LOCUS       sequence-217151-       12922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TevCas9 in Phaeodactylum tricornutum / Encodes elements 
            required for conjugation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12922)
  AUTHORS   Slattery SS, Diamond A, Wang H, Therrien JA, Lant JT, Jazey T, Lee 
            K, Klassen Z, Desgagne-Penix I, Karas BJ, Edgell DR
  TITLE     An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome 
            Editing and Stable Maintenance of Synthetic Pathways in 
            Phaeodactylum tricornutum.
  JOURNAL   ACS Synth Biol. 2018 Jan 8. doi: 10.1021/acssynbio.7b00191.
  PUBMED    29298053
REFERENCE   2  (bases 1 to 12922)
  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: "ACS Synth 
            Biol. 2018 Jan 8. doi: 10.1021/acssynbio.7b00191."
COMMENT     Sequence Label: pPtGE35
FEATURES             Location/Qualifiers
     source          1..12922
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(780..1151)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1184..1293
                     /note="incP origin of transfer"
     primer_bind     1570..1586
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1610..1628
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2249..2623
                     /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="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFVGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLNELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        3625..3810
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3811..4467
                     /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
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVL"
     CDS             5444..9544
                     /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             5444..9544
                     /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             9545..9592
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9599..9619
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9626..9691
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_RNA        10265..10340
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10687..10703)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10688..10704)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(10732..10750)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10771..10787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10771..10787)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10784..10806)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10795..10811
                     /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(10819..10825,10826..10843,10844..10849))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10864..10885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11002..11019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11173..11761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11253..11272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11932..12723,12724..12792))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     12555..12574
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12793..12897)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctgatcgtct
      661 tgccttgctc gtcggtgatg tacttcacca gctccgcgaa gtcgctcttc ttgatggagc
      721 gcatggggac gtgcttggca atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt
      781 catggctctg ccctcgggcg gaccacgccc atcatgacct tgccaagctc gtcctgcttc
      841 tcttcgatct tcgccagcag ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg
      901 tcgtcggtga gccagagttt cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg
      961 gccagctcgc ggacgtgctc atagtccacg acgcccgtga ttttgtagcc ctggccgacg
     1021 gccagcaggt aggccgacag gctcatgccg gccgccgccg ccttttcctc aatcgctctt
     1081 cgttcgtctg gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt
     1141 tcatcagcca tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga
     1201 gcaggattcc cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg
     1261 ctcgcgggtg ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga
     1321 aagtctacac gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc
     1381 gaaaaaatcg ctataatgac cccgaagcag ggttatgcag cggaagatgc cattcgccat
     1441 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     1501 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     1561 cacgacgttg taaaacgacg gccagtgacc atgattacgc caagctcgaa attaaccctc
     1621 actaaaggga acaaaagctg gtacctaacc ctgcgataga ccttttccaa actcacgcag
     1681 tccaagaaaa caaaggggtg agaagtatac gcacctttcg gtttcggcat aattcttaaa
     1741 ctcttgtggt cactttcttg tgaagaagct aggggcactc gttttccctc agagcctgca
     1801 aacacaaaat tcctgcagtc aattgtccca acactcggca agccgtatgc gcaagcaacg
     1861 atgcgcagaa ggccgtggat ggatggcgac tcgcgatatg gcttcttggg tcgccagtgt
     1921 ggtatgtccg gcgtatgtca atacgcgaat tcggacgact ggcatctcta ggaggaggat
     1981 tccttctttt atgacatgtt tattttatat acattgatgc tttccgacag tcggaagtaa
     2041 taaatgaatt tatttcaaga ctacctatac tcctttgact tgttcgacta atcttaccgc
     2101 ttactaaaat ctcgaaatca cgcttgacct ctcgcacgca aatttttgct gctggacgct
     2161 acgcactcgg cccaattctt ctcggtcctc gtcgtcgcaa ttgtcgttgc gttgatcttg
     2221 caccgaagga atcagagaat agaataccat ggccaagttg accagtgccg ttccggtgct
     2281 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     2341 ggacttcgtg gaggacgact tcgtcggtgt ggtccgggac gacgtgaccc tgttcatcag
     2401 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     2461 gaacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     2521 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     2581 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgaagataag aatatctcat
     2641 tgtgaacatc tatgatttac caattttatt ctttgtttac agttagacgc cagtaattgt
     2701 gctgtttctc tcaagtctgt gtcaatacaa actacgaaac ttggcaattt ttctcttgaa
     2761 tatgagcacg agattgaaac gcacaaagga aattagtttc catcctttga caaagtttgt
     2821 tgctgtttag agaacagatg tcaaaattaa cgtgccatgg aattgaacca tggtgcgcta
     2881 tccccaaatc acgtcgtttg acctcgtcac atttaaggta tatcaagcat attcacttat
     2941 atcttgacat cctccctgct tgatattcct ttgcccttga gccatcttcc ccacacgctg
     3001 gagatgaccc ctgcccctcc tttctttatc cacccgagta aggtgtcgac aagtcacttt
     3061 gcccctgagc catcctcccc actcaggaga tgacccctac ccctttcttc tttatccacc
     3121 cccacgtgct ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa
     3181 atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag
     3241 actctagggg gatcgccaac aaatactacc ttttaccttg ctcttcctgc tctcaggtat
     3301 taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt
     3361 tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata
     3421 aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt
     3481 cttcattccg taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat
     3541 aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg
     3601 cgtgtaagtt acaggcaagc gatcctagta cactctatat ttttttatgc ctcggtaatg
     3661 attttcattt ttttttccac ctagcggatg actctttttt tttcttagcg attggcatta
     3721 tcacataatg aattatacat tatataaagt aatgtgattt cttcgaagaa tatactaaaa
     3781 aatgagcagg caagataaac gaaggcaaag atgacagagc agaaagccct agtaaagcgt
     3841 attacaaatg aaaccaagat tcagattgcg atctctttaa agggtggtcc cctagcgata
     3901 gagcactcga tcttcccaga aaaagaggca gaagcagtag cagaacaggc cacacaatcg
     3961 caagtgatta acgtccacac aggtataggg tttctggacc atatgataca tgctctggcc
     4021 aagcattccg gctggtcgct aatcgttgag tgcattggtg acttacacat agacgaccat
     4081 cacaccactg aagactgcgg gattgctctc ggtcaagctt ttaaagaggc cctaggggcc
     4141 gtgcgtggag taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga
     4201 gcggtggtag atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag
     4261 aaagtaggag atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct
     4321 agcagaatta ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag
     4381 agtgcgttca aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac
     4441 gatgttccct ccaccaaagg tgttctttat gcaatctcgc ctattcatgg tgtataaaag
     4501 ttcaacatcc aaagctagaa cttttggaaa gagaaagaat atccgaatag ggcacggcgt
     4561 gccgtattgt tggagtggac tagcagaaag tgaggaaggc acaggatgag ttttctcgag
     4621 acacatagct tcagcgtcgt gtaggctagg cagaggtgag ttttctcgag acataccttc
     4681 agcgtcgtct tcactgtcac agtcaactga cagtaatcgt tgatccggag agattcaaaa
     4741 ttcaatctgt ttggacctgg ataagacaca agagcgacat cctgacatga acgccgtaaa
     4801 cagcaaatcc tggttgaaca cgtatccttt tgggggcctc cgctacgacg ctcgctccag
     4861 ctggggcttc cttactatac acagcgcgca tatttcacgg ttgccagaag tcaagatggg
     4921 aaagtccgga atttaccaga ttaagaacac cctcaacaac aaggtctacg tcggatccgc
     4981 caaggacttc gaaaagcgct ggaagcgtca cttcaaggac ctggaaaagg gatgccactc
     5041 ctccattaag ctccagcgtt ccttcaacaa gcacggaaac gtcttcgaat gctccattct
     5101 ggaagaaatt ccctacgaaa aggacctgat tattgaacgt gaaaacttct ggattaagga
     5161 actgaactcc aagattaacg gatacaacat tgccgacgcc accttcggag acacctgctc
     5221 cacccacccc ctgaaggaag aaattattaa gaagcgttcc gaaaccgtca aggccaagat
     5281 gctgaagctg ggacccgacg gacgtaaggc cctgtactcc aagcccggat ccaagaacgg
     5341 acgttggaac cccgaaaccc acaagttctg caagtgcgga gtccgtattc agacctccgc
     5401 ctacacctgc tccaagtgcc gtaacggtgg tagcggcgga tccgacaaga agtactcgat
     5461 tggattggac atcggcacca actctgttgg atgggccgtc atcacggacg agtacaaggt
     5521 gcccagcaag aaattcaaag tactgggaaa cactgaccgc cattcgatca agaagaattt
     5581 gattggagct ttgctgtttg acagcggcga aacagcggaa gccacgcggc ttaaacgaac
     5641 cgcgagacga agatatacgc gacggaagaa ccgtatctgc tatctacaag agattttctc
     5701 gaatgagatg gcaaaagtcg atgactcttt ctttcaccga ttggaagaat ccttcctcgt
     5761 tgaagaggat aagaaacatg agcggcaccc gatatttgga aacattgtgg acgaggtggc
     5821 ttaccatgag aaatacccga ctatttacca cctgcgaaag aaattggtgg actcgacgga
     5881 caaagcggat cttcgcctaa tctatttggc gctagcacat atgattaagt ttcgcggcca
     5941 cttcttgatc gaaggcgact tgaatccgga caacagtgat gttgacaaac tgtttattca
     6001 acttgtacaa acttacaacc agctctttga agaaaatccc attaacgctt cgggagtgga
     6061 cgcgaaagcc atcctttctg cccgactcag caagagccga cgcctagaaa atttgattgc
     6121 gcaacttccc ggcgagaaaa agaatggact ttttggaaat cttattgctt tgtcgctggg
     6181 ccttacgccg aactttaaga gcaacttcga ccttgcggaa gatgccaaat tgcaactgag
     6241 taaagacaca tatgacgacg acctcgacaa tcttttggca caaataggag atcagtacgc
     6301 cgacctcttt ttggcggcta agaacttgtc cgacgccatt ctgttgtcgg acatccttcg
     6361 agttaatacg gagattacta aggcgccctt gagcgcctct atgatcaaaa gatacgacga
     6421 gcatcaccaa gacctgaccc tccttaaagc tctcgtccgg caacagctgc ctgagaagta
     6481 caaagagatt ttctttgatc aaagcaaaaa cggatacgcg ggctacattg acgggggagc
     6541 ctcgcaagaa gagttctaca agtttattaa accgatcctg gaaaagatgg acggcacgga
     6601 ggaattactc gtgaaactta accgagagga ccttttgcgt aagcaacgga ctttcgacaa
     6661 cggatctatt ccgcaccaaa tccatctcgg agaattgcac gcaattctgc gccggcaaga
     6721 agatttttat ccatttctca aggacaaccg ggaaaagatt gagaaaatct taaccttccg
     6781 cataccctac tacgtcggcc ctttggccag gggaaattcg agatttgcct ggatgacgcg
     6841 aaagagtgag gaaactatta ctccctggaa tttcgaagaa gtggtagaca aaggagcttc
     6901 cgcgcaatcc tttatcgagc gcatgaccaa cttcgataag aacctcccga atgagaaagt
     6961 cctacccaag cattcgttgc tctacgaata ctttacggtg tataatgagc tgactaaagt
     7021 caaatacgtt acagagggaa tgcgaaaacc ggctttcctc tcgggagagc agaaaaaggc
     7081 catagtcgat cttctcttta aaacgaaccg taaagttacc gtgaaacaac taaaagagga
     7141 ctacttcaag aaaatcgaat gctttgactc cgttgaaatt tccggcgttg aagatcggtt
     7201 caatgcctcc ttgggaacat accacgatct cctgaaaatt atcaaagaca aggactttct
     7261 tgacaatgag gaaaacgagg acattcttga agatattgtc ttgacgctaa cactttttga
     7321 ggatcgagag atgattgagg aacgccttaa aacctatgcg catctgttcg acgacaaagt
     7381 tatgaagcaa ttgaagcgcc ggagatatac gggatgggga aggttgagtc ggaaactgat
     7441 aaatggcata cgcgataagc agtccggcaa aacaattctc gatttcttga agtccgacgg
     7501 atttgcgaat cgaaacttca tgcaactgat ccatgacgat tcgttgacct ttaaagagga
     7561 cattcaaaaa gcccaagtat ccggacaagg agattctttg cacgagcata ttgcgaatct
     7621 tgctggaagt ccggctatta aaaagggcat tctccagaca gtaaaggtcg tggatgaact
     7681 cgttaaagtt atgggccgac acaaacccga gaacatagtc atcgaaatgg cgagagagaa
     7741 ccagaccacc caaaagggac agaaaaattc acgcgagcga atgaagcgga ttgaagaggg
     7801 gatcaaagag cttggcagcc aaattctgaa agaacacccg gttgaaaaca cccaattgca
     7861 gaacgagaag ctctacctgt actatcttca aaatgggcgt gatatgtacg tcgatcaaga
     7921 attggacatc aaccggctat ccgattacga tgtggaccat attgttcctc agtcgtttct
     7981 caaagacgac tccatagaca ataaggttct aacgagaagc gacaagaacc gcggcaaatc
     8041 agacaacgtc ccctccgagg aagtagttaa gaaaatgaag aattactggc gccaattgct
     8101 gaacgctaag ctcattaccc aaagaaagtt tgacaatttg acgaaagccg agcgaggcgg
     8161 gctctcggaa ctggataagg ccggcttcat taaaagacaa ttggtggaaa cgcgacaaat
     8221 cacaaagcac gtcgcacaga ttctcgactc ccgcatgaac actaagtatg acgagaatga
     8281 caaactaatc cgggaagtca aagtgattac cttgaagtcc aagctggttt ccgattttcg
     8341 taaagatttt cagttttaca aagttcgcga gatcaacaac taccatcacg cgcacgacgc
     8401 ttacttgaac gctgtcgtgg gaacggcctt gattaaaaaa taccctaagc tggaaagcga
     8461 gttcgtttac ggcgattaca aggtgtatga cgtacgcaaa atgatcgcga agtcagagca
     8521 ggaaattggc aaagctactg cgaagtactt cttttatagc aacataatga attttttcaa
     8581 aacggagatt accctcgcta acggcgagat tcgcaagcgc cctcttatcg agacaaacgg
     8641 cgaaacgggg gagattgtct gggataaagg ccgcgatttt gcgactgtgc ggaaagtctt
     8701 gtcgatgccg caagtgaata tagtcaaaaa gacggaggtg caaacaggcg gcttttcgaa
     8761 agaatctatc cttcccaaaa ggaactctga taagttgatt gcgcgaaaaa aggattggga
     8821 ccctaagaag tatggcggct ttgacagccc gaccgtagca tattctgtcc ttgtggtggc
     8881 caaagtcgaa aagggcaaat ccaagaaact gaagagtgtg aaagaattgc ttgggattac
     8941 gatcatggaa agatcatctt ttgagaagaa tcccatcgac tttctcgaag ctaaaggcta
     9001 caaagaagtg aaaaaggacc tgattatcaa gttgcctaag tactccttgt ttgagctgga
     9061 aaatggccgt aagcgaatgc tcgcttctgc cggcgaactc cagaagggaa acgaattggc
     9121 gctgccctcc aaatatgtta actttcttta ccttgcctcg cactatgaga agctcaaggg
     9181 ctcccccgag gataatgagc aaaaacaact gtttgtagaa cagcataagc actacttgga
     9241 cgagatcatt gagcaaatta gcgagttctc caagcgagtc atccttgccg acgctaatct
     9301 ggacaaagtc ctgtccgcct acaataagca ccgcgataag ccaatccgag agcaagcgga
     9361 gaatattatc cacttgttta cgctcaccaa tcttggagcc cctgccgcct ttaagtactt
     9421 tgacaccacc atcgaccgca agaggtacac cagcaccaaa gaagtcttgg acgccaccct
     9481 cattcaccaa tcaatcaccg gcttgtacga gacacgcatc gacctgtctc aactcggagg
     9541 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatcccc
     9601 caagaagaag aggaaagtct cgagcgacta caaagaccat gacggtgatt ataaagatca
     9661 tgacatcgat tacaaggatg acgatgacaa gtgacggtcc gacgcggccc gacgggtccg
     9721 aggcctcgga gatctggatc cctacctcga ctttggctgg gacactttca gtgaggacaa
     9781 gaagctccag aagcgtgcta tcgaactcaa ccagggacgt gcggcacaaa tgggcatcct
     9841 tgctctcatg gtgcacgaac agttgggagt ctctatcctt ccttaaaaat ttaattttca
     9901 ttagttgcag tcactccgct ttggtttcac agtcaggaat aacactagct cgtcttcact
     9961 gcaggttggc tcggaagttg gtgttgacgg tgagctggaa attggttgtc ggtcactgct
    10021 agcgagaaga aaacggagga cagaaggaag tgaaactcgg ttcgttctcg acagcctcac
    10081 tgtcaatatg ctcattttca atccttagcg cttttaatgt cgaattgacg gtaaattgaa
    10141 taggatctat aatatctaca aggtactttg acacgccaag tattcattgt tagtcaacaa
    10201 tattttagag ctttataagg tcaaaaaaca ccttcaaagt cgaggagacc gagagagggt
    10261 ctcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
    10321 aagtggcacc gagtcggtgc ttttttttag aaccgctcac ccatgctatc gtatgtcatt
    10381 tacattgaca aactgctatg aatgcgtttg cgataacttc agctcgctga agccctagac
    10441 atttcgtttg actaaattgt tttccggttg ctttactacc gaccggtctg cgggtagcat
    10501 gtcttggctt ttgcgagttc gagagaattt atggacaagt gcgtttacag tttggaacac
    10561 gagtgtgagt gagcggaatt ggctatctat aacattctta tttcaggagg ctggtgatta
    10621 gcagaatttt caaagatctc gaatctaaaa tcgccatggg ctcgacgaac ttttctgcaa
    10681 gcttatcgat accgtcgacc tcgagggggg gcccggtacc agcttttgtt ccctttagtg
    10741 agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
    10801 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
    10861 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
    10921 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
    10981 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
    11041 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
    11101 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
    11161 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
    11221 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
    11281 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
    11341 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
    11401 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
    11461 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
    11521 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
    11581 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
    11641 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
    11701 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
    11761 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
    11821 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
    11881 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
    11941 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
    12001 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
    12061 aatgataccg cgagagccac gctcaccggc tccagattta tcagcaataa accagccagc
    12121 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
    12181 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
    12241 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
    12301 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
    12361 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
    12421 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
    12481 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
    12541 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
    12601 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
    12661 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
    12721 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
    12781 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
    12841 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
    12901 atttccccga aaagtgccac ct
//
