pDe-CAS9-D10A

LOCUS       pDe-CAS9-D10A           16467 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  based on pDe-CAS9, encodes Cas9 D10A nickase variant.
ACCESSION   .
VERSION     .
KEYWORDS    pDe-CAS9-D10A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16467)
  AUTHORS   Fauser F, Schiml S, Puchta H
  TITLE     Both CRISPR/Cas-based nucleases and nickases can be used efficiently
            for genome engineering in Arabidopsis thaliana.
  JOURNAL   Plant J. 2014 Jul;79(2):348-59. doi: 10.1111/tpj.12554. Epub 2014 
            Jun 17.
  PUBMED    24836556
REFERENCE   2  (bases 1 to 16467)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..16467
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1187..1816
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2250..3317
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     rep_origin      3383..3577
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(3813..3835)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3921..4061
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3976..3995
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4076..4093)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4247..4835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4327..4346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5079..5870)
                     /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_feature    6398..6422
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    6599..6620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6635..6665
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6673..6689
                     /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     6678..6700
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6697..6713
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6697..6713
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7714..11817
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             11830..11850
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    12362..12486
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        12511..12541
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             12595..13275
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(12661..12680)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             13595..13900
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     13779..13798
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(13941..14065)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        14261..14939
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     14876..14896
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             14999..15796
                     /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(15056..15075)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     15666..15685
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    15850..16024
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(16118..16135)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(16118..16134)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(16127..16149)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    16330..16354
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
ORIGIN
        1 agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag
       61 ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg
      121 cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac
      181 tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc
      241 cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc
      301 tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc
      361 aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg
      421 gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc
      481 ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg
      541 accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc
      601 gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg
      661 gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg
      721 gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa
      781 gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc
      841 gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg
      901 acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg
      961 ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg
     1021 gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc
     1081 gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg
     1141 cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc
     1201 tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc
     1261 cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg
     1321 cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac
     1381 gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc
     1441 ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt
     1501 gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg
     1561 cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg
     1621 cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga
     1681 gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct
     1741 tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa
     1801 atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta
     1861 agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac
     1921 acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag
     1981 atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag
     2041 ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg
     2101 cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg
     2161 aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg
     2221 aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg
     2281 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc
     2341 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc
     2401 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg
     2461 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc
     2521 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg
     2581 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg
     2641 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga
     2701 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg
     2761 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa
     2821 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg
     2881 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga
     2941 gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga
     3001 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc
     3061 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca
     3121 gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct
     3181 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg
     3241 aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag
     3301 catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa
     3361 aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca
     3421 ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca
     3481 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac
     3541 ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg
     3601 aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc
     3661 gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac
     3721 cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc
     3781 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     3841 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     3901 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     3961 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     4021 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     4081 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4141 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4201 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4261 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4321 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4381 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4441 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4501 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4561 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4621 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4681 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4741 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4801 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4861 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg catgatatat
     4921 ctcccaattt gtgtagggct tattatgcac gcttaaaaat aataaaagca gacttgacct
     4981 gatagtttgg ctgtgagcaa ttatgtgctt agtgcatcta atcgcttgag ttaacgccgg
     5041 cgaagcggcg tcggcttgaa cgaatttcta gctagacatt atttgccgac taccttggtg
     5101 atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga
     5161 tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc
     5221 ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact
     5281 gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg
     5341 ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga
     5401 accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct
     5461 tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga
     5521 atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga
     5581 atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca
     5641 ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc
     5701 cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact
     5761 gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca
     5821 actacctctg atagttgagt cgatacttcg gcgatcaccg cttcccccat gatgtttaac
     5881 tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctccataa catcaaacat
     5941 cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg taccccaaaa
     6001 aaacatgtca taacaagaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc
     6061 ggtcaaggtt ctggaccagt tgcgtgacgg cagttacgct acttgcatta cagcttacga
     6121 accgaacgag gcttatgtcc actgggttcg tgcccgaatt gatcacaggc agcaacgctc
     6181 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca
     6241 gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa
     6301 cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg
     6361 tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa
     6421 acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta
     6481 acgccgaatt gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6541 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6601 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     6661 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta
     6721 cgaattcaaa aattacggat atgaatatag gcatatccgt atccgaatta tccgtttgac
     6781 agctagcaac gattgtacaa ttgcttcttt aaaaaaggaa gaaagaaaga aagaaaagaa
     6841 tcaacatcag cgttaacaaa cggccccgtt acggcccaaa cggtcatata gagtaacggc
     6901 gttaagcgtt gaaagactcc tatcgaaata cgtaaccgca aacgtgtcat agtcagatcc
     6961 cctcttcctt caccgcctca aacacaaaaa taatcttcta cagcctatat atacaacccc
     7021 cccttctatc tctcctttct cacaattcat catctttctt tctctacccc caattttaag
     7081 aaatcctctc ttctcctctt cattttcaag gtaaatctct ctctctctct ctctctctgt
     7141 tattccttgt tttaattagg tatgtattat tgctagtttg ttaatctgct tatcttatgt
     7201 atgccttatg tgaatatctt tatcttgttc atctcatccg tttagaagct ataaatttgt
     7261 tgatttgact gtgtatctac acgtggttat gtttatatct aatcagatat gaatttcttc
     7321 atattgttgc gtttgtgtgt accaatccga aatcgttgat ttttttcatt taatcgtgta
     7381 gctaattgta cgtatacata tggatctacg tatcaattgt tcatctgttt gtgtttgtat
     7441 gtatacagat ctgaaaacat cacttctctc atctgattgt gttgttacat acatagatat
     7501 agatctgtta tatcattttt tttattaatt gtgtatatat atatgtgcat agatctggat
     7561 tacatgattg tgattattta catgattttg ttatttacgt atgtatatat gtagatctgg
     7621 actttttgga gttgttgact tgattgtatt tgtgtgtgta tatgtgtgtt ctgatcttga
     7681 tatgttatgt atgtgcagcg aattcggcgc gccatggata agaagtactc tatcggactc
     7741 gctatcggaa ctaactctgt gggatgggct gtgatcaccg atgagtacaa ggtgccatct
     7801 aagaagttca aggttctcgg aaacaccgat aggcactcta tcaagaaaaa ccttatcggt
     7861 gctctcctct tcgattctgg tgaaactgct gaggctacca gactcaagag aaccgctaga
     7921 agaaggtaca ccagaagaaa gaacaggatc tgctacctcc aagagatctt ctctaacgag
     7981 atggctaaag tggatgattc attcttccac aggctcgaag agtcattcct cgtggaagaa
     8041 gataagaagc acgagaggca ccctatcttc ggaaacatcg ttgatgaggt ggcataccac
     8101 gagaagtacc ctactatcta ccacctcaga aagaagctcg ttgattctac tgataaggct
     8161 gatctcaggc tcatctacct cgctctcgct cacatgatca agttcagagg acacttcctc
     8221 atcgagggtg atctcaaccc tgataactct gatgtggata agttgttcat ccagctcgtg
     8281 cagacctaca accagctttt cgaagagaac cctatcaacg cttcaggtgt ggatgctaag
     8341 gctatcctct ctgctaggct ctctaagtca agaaggcttg agaacctcat tgctcagctc
     8401 cctggtgaga agaagaacgg acttttcgga aacttgatcg ctctctctct cggactcacc
     8461 cctaacttca agtctaactt cgatctcgct gaggatgcaa agctccagct ctcaaaggat
     8521 acctacgatg atgatctcga taacctcctc gctcagatcg gagatcagta cgctgatttg
     8581 ttcctcgctg ctaagaacct ctctgatgct atcctcctca gtgatatcct cagagtgaac
     8641 accgagatca ccaaggctcc actctcagct tctatgatca agagatacga tgagcaccac
     8701 caggatctca cacttctcaa ggctcttgtt agacagcagc tcccagagaa gtacaaagag
     8761 attttcttcg atcagtctaa gaacggatac gctggttaca tcgatggtgg tgcatctcaa
     8821 gaagagttct acaagttcat caagcctatc ctcgagaaga tggatggaac cgaggaactc
     8881 ctcgtgaagc tcaatagaga ggatcttctc agaaagcaga ggaccttcga taacggatct
     8941 atccctcatc agatccacct cggagagttg cacgctatcc ttagaaggca agaggatttc
     9001 tacccattcc tcaaggataa cagggaaaag attgagaaga ttctcacctt cagaatccct
     9061 tactacgtgg gacctctcgc tagaggaaac tcaagattcg cttggatgac cagaaagtct
     9121 gaggaaacca tcaccccttg gaacttcgaa gaggtggtgg ataagggtgc tagtgctcag
     9181 tctttcatcg agaggatgac caacttcgat aagaaccttc caaacgagaa ggtgctccct
     9241 aagcactctt tgctctacga gtacttcacc gtgtacaacg agttgaccaa ggttaagtac
     9301 gtgaccgagg gaatgaggaa gcctgctttt ttgtcaggtg agcaaaagaa ggctatcgtt
     9361 gatctcttgt tcaagaccaa cagaaaggtg accgtgaagc agctcaaaga ggattacttc
     9421 aagaaaatcg agtgcttcga ttcagttgag atttctggtg ttgaggatag gttcaacgca
     9481 tctctcggaa cctaccacga tctcctcaag atcattaagg ataaggattt cttggataac
     9541 gaggaaaacg aggatatctt ggaggatatc gttcttaccc tcaccctctt tgaagataga
     9601 gagatgattg aagaaaggct caagacctac gctcatctct tcgatgataa ggtgatgaag
     9661 cagttgaaga gaagaagata cactggttgg ggaaggctct caagaaagct cattaacgga
     9721 atcagggata agcagtctgg aaagacaatc cttgatttcc tcaagtctga tggattcgct
     9781 aacagaaact tcatgcagct catccacgat gattctctca cctttaaaga ggatatccag
     9841 aaggctcagg tttcaggaca gggtgatagt ctccatgagc atatcgctaa cctcgctgga
     9901 tctcctgcaa tcaagaaggg aatcctccag actgtgaagg ttgtggatga gttggtgaag
     9961 gtgatgggaa ggcataagcc tgagaacatc gtgatcgaaa tggctagaga gaaccagacc
    10021 actcagaagg gacagaagaa ctctagggaa aggatgaaga ggatcgagga aggtatcaaa
    10081 gagcttggat ctcagatcct caaagagcac cctgttgaga acactcagct ccagaatgag
    10141 aagctctacc tctactacct ccagaacgga agggatatgt atgtggatca agagttggat
    10201 atcaacaggc tctctgatta cgatgttgat catatcgtgc cacagtcatt cttgaaggat
    10261 gattctatcg ataacaaggt gctcaccagg tctgataaga acaggggtaa gagtgataac
    10321 gtgccaagtg aagaggttgt gaagaaaatg aagaactatt ggaggcagct cctcaacgct
    10381 aagctcatca ctcagagaaa gttcgataac ttgactaagg ctgagagggg aggactctct
    10441 gaattggata aggcaggatt catcaagagg cagcttgtgg aaaccaggca gatcactaag
    10501 cacgttgcac agatcctcga ttctaggatg aacaccaagt acgatgagaa cgataagttg
    10561 atcagggaag tgaaggttat caccctcaag tcaaagctcg tgtctgattt cagaaaggat
    10621 ttccaattct acaaggtgag ggaaatcaac aactaccacc acgctcacga tgcttacctt
    10681 aacgctgttg ttggaaccgc tctcatcaag aagtatccta agctcgagtc agagttcgtg
    10741 tacggtgatt acaaggtgta cgatgtgagg aagatgatcg ctaagtctga gcaagagatc
    10801 ggaaaggcta ccgctaagta tttcttctac tctaacatca tgaatttctt caagaccgag
    10861 attaccctcg ctaacggtga gatcagaaag aggccactca tcgagacaaa cggtgaaaca
    10921 ggtgagatcg tgtgggataa gggaagggat ttcgctaccg ttagaaaggt gctctctatg
    10981 ccacaggtga acatcgttaa gaaaaccgag gtgcagaccg gtggattctc taaagagtct
    11041 atcctcccta agaggaactc tgataagctc attgctagga agaaggattg ggaccctaag
    11101 aaatacggtg gtttcgattc tcctaccgtg gcttactctg ttctcgttgt ggctaaggtt
    11161 gagaagggaa agagtaagaa gctcaagtct gttaaggaac ttctcggaat cactatcatg
    11221 gaaaggtcat ctttcgagaa gaacccaatc gatttcctcg aggctaaggg atacaaagag
    11281 gttaagaagg atctcatcat caagctccca aagtactcac tcttcgaact cgagaacggt
    11341 agaaagagga tgctcgcttc tgctggtgag cttcaaaagg gaaacgagct tgctctccca
    11401 tctaagtacg ttaactttct ttacctcgct tctcactacg agaagttgaa gggatctcca
    11461 gaagataacg agcagaagca acttttcgtt gagcagcaca agcactactt ggatgagatc
    11521 atcgagcaga tctctgagtt ctctaaaagg gtgatcctcg ctgatgcaaa cctcgataag
    11581 gtgttgtctg cttacaacaa gcacagagat aagcctatca gggaacaggc agagaacatc
    11641 atccatctct tcacccttac caacctcggt gctcctgctg ctttcaagta cttcgataca
    11701 accatcgata ggaagagata cacctctacc aaagaagtgc tcgatgctac cctcatccat
    11761 cagtctatca ctggactcta cgagactagg atcgatctct cacagctcgg tggtgattca
    11821 agggctgatc ctaagaagaa gaggaaggtt tgaggcgcgc cgagctccag gcctcccagc
    11881 tttcgtccgt atcatcggtt tcgacaacgt tcgtcaagtt caatgcatca gtttcattgc
    11941 ccacacacca gaatcctact aagtttgagt attatggcat tggaaaagct gttttcttct
    12001 atcatttgtt ctgcttgtaa tttactgtgt tctttcagtt tttgttttcg gacatcaaaa
    12061 tgcaaatgga tggataagag ttaataaatg atatggtcct tttgttcatt ctcaaattat
    12121 tattatctgt tgtttttact ttaatgggtt gaatttaagt aagaaaggaa ctaacagtgt
    12181 gatattaagg tgcaatgtta gacatataaa acagtctttc acctctcttt ggttatgtct
    12241 tgaattggtt tgtttcttca cttatctgtg taatcaagtt tactatgagt ctatgatcaa
    12301 gtaattatgc aatcaagtta agtacagtat aggcttgagc tccctaggcc tgttatccct
    12361 aacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt
    12421 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
    12481 actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
    12541 gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag
    12601 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
    12661 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
    12721 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
    12781 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg
    12841 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
    12901 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
    12961 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
    13021 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
    13081 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
    13141 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
    13201 agaatgctta atgaattaca acagtactgc gatgagtggc aggcggggcg taatctagag
    13261 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
    13321 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
    13381 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
    13441 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
    13501 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
    13561 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
    13621 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
    13681 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tcccccgtga
    13741 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
    13801 cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
    13861 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
    13921 cagccagtct gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag
    13981 tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc
    14041 gttcagcttt cttgtacaaa gtggttcgat aattccgatc cagcctaggc ccgggcctga
    14101 ggacgcgtcc atggttaatt aagacgtccg gaccgactag tggatcctct agagtcgacc
    14161 tgcaggcatg caagcttcag cttgccaaca tggtggagca cgacactctc gtctactcca
    14221 agaatatcaa agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg
    14281 taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcaaaagga
    14341 cagtagaaaa ggaaggtggc acctacaaat gccatcattg cgataaagga aaggctatcg
    14401 ttcaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg
    14461 tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gataacatgg
    14521 tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaaa
    14581 gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc
    14641 cagctatctg tcacttcatc aaaaggacag tagaaaagga aggtggcacc tacaaatgcc
    14701 atcattgcga taaaggaaag gctatcgttc aagatgcctc tgccgacagt ggtcccaaag
    14761 atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa
    14821 agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc
    14881 cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg acacgctgaa
    14941 atcaccagtc tctctctaca aatctatctc tctcgattcg cagatctgtc gatcgaccat
    15001 ggggattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
    15061 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
    15121 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
    15181 ccaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
    15241 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
    15301 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
    15361 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
    15421 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
    15481 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
    15541 cggcgaggat ctcgtcgtga cacatggcga tgcctgcttg ccgaatatca tggtggaaaa
    15601 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
    15661 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
    15721 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
    15781 tgacgagttc ttctgagcgg gactctgggg ttcggatcga tcctctagct agagtcgatc
    15841 gacatcgagt ttctccataa taatgtgtga gtagttccca gataagggaa ttagggttct
    15901 tatagggttt cgctcacgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt
    15961 aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagta ctaaaatcca
    16021 gatcacctaa agtccctata gatcccccga attaattcgg cgttaattca gtacattaaa
    16081 aacgtccgca atgtgttatt aagttgtcaa gcttggcact ggccgtcgtt ttacaacgtc
    16141 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
    16201 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
    16261 tgaatggcga atgagcttga gcttggatca gattgtcgtt tcccgccttc agtttaaact
    16321 atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa
    16381 taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc
    16441 caaccacagg gttcccctcg ggatcaa
//