LOCUS       sequence-182475-       16058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct Cloning, Type: T-DNA, Engineering Reagent: 
            35S:Csy4-P2A-AtCas9_dead + CmYLCV:gRNAs with Csy4 spacers, Plant 
            Selection: 2x35S:npt II.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16058)
  AUTHORS   Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, 
            Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF
  TITLE     A multi-purpose toolkit to enable advanced genome engineering in 
            plants.
  JOURNAL   Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 
            10.1105/tpc.16.00922.
  PUBMED    28522548
REFERENCE   2  (bases 1 to 16058)
  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 Cell.
            2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922."
COMMENT     Sequence Label: pDIRECT_22D
FEATURES             Location/Qualifiers
     source          1..16058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(291..1085)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    1510..1534
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    1612..1786
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(1843..2640)
                     /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(1954..1973)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2564..2583
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2703..3379)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     protein_bind    3570..3591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3606..3611,3612..3629,3630..3636)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3644..3660
                     /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     3649..3671
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3668..3684
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3668..3684
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4214..4559
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             5151..5207
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5208..5234,5235..5237,5238..7724,7725..7727,
                     7728..9311)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9324..9344
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      9355..9603
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    10108..10127
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        10128..10203
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    10204..10223
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             10543..10848
                     /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     10727..10746
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    10876..11050
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(11069..11086)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11069..11085)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11078..11100)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    11288..11312
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             12613..13242
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             13671..14744
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      14810..15004
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(15240..15262)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    15348..15488
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     15403..15422
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(15503..15520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(15674..16058,1..204))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(15754..15773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
       61 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      241 cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat
      301 tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca
      361 aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag
      421 gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact
      481 ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc
      541 tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg
      601 tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat
      661 tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga
      721 aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca
      781 tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca
      841 tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg
      901 tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa
      961 tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta
     1021 tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta
     1081 gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt
     1141 ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa
     1201 cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt
     1261 tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc
     1321 tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat
     1381 cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac
     1441 tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg
     1501 gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa
     1561 cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat
     1621 tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa
     1681 tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac
     1741 cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga
     1801 tcgactctag ctagaggatc gatccgaacc ccagagtccc gctcagaaga actcgtcaag
     1861 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     1921 gcggtcagcc cattcgccgc caagttcttc agcaatatca cgggtagcca acgctatgtc
     1981 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     2041 ttccaccatg atattcggca agcaggcatc gccatgtgtc acgacgagat cctcgccgtc
     2101 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgttcttc
     2161 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2221 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2281 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2341 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2401 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctggag
     2461 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2521 cagccgaaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2581 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatccccat
     2641 ggtcgatcga cagatctgcg aaagctcgag agagatagat ttgtagagag agactggtga
     2701 tttcagcgtg tcctctccaa atgaaatgaa cttccttata tagaggaagg gtcttgcgaa
     2761 ggatagtggg attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc
     2821 tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca
     2881 tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga
     2941 tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct
     3001 gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc
     3061 ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc
     3121 accatgttca catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac
     3181 gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac
     3241 gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt
     3301 ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac
     3361 cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt
     3421 ggagtagacg agagtgtcgt gctccaccat gttggcaagc tgctctagcc aatacgcaaa
     3481 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3541 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     3601 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     3661 tttcacacag gaaacagcta tgacatgatt acgaattcga gctcggtacc cggggatcgg
     3721 cgcgccagat ttgccttttc aatttcagaa agaatgctaa cccacagatg gttagagagg
     3781 cttacgcagc aggtatcatc aagacgatct acccgagcaa taatctccag gaaatcaaat
     3841 accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc atcaagaaca
     3901 cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg attcaaggct
     3961 tgcttcacaa accaaggcaa gtaatagaga ttggagtctc taaaaaggta gttcccactg
     4021 aatcaaaggc catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga
     4081 ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca
     4141 acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag
     4201 accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc
     4261 attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca
     4321 aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc
     4381 ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt
     4441 cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc
     4501 actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac
     4561 gggggactcc tgcaggaaaa atggatcatt atcttgatat tagacttaga cctgatccag
     4621 aatttccacc agctcaactt atgtctgttc tttttggaaa acttcatcaa gctcttgttg
     4681 ctcaaggagg agatagaatt ggagtttctt ttcctgatct tgatgaatca agatcaagac
     4741 ttggagaaag acttagaatt catgcttctg ctgatgatct tagagctttg cttgctagac
     4801 cttggcttga aggacttaga gatcatcttc aatttggaga accagctgtt gttccacatc
     4861 caactcctta tagacaagtt tcaagagttc aagctaaatc taatccagaa agacttagaa
     4921 gaagacttat gagaagacat gatctttctg aagaagaagc tagaaaaaga attcctgata
     4981 ctgttgctag agctttggat ttgccttttg ttacacttag atcacaatct actggacaac
     5041 attttagact ttttattaga catggaccac ttcaagttac tgctgaagaa ggaggattta
     5101 cttgttatgg actttctaag ggaggttttg ttccttggtt tggatctgga gctactaatt
     5161 tttctcttct taagcaagct ggagatgttg aagaaaatcc tggacccatg gataagaagt
     5221 actctatcgg actcgctatc ggaactaact ctgtgggatg ggctgtgatc accgatgagt
     5281 acaaggtgcc atctaagaag ttcaaggttc tcggaaacac cgataggcac tctatcaaga
     5341 aaaaccttat cggtgctctc ctcttcgatt ctggtgaaac tgctgaggct accagactca
     5401 agagaaccgc tagaagaagg tacaccagaa gaaagaacag gatctgctac ctccaagaga
     5461 tcttctctaa cgagatggct aaagtggatg attcattctt ccacaggctc gaagagtcat
     5521 tcctcgtgga agaagataag aagcacgaga ggcaccctat cttcggaaac atcgttgatg
     5581 aggtggcata ccacgagaag taccctacta tctaccacct cagaaagaag ctcgttgatt
     5641 ctactgataa ggctgatctc aggctcatct acctcgctct cgctcacatg atcaagttca
     5701 gaggacactt cctcatcgag ggtgatctca accctgataa ctctgatgtg gataagttgt
     5761 tcatccagct cgtgcagacc tacaaccagc ttttcgaaga gaaccctatc aacgcttcag
     5821 gtgtggatgc taaggctatc ctctctgcta ggctctctaa gtcaagaagg cttgagaacc
     5881 tcattgctca gctccctggt gagaagaaga acggactttt cggaaacttg atcgctctct
     5941 ctctcggact cacccctaac ttcaagtcta acttcgatct cgctgaggat gcaaagctcc
     6001 agctctcaaa ggatacctac gatgatgatc tcgataacct cctcgctcag atcggagatc
     6061 agtacgctga tttgttcctc gctgctaaga acctctctga tgctatcctc ctcagtgata
     6121 tcctcagagt gaacaccgag atcaccaagg ctccactctc agcttctatg atcaagagat
     6181 acgatgagca ccaccaggat ctcacacttc tcaaggctct tgttagacag cagctcccag
     6241 agaagtacaa agagattttc ttcgatcagt ctaagaacgg atacgctggt tacatcgatg
     6301 gtggtgcatc tcaagaagag ttctacaagt tcatcaagcc tatcctcgag aagatggatg
     6361 gaaccgagga actcctcgtg aagctcaata gagaggatct tctcagaaag cagaggacct
     6421 tcgataacgg atctatccct catcagatcc acctcggaga gttgcacgct atccttagaa
     6481 ggcaagagga tttctaccca ttcctcaagg ataacaggga aaagattgag aagattctca
     6541 ccttcagaat cccttactac gtgggacctc tcgctagagg aaactcaaga ttcgcttgga
     6601 tgaccagaaa gtctgaggaa accatcaccc cttggaactt cgaagaggtg gtggataagg
     6661 gtgctagtgc tcagtctttc atcgagagga tgaccaactt cgataagaac cttccaaacg
     6721 agaaggtgct ccctaagcac tctttgctct acgagtactt caccgtgtac aacgagttga
     6781 ccaaggttaa gtacgtgacc gagggaatga ggaagcctgc ttttttgtca ggtgagcaaa
     6841 agaaggctat cgttgatctc ttgttcaaga ccaacagaaa ggtgaccgtg aagcagctca
     6901 aagaggatta cttcaagaaa atcgagtgct tcgattcagt tgagatttct ggtgttgagg
     6961 ataggttcaa cgcatctctc ggaacctacc acgatctcct caagatcatt aaggataagg
     7021 atttcttgga taacgaggaa aacgaggata tcttggagga tatcgttctt accctcaccc
     7081 tctttgaaga tagagagatg attgaagaaa ggctcaagac ctacgctcat ctcttcgatg
     7141 ataaggtgat gaagcagttg aagagaagaa gatacactgg ttggggaagg ctctcaagaa
     7201 agctcattaa cggaatcagg gataagcagt ctggaaagac aatccttgat ttcctcaagt
     7261 ctgatggatt cgctaacaga aacttcatgc agctcatcca cgatgattct ctcaccttta
     7321 aagaggatat ccagaaggct caggtttcag gacagggtga tagtctccat gagcatatcg
     7381 ctaacctcgc tggatctcct gcaatcaaga agggaatcct ccagactgtg aaggttgtgg
     7441 atgagttggt gaaggtgatg ggaaggcata agcctgagaa catcgtgatc gaaatggcta
     7501 gagagaacca gaccactcag aagggacaga agaactctag ggaaaggatg aagaggatcg
     7561 aggaaggtat caaagagctt ggatctcaga tcctcaaaga gcaccctgtt gagaacactc
     7621 agctccagaa tgagaagctc tacctctact acctccagaa cggaagggat atgtatgtgg
     7681 atcaagagtt ggatatcaac aggctctctg attacgatgt tgatgctatc gtgccacagt
     7741 cattcttgaa ggatgattct atcgataaca aggtgctcac caggtctgat aagaacaggg
     7801 gtaagagtga taacgtgcca agtgaagagg ttgtgaagaa aatgaagaac tattggaggc
     7861 agctcctcaa cgctaagctc atcactcaga gaaagttcga taacttgact aaggctgaga
     7921 ggggaggact ctctgaattg gataaggcag gattcatcaa gaggcagctt gtggaaacca
     7981 ggcagatcac taagcacgtt gcacagatcc tcgattctag gatgaacacc aagtacgatg
     8041 agaacgataa gttgatcagg gaagtgaagg ttatcaccct caagtcaaag ctcgtgtctg
     8101 atttcagaaa ggatttccaa ttctacaagg tgagggaaat caacaactac caccacgctc
     8161 acgatgctta ccttaacgct gttgttggaa ccgctctcat caagaagtat cctaagctcg
     8221 agtcagagtt cgtgtacggt gattacaagg tgtacgatgt gaggaagatg atcgctaagt
     8281 ctgagcaaga gatcggaaag gctaccgcta agtatttctt ctactctaac atcatgaatt
     8341 tcttcaagac cgagattacc ctcgctaacg gtgagatcag aaagaggcca ctcatcgaga
     8401 caaacggtga aacaggtgag atcgtgtggg ataagggaag ggatttcgct accgttagaa
     8461 aggtgctctc tatgccacag gtgaacatcg ttaagaaaac cgaggtgcag accggtggat
     8521 tctctaaaga gtctatcctc cctaagagga actctgataa gctcattgct aggaagaagg
     8581 attgggaccc taagaaatac ggtggtttcg attctcctac cgtggcttac tctgttctcg
     8641 ttgtggctaa ggttgagaag ggaaagagta agaagctcaa gtctgttaag gaacttctcg
     8701 gaatcactat catggaaagg tcatctttcg agaagaaccc aatcgatttc ctcgaggcta
     8761 agggatacaa agaggttaag aaggatctca tcatcaagct cccaaagtac tcactcttcg
     8821 aactcgagaa cggtagaaag aggatgctcg cttctgctgg tgagcttcaa aagggaaacg
     8881 agcttgctct cccatctaag tacgttaact ttctttacct cgcttctcac tacgagaagt
     8941 tgaagggatc tccagaagat aacgagcaga agcaactttt cgttgagcag cacaagcact
     9001 acttggatga gatcatcgag cagatctctg agttctctaa aagggtgatc ctcgctgatg
     9061 caaacctcga taaggtgttg tctgcttaca acaagcacag agataagcct atcagggaac
     9121 aggcagagaa catcatccat ctcttcaccc ttaccaacct cggtgctcct gctgctttca
     9181 agtacttcga tacaaccatc gataggaaga gatacacctc taccaaagaa gtgctcgatg
     9241 ctaccctcat ccatcagtct atcactggac tctacgagac taggatcgat ctctcacagc
     9301 tcggtggtga ttcaagggct gatcctaaga agaagaggaa ggtttgactc gagatatgaa
     9361 gatgaagatg aaatatttgg tgtgtcaaat aaaaagcttg tgtgcttaag tttgtgtttt
     9421 tttcttggct tgttgtgtta tgaatttgtg gctttttcta atattaaatg aatgtaagat
     9481 cacattataa tgaataaaca aatgtttcta taatccattg tgaatgtttt gttggatctc
     9541 ttctgcagca tataactact gtatgtgcta tggtatggac tatggaatat gattaaagat
     9601 aaggagctcc ggtgacggac ggcgcgccga agagctggca gacatactgt cccacaaatg
     9661 aagatggaat ctgtaaaaga aaacgcgtga aataatgcgt ctgacaaagg ttaggtcggc
     9721 tgcctttaat caataccaaa gtggtcccta ccacgatgga aaaactgtgc agtcggtttg
     9781 gctttttctg acgaacaaat aagattcgtg gccgacaggt gggggtccac catgtgaagg
     9841 catcttcaga ctccaataat ggagcaatga cgtaagggct tacgaaataa gtaagggtag
     9901 tttgggaaat gtccactcac ccgtcagtct ataaatactt agcccctccc tcattgttaa
     9961 gggagcaaaa tctcagagag atagtcctag agagagaaag agagcaagta gcctagaagt
    10021 agtcaaggcg gcgaagtatt caggcacgtg gccaggaaga agaaaagcca agacgacgaa
    10081 aacaggtaag agctaagctt cctgcaggtt cactgccgta taggcaggtt ttagagctag
    10141 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
    10201 tgcgttcact gccgtatagg cagaagccag ataacagtat gcgtatttgc gcgctgattt
    10261 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaag gaggtatgct
    10321 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
    10381 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
    10441 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
    10501 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
    10561 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
    10621 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
    10681 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
    10741 gatatggcca gtgtgccggt ttccgttatc ggggaagaag tggctgatct cagccaccgc
    10801 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataagc tcttcagtcg
    10861 atcgacaagc tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag
    10921 ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt
    10981 atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa
    11041 aatccagatc ccccgaatta ccggtggcac tggccgtcgt tttacaacgt cgtgactggg
    11101 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
    11161 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
    11221 aatgctagag cagcttgagc ttggatcaga ttgtcgtttc ccgccttcag tttaaactat
    11281 cagtgtttga caggatatat tggcgggtaa acctaagaga aaagagcgtt tattagaata
    11341 atcggatatt taaaagggcg tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc
    11401 aaccacaggg ttcccctcgg gatcaaagta ctttgatcca acccctccgc tgctatagtg
    11461 cagtcggctt ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct
    11521 aagttacgcg acaggctgcc gccctgccct tttcctggcg ttttcttgtc gcgtgtttta
    11581 gtcgcataaa gtagaatact tgcgactaga accggagaca ttacgccatg aacaagagcg
    11641 ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc
    11701 aacgggccga actgcacgcg gccggctgca ccaagctgtt ttccgagaag atcaccggca
    11761 ccaggcgcga ccgcccggag ctggccagga tgcttgacca cctacgccct ggcgacgttg
    11821 tgacagtgac caggctagac cgcctggccc gcagcacccg cgacctactg gacattgccg
    11881 agcgcatcca ggaggccggc gcgggcctgc gtagcctggc agagccgtgg gccgacacca
    11941 ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt
    12001 ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt
    12061 ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag ctgatcgacc
    12121 aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt
    12181 accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct
    12241 tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat gaacgccaag
    12301 aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca ttaccgaaga
    12361 gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc acggctcaac
    12421 cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg cctggccggc
    12481 cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta
    12541 aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata aacaaatacg
    12601 caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc aggcaagacg
    12661 accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt tctgttagtc
    12721 gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta
    12781 accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat cggccggcgc
    12841 gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc cgcgatcaag
    12901 gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg ggccaccgcc
    12961 gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct acaagcggcc
    13021 tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg
    13081 gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc
    13141 actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag
    13201 gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga ggtaaagaga
    13261 aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg
    13321 ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac tttcagttgc
    13381 cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag accattaccg
    13441 agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga ataaatgagt
    13501 agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc aggcaccgac
    13561 gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag cggctgggtt
    13621 gcctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc gtgagcggtc
    13681 gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt
    13741 tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat
    13801 cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg
    13861 cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc
    13921 tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt
    13981 cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag
    14041 aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg
    14101 cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg
    14161 gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg
    14221 gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca
    14281 tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct
    14341 tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg
    14401 agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg
    14461 ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac
    14521 gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg
    14581 gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg
    14641 acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc
    14701 gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc
    14761 tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg cctctttcct gtggatagca
    14821 cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa
    14881 agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag aaaaaaggcg
    14941 atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg
    15001 cataactgtc tggccagcgc acagcccaag agctgcaaaa agcgcctacc cttcggtcgc
    15061 tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa
    15121 tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg
    15181 accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac
    15241 ctctgacaca tgcagctccc ggaaacggtc acagcttgtc tgtaagcgga tgccgggagc
    15301 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
    15361 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
    15421 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
    15481 gcatcaggcc ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
    15541 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
    15601 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    15661 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    15721 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    15781 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    15841 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    15901 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    15961 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    16021 cagcagccac tggtaacagg attagcagag cgaggtat
//
