LOCUS       sequence-260727-       12824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Episome based vector with CRISPR/Cas9_sgRNA module for genome 
            editing in Phaeodactylum tricornutum.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12824)
  AUTHORS   Sharma AK, Nymark M, Sparstad T, Bones AM, Winge P
  TITLE     Transgene-free genome editing in marine algae by bacterial 
            conjugation - comparison with biolistic CRISPR/Cas9 transformation.
  JOURNAL   Sci Rep. 2018 Sep 26;8(1):14401. doi: 10.1038/s41598-018-32342-0.
  PUBMED    30258061
REFERENCE   2  (bases 1 to 12824)
  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: "Sci Rep."; 
            date: "2018-09-26"; volume: "8(1):14401. doi"; pages: " 
            10.1038/s41598-018-32342-0"
COMMENT     Sequence Label: PtPuc3_diaCas9_sgRNA
FEATURES             Location/Qualifiers
     source          1..12824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        477..663
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             664..1326
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     CDS             1818..1883
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1890..1910
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1935..6035
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1935..6035
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6036..6083
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_RNA        6619..6694
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(7062..7078)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7062..7078)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7075..7097)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7086..7102
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7110..7116,7117..7134,7135..7140))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7155..7176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7293..7310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7464..8052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7544..7563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(8889..8908)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             8900..9715
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMHNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(8973..8992)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     9816..9835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10054..10158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10226..10244
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(10282..10304)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     10404..10423
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(10565..10936)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            10969..11078
                     /note="incP origin of transfer"
     primer_bind     11340..11362
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11354..11371
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11355..11371
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        11395..11413
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             12117..12491
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    join(12797..12824,1..476)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
       61 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
      121 ccttttacct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
      181 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
      241 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
      301 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
      361 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
      421 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatcctag
      481 tacactctat atttttttat gcctcggtaa tgattttcat tttttttttc cacctagcgg
      541 atgactcttt ttttttctta gcgattggca ttatcacata atgaattata cattatataa
      601 agtaatgtga tttcttcgaa gaatatacta aaaaatgagc aggcaagata aacgaaggca
      661 aagatgacag agcagaaagc cctagtaaag cgtattacaa atgaaaccaa gattcagatt
      721 gcgatctctt taaagggtgg tcccctagcg atagagcact cgatcttccc agaaaaagag
      781 gcagaagcag tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca cacaggtata
      841 gggtttctgg accatatgat acatgctctg gccaagcatt ccggctggtc gctaatcgtt
      901 gagtgcattg gtgacttaca catagacgac catcacacca ctgaagactg cgggattgct
      961 ctcggtcaag cttttaaaga ggccctaggg gccgtgcgtg gagtaaaaag gtttggatca
     1021 ggatttgcgc ctttggatga ggcactttcc agagcggtgg tagatctttc gaacaggccg
     1081 tacgcagttg tcgaacttgg tttgcaaagg gagaaagtag gagatctctc ttgcgagatg
     1141 atcccgcatt ttcttgaaag ctttgcagag gctagcagaa ttaccctcca cgttgattgt
     1201 ctgcgaggca agaatgatca tcaccgtagt gagagtgcgt tcaaggctct tgcggttgcc
     1261 ataagagaag ccacctcgcc caatggtacc aacgatgttc cctccaccaa aggtgttctt
     1321 atgtagtttt acacaggagt ctggacttga cctgagctca atctcgccta ttcatggtgt
     1381 ataaaagttc aacatccaaa gctagaactt ttggaaagag aaagaatatc cgaatagggc
     1441 acggcgtgcc gtattgttgg agtggactag cagaaagtga ggaaggcaca ggatgagttt
     1501 tctcgagaca catagcttca gcgtcgtgta ggctaggcag aggtgagttt tctcgagaca
     1561 taccttcagc gtcgtcttca ctgtcacagt caactgacag taatcgttga tccggagaga
     1621 ttcaaaattc aatctgtttg gacctggata agacacaaga gcgacatcct gacatgaacg
     1681 ccgtaaacag caaatcctgg ttgaacacgt atccttttgg gggcctccgc tacgacgctc
     1741 gctccagctg gggcttcctt actatacaca gcgcgcatat ttcacggttg ccagaagtca
     1801 agatggcctc tagaatggac tataaggacc acgacggaga ttacaaggat catgatattg
     1861 attacaaaga cgatgatgat aaaatggccc caaagaagaa gcggaaagtc ggtatacacg
     1921 gagtcccagc agccgacaag aagtactcga ttggattgga catcggcacc aactctgttg
     1981 gatgggccgt catcacggac gagtacaagg tgcccagcaa gaaattcaaa gtactgggaa
     2041 acactgaccg ccattcgatc aagaagaatt tgattggagc tttgctgttt gacagcggcg
     2101 aaacagcgga agccacgcgg cttaaacgaa ccgcgagacg aagatatacg cgacggaaga
     2161 accgtatctg ctatctacaa gagattttct cgaatgagat ggcaaaagtc gatgactctt
     2221 tctttcaccg attggaagaa tccttcctcg ttgaagagga taagaaacat gagcggcacc
     2281 cgatatttgg aaacattgtg gacgaggtgg cttaccatga gaaatacccg actatttacc
     2341 acctgcgaaa gaaattggtg gactcgacgg acaaagcgga tcttcgccta atctatttgg
     2401 cgctagcaca tatgattaag tttcgcggcc acttcttgat cgaaggcgac ttgaatccgg
     2461 acaacagtga tgttgacaaa ctgtttattc aacttgtaca aacttacaac cagctctttg
     2521 aagaaaatcc cattaacgct tcgggagtgg acgcgaaagc catcctttct gcccgactca
     2581 gcaagagccg acgcctagaa aatttgattg cgcaacttcc cggcgagaaa aagaatggac
     2641 tttttggaaa tcttattgct ttgtcgctgg gccttacgcc gaactttaag agcaacttcg
     2701 accttgcgga agatgccaaa ttgcaactga gtaaagacac atatgacgac gacctcgaca
     2761 atcttttggc acaaatagga gatcagtacg ccgacctctt tttggcggct aagaacttgt
     2821 ccgacgccat tctgttgtcg gacatccttc gagttaatac ggagattact aaggcgccct
     2881 tgagcgcctc tatgatcaaa agatacgacg agcatcacca agacctgacc ctccttaaag
     2941 ctctcgtccg gcaacagctg cctgagaagt acaaagagat tttctttgat caaagcaaaa
     3001 acggatacgc gggctacatt gacgggggag cctcgcaaga agagttctac aagtttatta
     3061 aaccgatcct ggaaaagatg gacggcacgg aggaattact cgtgaaactt aaccgagagg
     3121 accttttgcg taagcaacgg actttcgaca acggatctat tccgcaccaa atccatctcg
     3181 gagaattgca cgcaattctg cgccggcaag aagattttta tccatttctc aaggacaacc
     3241 gggaaaagat tgagaaaatc ttaaccttcc gcatacccta ctacgtcggc cctttggcca
     3301 ggggaaattc gagatttgcc tggatgacgc gaaagagtga ggaaactatt actccctgga
     3361 atttcgaaga agtggtagac aaaggagctt ccgcgcaatc ctttatcgag cgcatgacca
     3421 acttcgataa gaacctcccg aatgagaaag tcctacccaa gcattcgttg ctctacgaat
     3481 actttacggt gtataatgag ctgactaaag tcaaatacgt tacagaggga atgcgaaaac
     3541 cggctttcct ctcgggagag cagaaaaagg ccatagtcga tcttctcttt aaaacgaacc
     3601 gtaaagttac cgtgaaacaa ctaaaagagg actacttcaa gaaaatcgaa tgctttgact
     3661 ccgttgaaat ttccggcgtt gaagatcggt tcaatgcctc cttgggaaca taccacgatc
     3721 tcctgaaaat tatcaaagac aaggactttc ttgacaatga ggaaaacgag gacattcttg
     3781 aagatattgt cttgacgcta acactttttg aggatcgaga gatgattgag gaacgcctta
     3841 aaacctatgc gcatctgttc gacgacaaag ttatgaagca attgaagcgc cggagatata
     3901 cgggatgggg aaggttgagt cggaaactga taaatggcat acgcgataag cagtccggca
     3961 aaacaattct cgatttcttg aagtccgacg gatttgcgaa tcgaaacttc atgcaactga
     4021 tccatgacga ttcgttgacc tttaaagagg acattcaaaa agcccaagta tccggacaag
     4081 gagattcttt gcacgagcat attgcgaatc ttgctggaag tccggctatt aaaaagggca
     4141 ttctccagac agtaaaggtc gtggatgaac tcgttaaagt tatgggccga cacaaacccg
     4201 agaacatagt catcgaaatg gcgagagaga accagaccac ccaaaaggga cagaaaaatt
     4261 cacgcgagcg aatgaagcgg attgaagagg ggatcaaaga gcttggcagc caaattctga
     4321 aagaacaccc ggttgaaaac acccaattgc agaacgagaa gctctacctg tactatcttc
     4381 aaaatgggcg tgatatgtac gtcgatcaag aattggacat caaccggcta tccgattacg
     4441 atgtggacca tattgttcct cagtcgtttc tcaaagacga ctccatagac aataaggttc
     4501 taacgagaag cgacaagaac cgcggcaaat cagacaacgt cccctccgag gaagtagtta
     4561 agaaaatgaa gaattactgg cgccaattgc tgaacgctaa gctcattacc caaagaaagt
     4621 ttgacaattt gacgaaagcc gagcgaggcg ggctctcgga actggataag gccggcttca
     4681 ttaaaagaca attggtggaa acgcgacaaa tcacaaagca cgtcgcacag attctcgact
     4741 cccgcatgaa cactaagtat gacgagaatg acaaactaat ccgggaagtc aaagtgatta
     4801 ccttgaagtc caagctggtt tccgattttc gtaaagattt tcagttttac aaagttcgcg
     4861 agatcaacaa ctaccatcac gcgcacgacg cttacttgaa cgctgtcgtg ggaacggcct
     4921 tgattaaaaa ataccctaag ctggaaagcg agttcgttta cggcgattac aaggtgtatg
     4981 acgtacgcaa aatgatcgcg aagtcagagc aggaaattgg caaagctact gcgaagtact
     5041 tcttttatag caacataatg aattttttca aaacggagat taccctcgct aacggcgaga
     5101 ttcgcaagcg ccctcttatc gagacaaacg gcgaaacggg ggagattgtc tgggataaag
     5161 gccgcgattt tgcgactgtg cggaaagtct tgtcgatgcc gcaagtgaat atagtcaaaa
     5221 agacggaggt gcaaacaggc ggcttttcga aagaatctat ccttcccaaa aggaactctg
     5281 ataagttgat tgcgcgaaaa aaggattggg accctaagaa gtatggcggc tttgacagcc
     5341 cgaccgtagc atattctgtc cttgtggtgg ccaaagtcga aaagggcaaa tccaagaaac
     5401 tgaagagtgt gaaagaattg cttgggatta cgatcatgga aagatcatct tttgagaaga
     5461 atcccatcga ctttctcgaa gctaaaggct acaaagaagt gaaaaaggac ctgattatca
     5521 agttgcctaa gtactccttg tttgagctgg aaaatggccg taagcgaatg ctcgcttctg
     5581 ccggcgaact ccagaaggga aacgaattgg cgctgccctc caaatatgtt aactttcttt
     5641 accttgcctc gcactatgag aagctcaagg gctcccccga ggataatgag caaaaacaac
     5701 tgtttgtaga acagcataag cactacttgg acgagatcat tgagcaaatt agcgagttct
     5761 ccaagcgagt catccttgcc gacgctaatc tggacaaagt cctgtccgcc tacaataagc
     5821 accgcgataa gccaatccga gagcaagcgg agaatattat ccacttgttt acgctcacca
     5881 atcttggagc ccctgccgcc tttaagtact ttgacaccac catcgaccgc aagaggtaca
     5941 ccagcaccaa agaagtcttg gacgccaccc tcattcacca atcaatcacc ggcttgtacg
     6001 agacacgcat cgacctgtct caactcggag gcgacaaaag gccggcggcc acgaaaaagg
     6061 ccggccaggc aaaaaagaaa aagtaatctg gatccctacc tcgactttgg ctgggacact
     6121 ttcagtgagg acaagaagct ccagaagcgt gctatcgaac tcaaccaggg acgtgcggca
     6181 caaatgggca tccttgctct catggtgcac gaacagttgg gagtctctat ccttccttaa
     6241 aaatttaatt ttcattagtt gcagtcactc cgctttggtt tcacagtcag gaataacact
     6301 agctcgtctt cactgcaggt tggctcggaa gttggtgttg acggtgagct ggaaattggt
     6361 tgtcggtcac tgctagcgag aagaaaacgg aggacagaag gaagtgaaac tcggttcgtt
     6421 ctcgacagcc tcactgtcaa tatgctcatt ttcaatcctt agcgctttta atgtcgaatt
     6481 gacggtaaat tgaataggat ctataatatc tacaaggtac tttgacacgc caagtattca
     6541 ttgttagtca acaatatttt agagctttat aaggtcaaaa aacaccttca aagtcgagga
     6601 gaccgagaga gggtctcagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     6661 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttagaaccgc tcacccatgc
     6721 tatcgtatgt catttacatt gacaaactgc tatgaatgcg tttgcgataa cttcagctcg
     6781 ctgaagccct agacatttcg tttgactaaa ttgttttccg gttgctttac taccgaccgg
     6841 tctgcgggta gcatgtcttg gcttttgcga gttcgagaga atttatggac aagtgcgttt
     6901 acagtttgga acacgagtgt gagtgagcgg aattggctat ctataacatt cttatttcag
     6961 gaggctggtg attagcagaa ttttcaaaga tctcgaatct aaaatcgcca tgggctcgac
     7021 gaacttttct gcaagcttca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     7081 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     7141 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     7201 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7261 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     7321 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     7381 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     7441 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     7501 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     7561 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     7621 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     7681 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     7741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7801 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7861 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7921 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7981 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     8041 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     8101 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     8161 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     8221 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     8281 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     8341 cccagtgctg caatgatacc gcgataccca cgctcaccgg ctccagattt atcagcaata
     8401 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     8461 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     8521 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     8581 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     8641 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     8701 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     8761 tctgtgactg gtgagtcgat ttattcaaca aagccacgtt gtgtctcaaa atctctgatg
     8821 ttgcattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     8881 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct cgaggccgcg
     8941 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     9001 gcaatcaggt gcgacaatct atcgattgta cgggaagccc gatgcgccag agttgtttct
     9061 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     9121 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     9181 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     9241 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     9301 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     9361 acgaatgcat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     9421 tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt
     9481 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     9541 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     9601 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     9661 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     9721 attggttaat tggttgtaac actggcactc aaccaagtca ttctgagaat agtgtatgcg
     9781 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     9841 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     9901 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     9961 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    10021 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    10081 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    10141 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
    10201 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
    10261 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
    10321 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
    10381 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccagat
    10441 cgtcttgcct tgctcgtcgg tgatgtactt caccagctcc gcgaagtcgc tcttcttgat
    10501 ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc cggccgtttt agcggctaaa
    10561 aaagtcatgg ctctgccctc gggcggacca cgcccatcat gaccttgcca agctcgtcct
    10621 gcttctcttc gatcttcgcc agcagggcga ggatcgtggc atcaccgaac cgcgccgtgc
    10681 gcgggtcgtc ggtgagccag agtttcagca ggccgcccag gcggcccagg tcgccattga
    10741 tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc cgtgattttg tagccctggc
    10801 cgacggccag caggtaggcc gacaggctca tgccggccgc cgccgccttt tcctcaatcg
    10861 ctcttcgttc gtctggaagg cagtacacct tgataggtgg gctgcccttc ctggttggct
    10921 tggtttcatc agccatccgc ttgccctcat ctgttacgcc ggcggtagcc ggccagcctc
    10981 gcagagcagg attcccgttg agcaccgcca ggtgcgaata agggacagtg aagaaggaac
    11041 acccgctcgc gggtgggcct acttcaccta tcctgcccgg ctgacgccgt tggatacacc
    11101 aaggaaagtc tacacgaacc ctttggcaaa atcctgtata tcgtgcgaaa aaggatggat
    11161 ataccgaaaa aatcgctata atgaccccga agcagggtta tgcagcggaa gatgccattc
    11221 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
    11281 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
    11341 ccagtcacga cgttgtaaaa cgacggccag tgaccatgat tacgccaagc tcgaaattaa
    11401 ccctcactaa agggaacaaa agctggtacc taacaggatt agtgcaattc gagttgaatc
    11461 actgggaaaa acattgtctt cttttttata ttatcatttg cattagtgct gcagtcgtag
    11521 atacttgttg gttgaaagac atcagctggg agggactgga ctagcgtttg gtaaggagac
    11581 atacctgtta acgttggttg caaaattcca tttcgcgatt tatgttatct gtaaatcctg
    11641 atttgtctgg aattcttgat acttccgttt ttttagaggc caatgattag catcggcgat
    11701 tctcaaaata gcattttcga catgcggtgc tgatttcata aacatagaca acgcttttac
    11761 atgtaaaagt aacttgcgga cttggaacag tgctctgttt ttggtgtgaa cgtaactcag
    11821 caatatttct gtgctagcaa ggttttttat gatcgaccga agatctcaaa actccgggtc
    11881 tttcaactgt ctgactagac catgttcgta acgtcggaca gcagctttcg ttgtactggt
    11941 agaatttcta cgtgcgaagc acgtgtaggc aggttgaacg acgatccctg ccgatggatg
    12001 gattggcacg cggcggaacg ctttcgtgat ctacaccacc tggatcttca catatcttcg
    12061 aaatcgaaaa attaaccaag tcgacggtat cgataatatt ctagctgagg gtacccatgg
    12121 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
    12181 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
    12241 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
    12301 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
    12361 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
    12421 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
    12481 agcaggactg accgacgccg accaacaccg ccggtccgac gcggcccgac gggtccgagg
    12541 cctcggagat ctgggcccat gcggccgcaa caactacctc gactttggct gggacacttt
    12601 cagtgaggac aagaagcttc agaagcgtgc tatcgaactc aaccagggac gtgcggcaca
    12661 aatgggcatc cttgctctca tggtgcacga acagttggga gtctctatcc ttccttaaaa
    12721 atttaatttt cattagttgc agtcactccg ctttggtttc acagtcagga ataacactag
    12781 ctcgtcttca gcgagcatca cgtgctataa aaataattat aatt
//
