
    14461 cgtgcgctcg gacggccggc acttagcgtg tttgtgcttt tgctcatttt ctctttacct
    14521 cattaactca aatgagtttt gatttaattt cagcggccag cgcctggacc tcgcgggcag
    14581 cgtcgccctc gggttctgat tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc
    14641 tcacgcgctg cgtgatacgg gactcaagaa tgggcagctc gtacccggcc agcgcctcgg
    14701 caacctcacc gccgatgcgc gtgcctttga tcgcccgcga cacgacaaag gccgcttgta
    14761 gccttccatc cgtgacctca atgcgctgct taaccagctc caccaggtcg gcggtggccc
    14821 atatgtcgta agggcttggc tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg
    14881 acacagccaa gtccgccgcc tggggcgctc cgtcgatcac tacgaagtcg cgccggccga
    14941 tggccttcac gtcgcggtca atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat
    15001 cttcccgcac ggccgcccaa tcgcgggcac tgccctgggg atcggaatcg actaacagaa
    15061 catcggcccc ggcgagttgc agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg
    15121 acccgccttt ctggttaagt acagcgataa ccttcatgcg ttccccttgc gtatttgttt
    15181 atttactcat cgcatcatat acgcagcgac cgcatgacgc aagctgtttt actcaaatac
    15241 acatcacctt tttagacggc ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc
    15301 cgccagcttg gcatcagaca aaccggccag gatttcatgc agccgcacgg ttgagacgtg
    15361 cgcgggcggc tcgaacacgt acccggccgc gatcatctcc gcctcgatct cttcggtaat
    15421 gaaaaacggt tcgtcctggc cgtcctggtg cggtttcatg cttgttcctc ttggcgttca
    15481 ttctcggcgg ccgccagggc gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc
    15541 cgcctggcct cggtgggcgt cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag
    15601 cgatgcacgc caagcagtgc agccgcctct ttcacggtgc ggccttcctg gtcgatcagc
    15661 tcgcgggcgt gcgcgatctg tgccggggtg agggtagggc gggggccaaa cttcacgcct
    15721 cgggccttgg cggcctcgcg cccgctccgg gtgcggtcga tgattaggga acgctcgaac
    15781 tcggcaatgc cggcgaacac ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc
    15841 cacggctctg ccaggctacg caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc
    15901 agtaggtcgc gggtgctgcg ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca
    15961 gggcgtaggt ggtcaagcat cctggccagc tccgggcggt cgcgcctggt gccggtgatc
    16021 ttctcggaaa acagcttggt gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag
    16081 tcctggtcgt cggtgctgac gcgggcatag cccagcaggc cagcggcggc gctcttgttc
    16141 atggcgtaat gtctccggtt ctagtcgcaa gtattctact ttatgcgact aaaacacgcg
    16201 acaagaaaac gccaggaaaa gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc
    16261 gacatgtcgt tttcagaaga cggctgcact gaacgtcaga agccgactgc actatagcag
    16321 cggaggggtt ggatcaaagt actttgatcc cgaggggaac cctgtggttg gcatgcacat
    16381 acaaatggac gaacggataa accttttcac gcccttttaa atatccgatt attctaa
//