LOCUS       sequence-220638-       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(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(782..1153)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1186..1295
                     /note="incP origin of transfer"
     primer_bind     1572..1588
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1612..1630
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2251..2625
                     /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        3627..3812
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3813..4469
                     /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             5446..9546
                     /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             5446..9546
                     /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             9547..9594
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9601..9621
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9628..9693
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_RNA        10267..10342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10689..10705)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10690..10706)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(10734..10752)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10773..10789)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10773..10789)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10786..10808)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10797..10813
                     /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(10821..10827,10828..10845,10846..10851))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10866..10887
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11004..11021)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11175..11763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11255..11274)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11934..12725,12726..12794))
                     /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     12557..12576
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12795..12899)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctgatcgt
      661 cttgccttgc tcgtcggtga tgtacttcac cagctccgcg aagtcgctct tcttgatgga
      721 gcgcatgggg acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa
      781 gtcatggctc tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct
      841 tctcttcgat cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg
      901 ggtcgtcggt gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc
      961 gggccagctc gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga
     1021 cggccagcag gtaggccgac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc
     1081 ttcgttcgtc tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg
     1141 tttcatcagc catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca
     1201 gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc
     1261 cgctcgcggg tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag
     1321 gaaagtctac acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata
     1381 ccgaaaaaat cgctataatg accccgaagc agggttatgc agcggaagat gccattcgcc
     1441 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     1501 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     1561 gtcacgacgt tgtaaaacga cggccagtga ccatgattac gccaagctcg aaattaaccc
     1621 tcactaaagg gaacaaaagc tggtacctaa ccctgcgata gaccttttcc aaactcacgc
     1681 agtccaagaa aacaaagggg tgagaagtat acgcaccttt cggtttcggc ataattctta
     1741 aactcttgtg gtcactttct tgtgaagaag ctaggggcac tcgttttccc tcagagcctg
     1801 caaacacaaa attcctgcag tcaattgtcc caacactcgg caagccgtat gcgcaagcaa
     1861 cgatgcgcag aaggccgtgg atggatggcg actcgcgata tggcttcttg ggtcgccagt
     1921 gtggtatgtc cggcgtatgt caatacgcga attcggacga ctggcatctc taggaggagg
     1981 attccttctt ttatgacatg tttattttat atacattgat gctttccgac agtcggaagt
     2041 aataaatgaa tttatttcaa gactacctat actcctttga cttgttcgac taatcttacc
     2101 gcttactaaa atctcgaaat cacgcttgac ctctcgcacg caaatttttg ctgctggacg
     2161 ctacgcactc ggcccaattc ttctcggtcc tcgtcgtcgc aattgtcgtt gcgttgatct
     2221 tgcaccgaag gaatcagaga atagaatacc atggccaagt tgaccagtgc cgttccggtg
     2281 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     2341 cgggacttcg tggaggacga cttcgtcggt gtggtccggg acgacgtgac cctgttcatc
     2401 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     2461 ctgaacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     2521 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     2581 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaagata agaatatctc
     2641 attgtgaaca tctatgattt accaatttta ttctttgttt acagttagac gccagtaatt
     2701 gtgctgtttc tctcaagtct gtgtcaatac aaactacgaa acttggcaat ttttctcttg
     2761 aatatgagca cgagattgaa acgcacaaag gaaattagtt tccatccttt gacaaagttt
     2821 gttgctgttt agagaacaga tgtcaaaatt aacgtgccat ggaattgaac catggtgcgc
     2881 tatccccaaa tcacgtcgtt tgacctcgtc acatttaagg tatatcaagc atattcactt
     2941 atatcttgac atcctccctg cttgatattc ctttgccctt gagccatctt ccccacacgc
     3001 tggagatgac ccctgcccct cctttcttta tccacccgag taaggtgtcg acaagtcact
     3061 ttgcccctga gccatcctcc ccactcagga gatgacccct acccctttct tctttatcca
     3121 cccccacgtg ctataaaaat aattataatt taaatttttt aatataaata tataaattaa
     3181 aaatagaaag taaaaaaaga aattaaagaa aaaatagttt ttgttttccg aagatgtaaa
     3241 agactctagg gggatcgcca acaaatacta ccttttacct tgctcttcct gctctcaggt
     3301 attaatgccg aattgtttca tcttgtctgt gtagaagacc acacacgaaa atcctgtgat
     3361 tttacatttt acttatcgtt aatcgaatgt atatctattt aatctgcttt tcttgtctaa
     3421 taaatatata tgtaaagtac gctttttgtt gaaatttttt aaacctttgt ttattttttt
     3481 ttcttcattc cgtaactctt ctaccttctt tatttacttt ctaaaatcca aatacaaaac
     3541 ataaaaataa ataaacacag agtaaattcc caaattattc catcattaaa agatacgagg
     3601 cgcgtgtaag ttacaggcaa gcgatcctag tacactctat atttttttat gcctcggtaa
     3661 tgattttcat ttttttttcc acctagcgga tgactctttt tttttcttag cgattggcat
     3721 tatcacataa tgaattatac attatataaa gtaatgtgat ttcttcgaag aatatactaa
     3781 aaaatgagca ggcaagataa acgaaggcaa agatgacaga gcagaaagcc ctagtaaagc
     3841 gtattacaaa tgaaaccaag attcagattg cgatctcttt aaagggtggt cccctagcga
     3901 tagagcactc gatcttccca gaaaaagagg cagaagcagt agcagaacag gccacacaat
     3961 cgcaagtgat taacgtccac acaggtatag ggtttctgga ccatatgata catgctctgg
     4021 ccaagcattc cggctggtcg ctaatcgttg agtgcattgg tgacttacac atagacgacc
     4081 atcacaccac tgaagactgc gggattgctc tcggtcaagc ttttaaagag gccctagggg
     4141 ccgtgcgtgg agtaaaaagg tttggatcag gatttgcgcc tttggatgag gcactttcca
     4201 gagcggtggt agatctttcg aacaggccgt acgcagttgt cgaacttggt ttgcaaaggg
     4261 agaaagtagg agatctctct tgcgagatga tcccgcattt tcttgaaagc tttgcagagg
     4321 ctagcagaat taccctccac gttgattgtc tgcgaggcaa gaatgatcat caccgtagtg
     4381 agagtgcgtt caaggctctt gcggttgcca taagagaagc cacctcgccc aatggtacca
     4441 acgatgttcc ctccaccaaa ggtgttcttt atgcaatctc gcctattcat ggtgtataaa
     4501 agttcaacat ccaaagctag aacttttgga aagagaaaga atatccgaat agggcacggc
     4561 gtgccgtatt gttggagtgg actagcagaa agtgaggaag gcacaggatg agttttctcg
     4621 agacacatag cttcagcgtc gtgtaggcta ggcagaggtg agttttctcg agacatacct
     4681 tcagcgtcgt cttcactgtc acagtcaact gacagtaatc gttgatccgg agagattcaa
     4741 aattcaatct gtttggacct ggataagaca caagagcgac atcctgacat gaacgccgta
     4801 aacagcaaat cctggttgaa cacgtatcct tttgggggcc tccgctacga cgctcgctcc
     4861 agctggggct tccttactat acacagcgcg catatttcac ggttgccaga agtcaagatg
     4921 ggaaagtccg gaatttacca gattaagaac accctcaaca acaaggtcta cgtcggatcc
     4981 gccaaggact tcgaaaagcg ctggaagcgt cacttcaagg acctggaaaa gggatgccac
     5041 tcctccatta agctccagcg ttccttcaac aagcacggaa acgtcttcga atgctccatt
     5101 ctggaagaaa ttccctacga aaaggacctg attattgaac gtgaaaactt ctggattaag
     5161 gaactgaact ccaagattaa cggatacaac attgccgacg ccaccttcgg agacacctgc
     5221 tccacccacc ccctgaagga agaaattatt aagaagcgtt ccgaaaccgt caaggccaag
     5281 atgctgaagc tgggacccga cggacgtaag gccctgtact ccaagcccgg atccaagaac
     5341 ggacgttgga accccgaaac ccacaagttc tgcaagtgcg gagtccgtat tcagacctcc
     5401 gcctacacct gctccaagtg ccgtaacggt ggtagcggcg gatccgacaa gaagtactcg
     5461 attggattgg acatcggcac caactctgtt ggatgggccg tcatcacgga cgagtacaag
     5521 gtgcccagca agaaattcaa agtactggga aacactgacc gccattcgat caagaagaat
     5581 ttgattggag ctttgctgtt tgacagcggc gaaacagcgg aagccacgcg gcttaaacga
     5641 accgcgagac gaagatatac gcgacggaag aaccgtatct gctatctaca agagattttc
     5701 tcgaatgaga tggcaaaagt cgatgactct ttctttcacc gattggaaga atccttcctc
     5761 gttgaagagg ataagaaaca tgagcggcac ccgatatttg gaaacattgt ggacgaggtg
     5821 gcttaccatg agaaataccc gactatttac cacctgcgaa agaaattggt ggactcgacg
     5881 gacaaagcgg atcttcgcct aatctatttg gcgctagcac atatgattaa gtttcgcggc
     5941 cacttcttga tcgaaggcga cttgaatccg gacaacagtg atgttgacaa actgtttatt
     6001 caacttgtac aaacttacaa ccagctcttt gaagaaaatc ccattaacgc ttcgggagtg
     6061 gacgcgaaag ccatcctttc tgcccgactc agcaagagcc gacgcctaga aaatttgatt
     6121 gcgcaacttc ccggcgagaa aaagaatgga ctttttggaa atcttattgc tttgtcgctg
     6181 ggccttacgc cgaactttaa gagcaacttc gaccttgcgg aagatgccaa attgcaactg
     6241 agtaaagaca catatgacga cgacctcgac aatcttttgg cacaaatagg agatcagtac
     6301 gccgacctct ttttggcggc taagaacttg tccgacgcca ttctgttgtc ggacatcctt
     6361 cgagttaata cggagattac taaggcgccc ttgagcgcct ctatgatcaa aagatacgac
     6421 gagcatcacc aagacctgac cctccttaaa gctctcgtcc ggcaacagct gcctgagaag
     6481 tacaaagaga ttttctttga tcaaagcaaa aacggatacg cgggctacat tgacggggga
     6541 gcctcgcaag aagagttcta caagtttatt aaaccgatcc tggaaaagat ggacggcacg
     6601 gaggaattac tcgtgaaact taaccgagag gaccttttgc gtaagcaacg gactttcgac
     6661 aacggatcta ttccgcacca aatccatctc ggagaattgc acgcaattct gcgccggcaa
     6721 gaagattttt atccatttct caaggacaac cgggaaaaga ttgagaaaat cttaaccttc
     6781 cgcataccct actacgtcgg ccctttggcc aggggaaatt cgagatttgc ctggatgacg
     6841 cgaaagagtg aggaaactat tactccctgg aatttcgaag aagtggtaga caaaggagct
     6901 tccgcgcaat cctttatcga gcgcatgacc aacttcgata agaacctccc gaatgagaaa
     6961 gtcctaccca agcattcgtt gctctacgaa tactttacgg tgtataatga gctgactaaa
     7021 gtcaaatacg ttacagaggg aatgcgaaaa ccggctttcc tctcgggaga gcagaaaaag
     7081 gccatagtcg atcttctctt taaaacgaac cgtaaagtta ccgtgaaaca actaaaagag
     7141 gactacttca agaaaatcga atgctttgac tccgttgaaa tttccggcgt tgaagatcgg
     7201 ttcaatgcct ccttgggaac ataccacgat ctcctgaaaa ttatcaaaga caaggacttt
     7261 cttgacaatg aggaaaacga ggacattctt gaagatattg tcttgacgct aacacttttt
     7321 gaggatcgag agatgattga ggaacgcctt aaaacctatg cgcatctgtt cgacgacaaa
     7381 gttatgaagc aattgaagcg ccggagatat acgggatggg gaaggttgag tcggaaactg
     7441 ataaatggca tacgcgataa gcagtccggc aaaacaattc tcgatttctt gaagtccgac
     7501 ggatttgcga atcgaaactt catgcaactg atccatgacg attcgttgac ctttaaagag
     7561 gacattcaaa aagcccaagt atccggacaa ggagattctt tgcacgagca tattgcgaat
     7621 cttgctggaa gtccggctat taaaaagggc attctccaga cagtaaaggt cgtggatgaa
     7681 ctcgttaaag ttatgggccg acacaaaccc gagaacatag tcatcgaaat ggcgagagag
     7741 aaccagacca cccaaaaggg acagaaaaat tcacgcgagc gaatgaagcg gattgaagag
     7801 gggatcaaag agcttggcag ccaaattctg aaagaacacc cggttgaaaa cacccaattg
     7861 cagaacgaga agctctacct gtactatctt caaaatgggc gtgatatgta cgtcgatcaa
     7921 gaattggaca tcaaccggct atccgattac gatgtggacc atattgttcc tcagtcgttt
     7981 ctcaaagacg actccataga caataaggtt ctaacgagaa gcgacaagaa ccgcggcaaa
     8041 tcagacaacg tcccctccga ggaagtagtt aagaaaatga agaattactg gcgccaattg
     8101 ctgaacgcta agctcattac ccaaagaaag tttgacaatt tgacgaaagc cgagcgaggc
     8161 gggctctcgg aactggataa ggccggcttc attaaaagac aattggtgga aacgcgacaa
     8221 atcacaaagc acgtcgcaca gattctcgac tcccgcatga acactaagta tgacgagaat
     8281 gacaaactaa tccgggaagt caaagtgatt accttgaagt ccaagctggt ttccgatttt
     8341 cgtaaagatt ttcagtttta caaagttcgc gagatcaaca actaccatca cgcgcacgac
     8401 gcttacttga acgctgtcgt gggaacggcc ttgattaaaa aataccctaa gctggaaagc
     8461 gagttcgttt acggcgatta caaggtgtat gacgtacgca aaatgatcgc gaagtcagag
     8521 caggaaattg gcaaagctac tgcgaagtac ttcttttata gcaacataat gaattttttc
     8581 aaaacggaga ttaccctcgc taacggcgag attcgcaagc gccctcttat cgagacaaac
     8641 ggcgaaacgg gggagattgt ctgggataaa ggccgcgatt ttgcgactgt gcggaaagtc
     8701 ttgtcgatgc cgcaagtgaa tatagtcaaa aagacggagg tgcaaacagg cggcttttcg
     8761 aaagaatcta tccttcccaa aaggaactct gataagttga ttgcgcgaaa aaaggattgg
     8821 gaccctaaga agtatggcgg ctttgacagc ccgaccgtag catattctgt ccttgtggtg
     8881 gccaaagtcg aaaagggcaa atccaagaaa ctgaagagtg tgaaagaatt gcttgggatt
     8941 acgatcatgg aaagatcatc ttttgagaag aatcccatcg actttctcga agctaaaggc
     9001 tacaaagaag tgaaaaagga cctgattatc aagttgccta agtactcctt gtttgagctg
     9061 gaaaatggcc gtaagcgaat gctcgcttct gccggcgaac tccagaaggg aaacgaattg
     9121 gcgctgccct ccaaatatgt taactttctt taccttgcct cgcactatga gaagctcaag
     9181 ggctcccccg aggataatga gcaaaaacaa ctgtttgtag aacagcataa gcactacttg
     9241 gacgagatca ttgagcaaat tagcgagttc tccaagcgag tcatccttgc cgacgctaat
     9301 ctggacaaag tcctgtccgc ctacaataag caccgcgata agccaatccg agagcaagcg
     9361 gagaatatta tccacttgtt tacgctcacc aatcttggag cccctgccgc ctttaagtac
     9421 tttgacacca ccatcgaccg caagaggtac accagcacca aagaagtctt ggacgccacc
     9481 ctcattcacc aatcaatcac cggcttgtac gagacacgca tcgacctgtc tcaactcgga
     9541 ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc
     9601 cccaagaaga agaggaaagt ctcgagcgac tacaaagacc atgacggtga ttataaagat
     9661 catgacatcg attacaagga tgacgatgac aagtgacggt ccgacgcggc ccgacgggtc
     9721 cgaggcctcg gagatctgga tccctacctc gactttggct gggacacttt cagtgaggac
     9781 aagaagctcc agaagcgtgc tatcgaactc aaccagggac gtgcggcaca aatgggcatc
     9841 cttgctctca tggtgcacga acagttggga gtctctatcc ttccttaaaa atttaatttt
     9901 cattagttgc agtcactccg ctttggtttc acagtcagga ataacactag ctcgtcttca
     9961 ctgcaggttg gctcggaagt tggtgttgac ggtgagctgg aaattggttg tcggtcactg
    10021 ctagcgagaa gaaaacggag gacagaagga agtgaaactc ggttcgttct cgacagcctc
    10081 actgtcaata tgctcatttt caatccttag cgcttttaat gtcgaattga cggtaaattg
    10141 aataggatct ataatatcta caaggtactt tgacacgcca agtattcatt gttagtcaac
    10201 aatattttag agctttataa ggtcaaaaaa caccttcaaa gtcgaggaga ccgagagagg
    10261 gtctcagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
    10321 aaaagtggca ccgagtcggt gctttttttt agaaccgctc acccatgcta tcgtatgtca
    10381 tttacattga caaactgcta tgaatgcgtt tgcgataact tcagctcgct gaagccctag
    10441 acatttcgtt tgactaaatt gttttccggt tgctttacta ccgaccggtc tgcgggtagc
    10501 atgtcttggc ttttgcgagt tcgagagaat ttatggacaa gtgcgtttac agtttggaac
    10561 acgagtgtga gtgagcggaa ttggctatct ataacattct tatttcagga ggctggtgat
    10621 tagcagaatt ttcaaagatc tcgaatctaa aatcgccatg ggctcgacga acttttctgc
    10681 aagcttatcg ataccgtcga cctcgagggg gggcccggta ccagcttttg ttccctttag
    10741 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
    10801 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
    10861 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
    10921 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
    10981 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    11041 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    11101 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    11161 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    11221 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    11281 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    11341 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    11401 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    11461 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    11521 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    11581 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    11641 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    11701 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    11761 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    11821 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    11881 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    11941 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    12001 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    12061 gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    12121 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    12181 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    12241 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    12301 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    12361 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    12421 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    12481 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    12541 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    12601 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    12661 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    12721 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    12781 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    12841 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    12901 acatttcccc gaaaagtgcc ac
//
