LOCUS       sequence-326166-       10847 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR/RNA scaffold based transcription regulation in Pichia 
            pastoris 
            BB3rN_pTEF2_dCAS9(3xFLAG_SV40NLS)_pPOR1_MS2bind_VP64_SV40NLS_pGAP_RI
            B1_R3_gRNA_MS2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10847)
  AUTHORS   Baumschabl M, Prielhofer R, Mattanovich D, Steiger MG
  TITLE     Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA 
            Scaffolds.
  JOURNAL   ACS Synth Biol. 2020 Dec 18;9(12):3202-3209. doi: 
            10.1021/acssynbio.0c00214. Epub 2020 Nov 12.
  PUBMED    33180466
REFERENCE   2  (bases 1 to 10847)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.0c00214"; journalName: "ACS Synth Biol"; date: 
            "2020-12-18- 18"; volume: "9"; issue: "12"; pages: "3202-3209"
COMMENT     Sequence Label: RIB VP64 R3
FEATURES             Location/Qualifiers
     source          1..10847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             1303..1368
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(1372..1398,1399..1401,1402..3885,3886..3891,3892..3957,
                     3958..3960,3961..5475)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /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             join(1372..1398,1399..1401,1402..3888,3889..3891,
                     3892..5475)
                     /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             1372..5475
                     /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="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             5488..5508
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6699..6719
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6729..7115
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2
                     /note="binds to a specific stem-loop structure in the viral
                     RNA (Peabody, 1993)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             7131..7280
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     promoter        7478..7954
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     ncRNA           7973..8009
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        8030..8105
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        8110..8128
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           8135..8202
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     gene            8715..9834
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        8715..9058
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             9059..9631
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      9637..9834
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
      721 gcgatcggag cggtactacc attgacacta cacatcctta attccaatcc tgtctggcct
      781 ccttcacctt ttaaccatct tgcccattcc aactcgtgtc agattgcgta tcaagtgaaa
      841 aaaaaaaaat tttaaatctt taacccaatc aggtaataac tgtcgcctct tttatctgcc
      901 gcactgcatg aggtgtcccc ttagtgggaa agagtactga gccaaccctg gaggacagca
      961 agggaaaaat acctacaact tgcttcataa tggtcgtaaa aacaatcctt gtcggatata
     1021 agtgttgtag actgtccctt atcctctgcg atgttcttcc tctcaaagtt tgcgatttct
     1081 ctctatcaga attgccatca agagactcag gactaatttc gcagtcccac acgcactcgt
     1141 acatgattgg ctgaaatttc cctaaagaat ttctttttca cgaaaatttt ttttttacac
     1201 aagattttca gcagatataa aatggagagc aggacctccg ctgtgactct tctttttttt
     1261 cttttattct cactacatac attttagtta ttcgccaaca tggactacaa agaccatgac
     1321 ggtgattata aagatcatga catcgattac aaggatgacg atgacaagct catggacaag
     1381 aagtactcca ttgggctcgc tatcggcaca aacagcgtcg gctgggccgt cattacggac
     1441 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata
     1501 aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg
     1561 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag
     1621 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag
     1681 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg
     1741 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta
     1801 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa
     1861 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa
     1921 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca
     1981 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa
     2041 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc
     2101 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag
     2161 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc
     2221 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt
     2281 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag
     2341 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg
     2401 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt
     2461 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg
     2521 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc
     2581 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc
     2641 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc
     2701 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg
     2761 tggatgactc gcaaatcaga agagactatc actccctgga acttcgagga agtcgtggat
     2821 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct
     2881 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag
     2941 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag
     3001 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag
     3061 ctcaaagagg actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg
     3121 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac
     3181 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt
     3241 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc
     3301 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca
     3361 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt
     3421 aagtccgatg gatttgccaa caggaacttc atgcagttga tccatgatga ctctctcacc
     3481 tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct gcacgagcac
     3541 atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc
     3601 gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg
     3661 gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg
     3721 attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac
     3781 acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac
     3841 gtggatcagg aactggacat caatcggctc tccgactacg acgtggatgc catcgtgccc
     3901 cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat
     3961 agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg
     4021 cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct
     4081 gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag
     4141 acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac
     4201 gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctagtc
     4261 tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat
     4321 gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag
     4381 cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca
     4441 aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg
     4501 aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc
     4561 gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc
     4621 cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga
     4681 ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa
     4741 aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta
     4801 ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg
     4861 ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag
     4921 gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc
     4981 tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt
     5041 aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa
     5101 aagctcaaag ggtcccccga agataatgag cagaagcagc tgttcgtgga acaacacaaa
     5161 cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc
     5221 gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg
     5281 gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc tcctgcagcc
     5341 ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg
     5401 gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct
     5461 cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtgtg agctttagtc
     5521 aaatattaat ctatttcacc tgttcaaact ttacttaatg tacaaatgtg gtagttatta
     5581 gttttgcaac ggaacttgtt ccataatctg gtcctctggg acagcaaact gtctttcact
     5641 agtagcgcca gtttcgggag tccacacagc attagtcacc ggtgcaccag cactaatctc
     5701 acgaccttct gggtgtttaa atgggcagtt agggttgcgg catccagctg caaacttaca
     5761 atcctcatca attggatgag tgaaaaaaca gtttggtctg gtacaactgt tgccttcacg
     5821 acacagtaca ggagtagttg cgtgacgtct tgggcacttg taattacggc atgatttacc
     5881 aaatcgacat tgttccaaag ccctctgttg tttttgttgt ttctcttctt cggtgatctt
     5941 gtgttcaggt gatcgatgag cctttggaca gtccggatta gagcgctccg gggagcgcat
     6001 tatttgggtc ggctactgtc attggttatg atggccttac cgagcacgaa ggtggttgga
     6061 tagaccacaa ctggaaacag ttgtacaaac agattgcatt catttttgct tgtattggat
     6121 actcgatggc catcaccgct cttatctgtt tcatcctcaa ccgtattcca tttttgcaac
     6181 tgcgagcttc agaagaggct gaggagaaag gtatggatga ggatcagatt ggagagttcg
     6241 cttatgacta cgtggaagta cgtcgtgatt ttttggcttg gggatcaggc ccaaacaatg
     6301 gcttcaagga gccggaagtt ctggatcagg tagttccggt taatgatttc agcagtgacc
     6361 agaatgtgac taatcagacc aacgaatctg agaagcagta gagtaaatat agagatgata
     6421 tttagtgtat tctaatgctt atgtaatgta ttaagcaaaa agttgtgttt atgagttagc
     6481 atttgtctta gcaaacataa aattatgtcg acatttgcaa cccgcatgtc tagtgttttt
     6541 agatcgatct tcgatgtgta gaataatgcc tccacgtgat gccccgcgat tttgttgggt
     6601 cgcaatgcct ccaacataaa cccatcacgt ataaaaagcc ctcttaaccc tcccccctgt
     6661 ttcgtttgct tcatcactta acctgaacta tcaacatgcc caaaaagaaa agaaaagtgg
     6721 gtagtatggc gtctaacttc acccagttcg ttctggttga caacggtggt accggtgacg
     6781 ttaccgttgc gccgtctaac ttcgcgaacg gtgttgcgga atggatctct tctaactctc
     6841 gttctcaggc gtacaaagtt acctgctctg ttcgtcaatc ctccgcgcag aaccgtaaat
     6901 acaccatcaa agttgaagtt ccgaaagttg cgacccagac cgttggtggt gttgaactgc
     6961 cggttgcggc gtggcgttct tacctgaaca tggaactgac catcccgatc ttcgcgacca
     7021 actctgactg cgaactgatc gttaaagcga tgcagggtct gctgaaagac ggtaacccga
     7081 tcccgtctgc gatcgcggcg aactctggta tctacggctc cggacgagcc gatgcccttg
     7141 acgattttga cttggacatg ttgggttctg acgctttgga tgactttgat cttgatatgc
     7201 ttggttccga cgctctagat gatttcgact tggatatgct gggatccgat gccttggacg
     7261 atttcgactt ggatatgttg atcaactgag cttctgttta attctgtagg ataaacgaaa
     7321 taactgtatc tttataatca tccttaagat agccctggac tcgtcagaca ttagttcgcg
     7381 cacattattt tcctccactg tctgcaaaca gccacacggc atttccctct ctagtttcat
     7441 ttatcaaagt agcacaacac gctaattgga ggatcctttt ttgtagaaat gtcttggtgt
     7501 cctcgtccaa tcaggtagcc atctctgaaa tatctggctc cgttgcaact ccgaacgacc
     7561 tgctggcaac gtaaaattct ccggggtaaa acttaaatgt ggagtaatgg aaccagaaac
     7621 gtctcttccc ttctctctcc ttccaccgcc cgttaccgtc cctaggaaat tttactctgc
     7681 tggagagctt cttctacggc ccccttgcag caatgctctt cccagcatta cgttgcgggt
     7741 aaaacggagg tcgtgtaccc gacctagcag cccagggatg gaaaagtccc ggccgtcgct
     7801 ggcaataata gcgggcggac gcatgtcatg agattattgg aaaccaccag aatcgaatat
     7861 aaaaggcgaa cacctttccc aattttggtt tctcctgacc caaagacttt aaatttaatt
     7921 tatttgtccc tatttcaatc aattgaacaa ctatcaaaac accatgcatg tgctgatgag
     7981 tccgtgagga cgaaacgagt aagctcgtcc acatgactag tcacatggcg ttttagagct
     8041 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     8101 ggtgcgcgca catgaggatc acccatgtgc ttttggccgg catggtccca gcctcctcgc
     8161 tggcgccggc tgggcaacat gcttcggcat ggcgaatggg acagctttgg actgctttta
     8221 gtgtacatct gataatatag tactaccacg tatgataatg tagagaatag gcttccttgt
     8281 cgagtgtgtt tgcagttttc ttgagtttca aggtttaaat gctggtatat tagttcatcg
     8341 aaggtttcag ccaatagcac cttaaatcaa tcaaactaat tcgactctta cgaaagagcc
     8401 tactgtgttt agtatcgaag tcgtttacct ttcatgttga atagcttcct ctctgaccct
     8461 aacatttcaa gatcctccta aagttacccg gattgtgaaa ttctaatgat ccacctgccc
     8521 aatgcatttt ttctttattc agtttacctt ttttacctaa tatacgagct tgttaaagta
     8581 agtggcactg caatactagg cttattgttg atattatgat gaatcgtttt cacaaacttg
     8641 atttcctgtg aactcaccat gtactaagga aaaaaacatg catcaccatc tgaatatttg
     8701 accgctagct catggacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca
     8761 gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat
     8821 catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc
     8881 tgcagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg
     8941 cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac
     9001 ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat
     9061 gggtaccact cttgacgaca cggcttaccg gtaccgcacc agtgtcccgg gggacgccga
     9121 ggccatcgag gcactggatg ggtccttcac caccgacacc gtttttcgcg tcaccgccac
     9181 cggggacggc ttcaccctgc gggaggtgcc ggtggacccg cccctgacca aggtgttccc
     9241 cgacgacgaa tcggacgacg aatcggacga cggggaggac ggcgacccgg actcccggac
     9301 gttcgtcgcg tacggggacg acggcgacct ggcgggcttc gtggtcgtct cgtactccgg
     9361 ctggaaccgc cggctgaccg tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg
     9421 ggtcgggcgc gcgttgatgg ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca
     9481 cctctggctg gaggtcacca acgtcaacgc accggcgatc cacgcgtacc ggcggatggg
     9541 gttcaccctc tgcggcctgg acaccgccct gtacgacggc accgcctcgg acggcgagca
     9601 ggcgctctac atgagcatgc cctgccccta atcagtactg acaataaaaa gattcttgtt
     9661 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     9721 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     9781 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actggcttca
     9841 tacccagcat tgacctttgg tatgagcatc tgaaaaacaa ccaggtgttg caaagttaaa
     9901 catccttctt tgttcatata gaacccacta ttcatggtac tccccaatcg aatttcacat
     9961 tctggttttg aaattacaca ccacgttagc ttataagatt tcatataact tattgatata
    10021 cggtttccat tgttcgaata gttgaggttg tatgtaattc gattgaaggg gccatttttg
    10081 tttcctactt ttcctgggag cttatccgat gcgcttcaaa gctggaattg taaatataga
    10141 gaaaaagaag gatgttgttt tattcttgaa agagtataat tttacttcta gcaactctcc
    10201 cacttcgctt gacttcattt atttcttggg cacataggcg tagtaatcta gaccaacaga
    10261 taatttgccg gaatgatata gcgattggaa aatgaactga aattttttgc tgtctttcaa
    10321 tttgacgggc agttcatcag gcgcgccgtg accgaccata taaatacgtt gagaatgtta
    10381 ttcttcctcg tagttgaagt ggcttcataa tttcagaact caatagataa actaggatgt
    10441 tttaaagcaa ttaatgctca caagtaagga gcgactctct tgcttttcga atactaaaag
    10501 tatcgtccca acccagaaaa aaagacctct taactgcaaa ataaactcta tatatttctt
    10561 ctaaaacagt ttcaggttgg atagtatcgc attctcatca cttctaacta gtaggccatg
    10621 agatatatta acgtttactt gagttctaag ttctccgaat tagatgcaca gcacaaacaa
    10681 gattaggttt cacttggtac aaaatacgaa cagagtttaa ggtcgtaatt tcatttcgtt
    10741 attgatcccc acaatctatt cttatcacag tcatcagata gtcgcgaaaa agcatgcaga
    10801 aaagggggtc gtccctatct aagttgtagc attacaacaa atatgac
//
