LOCUS       sequence-250941-       11667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CRISPR activation device with the necessary genes (dxcas9 
            controlled by Ptet, pspF-Delta-HTH::lambda-N22plus controlled by 
            promoter PrhaB), and the reporter part (PpspA-LEA3B3 with 
            sfgfp::ASV)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11667)
  AUTHORS   Liu Y, Wan X, Wang B
  TITLE     Engineered CRISPRa enables programmable eukaryote-like gene 
            activation in bacteria.
  JOURNAL   Nat Commun. 2019 Aug 26;10(1):3693. doi: 10.1038/s41467-019-11479-0.
  PUBMED    31451697
REFERENCE   2  (bases 1 to 11667)
  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: "Nat 
            Commun."; date: "2019-08-26"; volume: "10(1):3693. doi"; pages: " 
            10.1038/s41467-019-11479-0"
COMMENT     Sequence Label: pLY70
FEATURES             Location/Qualifiers
     source          1..11667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..59
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(362..1312)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1360..1582
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(2074..2168)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2198..2989,2990..3058))
                     /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     2821..2840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3059..3163)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3228..3246
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3250..3293)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3296..3317
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(join(3381..4967,4968..4970,4971..7457,7458..7460,
                     7461..7487))
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(3384..7487)
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             7494..7505
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        7523..7578
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7524..7542
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7594..8220
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(7670..7689)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             8235..8246
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(8390..9226)
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     promoter        complement(9244..9278)
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     terminator      9294..9321
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        9379..9497
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             10495..10506
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_RNA        10728..10777
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             10810..10821
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             10828..11541
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     misc_feature    join(11648..11667,1)
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
ORIGIN
        1 gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat
       61 tacttcgcgt tatgcaggct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatctcgag gttacattgt cgatctgttc
      181 atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt
      241 tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac ggtgaacagt
      301 tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag ccataagaac
      361 ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga
      421 gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa aattttgcct
      481 caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt cttagtccgt
      541 tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc atttttatct
      601 ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact tggaaaatca
      661 acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg taagtgttta
      721 aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca tggtagttat
      781 tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt gccttgtgag
      841 ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag tatttgtttt
      901 caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg aaaagataag
      961 gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg gcatagtttg
     1021 tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca gttctcgtca
     1081 tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg atgttcatca
     1141 tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta gggttttcaa
     1201 tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc tccgttaagt
     1261 catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac atacatctca
     1321 attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa tgataattac
     1381 atgtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt gctggaaaac
     1441 ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt
     1501 atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata aaaagaatag
     1561 atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac aaaaggatgt
     1621 cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca
     1681 ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat caggcacctg
     1741 agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc agtgaatggg
     1801 ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc cataatacaa
     1861 gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg tggtgctatc
     1921 tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc acttcggatt
     1981 atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg tatcatcaac
     2041 aggcttaccc gtcttactgt ccctagtgct tggattctca ccaataaaaa acgcccggcg
     2101 gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa
     2161 caggagtcca agcgagctcg taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2221 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2281 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2341 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2401 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2461 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2521 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2581 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2641 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2701 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2761 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2821 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2881 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2941 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3001 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3061 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3121 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt cccatggtgc
     3181 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3241 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggaatt
     3301 cgcggccgct tctagagggg agaccagaaa caaaaaaagg ggagcggttt cccgctcccc
     3361 ttcaataatt ggctctagta ttagtcacct cctagctgac tcaaatcaat gcgtgtttca
     3421 taaagaccag tgatggattg atggataaga gtggcatcta aaacttcttt tgtagacgta
     3481 tatcgtttac gatcaattgt tgtatcaaaa tatttaaaag cagcgggagc tccaagattc
     3541 gtcaacgtaa ataaatgaat aatattttct gcttgttcac gtattggttt gtctctatgt
     3601 ttgttatatg cactaagaac tttatctaaa ttggcatctg ctaaaataac acgcttagaa
     3661 aattcactga tttgctcaat aatctcatct aaataatgct tatgctgctc cacaaacaat
     3721 tgtttttgtt cgttatcttc tggactaccc ttcaactttt cataatgact agctaaatat
     3781 aaaaaattca catatttgct tggcagagcc agctcatttc ctttttgtaa aactccggca
     3841 ctagccagca tccgtttacg accgttttct aactcaaaaa gactatattt aggtagttta
     3901 atgattaagt cttttttaac ttccttatat cctttagctt ctaaaaagtc aatcggattt
     3961 ttttcaaagg aacttctttc cataattgtg atccctagta actctttaac ggattttaac
     4021 ttcttcgatt tccctttttc caccttagca accactagga ctgaataagc taccgttgga
     4081 ctatcaaaac caccatattt ttttggatcc cagtcttttt tacgagcaat aagcttgtcc
     4141 gaatttcttt ttggtaaaat tgactccttg gagaatccgc ctgtctgtac ttctgttttc
     4201 ttgacaatat tgacttgggg catggacaat actttgcgca ctgtggcaaa atctcgccct
     4261 ttatcccaga caatttctcc agtttcccca ttagtttcga ttagagggcg tttgcgaatc
     4321 tctccatttg caagtgtaat ttctgttttg aagaagttca tgatattaga gtaaaagaaa
     4381 tattttgcgg ttgctttgcc tatttcttgc tcagacttag caatcatttt acgaacatca
     4441 taaactttat aatcaccata gacaaactcc gattcaagtt ttggatattt cttaatcaaa
     4501 gcagttccaa cgacggcatt tagatacgca tcatgggcat gatggtaatt gttaatctca
     4561 cgtactttat agaattggaa atcttttcgg aagtcagaaa ctaatttaga ttttaaggta
     4621 atcactttaa cctctcgaat aagtttatca ttttcatcgt atttagtatt catgcgacta
     4681 tccaaaattt gtgccacatg cttagtgatt tggcgagttt caaccaattg gcgtttgata
     4741 aaaccagctt tatcaagttc actcaaacct ccacgttcag ctttcgttaa attatcaaac
     4801 ttacgttgag tgattaactt ggcgtttaga agttgtctcc aatagttttt catctttttg
     4861 actacttctt cacttggaac gttatccgat ttaccacgat ttttatcaga acgcgttaag
     4921 accttattgt ctattgaatc gtctttaagg aaactttgtg gaacaatggc atcgacatca
     4981 taatcactta aacgattaat atctaattct tggtccacat acatgtctct tccattttgg
     5041 agataataga gatagagctt ttcattttgc aattgagtat tttcaacagg atgctcttta
     5101 agaatctgac ttcctaattc tttgatacct tcttcgattc gtttcatacg ctctcgcgaa
     5161 tttttctggc ccttttgagt tgtctgattt tcacgtgcca tttcaataac gatattttct
     5221 ggcttatgcc gccccattac tttgaccaat tcatcaacaa cttttacagt ctgtaaaata
     5281 ccttttttaa tagcagggct accagctaaa tttgcaatat gttcatgtaa actatcgcct
     5341 tgtccagaca cttgtgcttt ttgaatgtct tctttaaatg tcaaactatc atcatggatc
     5401 agctgaataa aattgcgatt ggcaaaacca tctgatttca aaaaatctaa tattgttttg
     5461 ccagattgct tatccctaat accattaatc aattttcgag acaaacgtcc ccaaccagta
     5521 taacggcgac gtttaagctg tttcatcacc ttatcatcaa agaggtgagc atatgtttta
     5581 agtctttcct caatcatctc cctatcttca aataaggtca atgttaaaac aatatcctct
     5641 aagatatctt cattttcttc attatccaaa aaatctttat ctttaataat ttttagcaaa
     5701 tcatggtagg tacctaatga agcattaaat ctatcttcaa ctcctgaaat ttcaacacta
     5761 tcaaaacatt ctattttttt gaaataatct tcttttaatt gcttaacggt tacttttcga
     5821 tttgttttga agagtaaatc aacaatggct ttcttctgat cacctgaaag aaatgctggt
     5881 tttcgcattc cttcagtaac atatttgacc tttgtcaatt cgttataaac cgtaaaatac
     5941 tcataaagca aactatgttt tggtagtact ttttcatttg gaagattttt atcaaagttt
     6001 gtcatgcgtt caataaatga ttgagctgaa gcacctttat cgacaacttt ttcaaaattc
     6061 catggggtaa ttgtttcttc agacttccga gtcatccatg caaaacgact attgccacgc
     6121 gccaatggac caacataata aggaatacga aaagtcaaga ttttttcaat cttctcacga
     6181 ttgtctttta aaaatggata aaagtcttct tgtcttctca aaatagcatg cagctcaccc
     6241 aagtgaattt gatggggaat aatgccgttg tcaaaggtcc gttgcttgcg cagcaaatct
     6301 tcacgattta gtttcaccaa taattcctca gtaccatcca ttttttctaa aattggtttg
     6361 ataaatttat aaaattcttc ttggctagct cccccatcaa tataacctgc atatccgttt
     6421 tttgattgat caaaaaagat ttctttatac ttttctggaa gttgttgtcg aactaaagct
     6481 tttaaaagag tcaagtcttg atgatgttca tcgtataatt taatcattga agctgatagg
     6541 ggagccttag ttatttcagt atttactctt aggatatctg aaagtaaaat agcatctgat
     6601 aaattcttag ctgccaaaaa caaatcagca tattgatctc caatttgcgc caataaatta
     6661 tctaaatcat catcgtaagt atcttttgaa agctgtaatt tggtatcttc tgccaaatca
     6721 aaatttgatt taaaattagg ggtcaaaccc aatgacaaag caatgagatt cccaaataag
     6781 ccatttttct tctcaccggg gagctgagca atgagatttt ctaatcgtct tgatttactc
     6841 aatcgtgcag aaagaatcgc tttagcatct actccacttg cgttaatagg gttttcttca
     6901 aataattgat tgtaggtttg taccaactgg ataaatagtt tgtccacatc actattatca
     6961 ggatttaaat ctccctcaat caaaaaatga ccacgaaact taatcatatg cgctaaggcc
     7021 aaatagatta agcgcaaatc cgctttatca gtagaatcta ccaatttttt tcgcagatga
     7081 tagatagttg gatatttctc atgataagca acttcatcta ctatatttcc aaaaatagga
     7141 tgacgttcat gcttcttgtc ttcttccacc aaaaaagact cttcaagtcg atgaaagaaa
     7201 ctatcatcta ctttcgccat ctcatttgaa aaaatctcct gtagataaca aatacgattc
     7261 ttccgacgtg tataccttct acgagctgtc cgtttgaggc gagtcgcttc cgctgtctct
     7321 ccactgtcaa ataaaagagc ccctataaga ttttttttga tactgtggcg gtctgtattt
     7381 cccagaacct tgaacttttt agacggaacc ttatattcat cagtgatcac cgcccatccg
     7441 acgctatttg tgccgatagc taagcctatt gagtatttct tatccataga tcctttctcc
     7501 tctttagatc ttttcaattc ttttctctat cactgatagg gagtggtaaa ataactctat
     7561 caacgataga gtgtcaacaa aaattaggaa ttaatgatgt caagattaga taaaagtaaa
     7621 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
     7681 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
     7741 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
     7801 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
     7861 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
     7921 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
     7981 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
     8041 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
     8101 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
     8161 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcttaa
     8221 tactagagcc aattattgaa ggccgctaac gcggcctttt tttgtttctg gtctccctac
     8281 tagagcctag ttgtcttcat gcatgaagac aaaattaata ctagagggac caaaacgaaa
     8341 aaagacgctt ttcagcgtct tattgttcgt ctttggtacc gagctagtat tattgcagaa
     8401 agccatcccg tccctggcga atatcacgcg gtgaccagtt aaactctcgg cgaaaaagcg
     8461 tcgaaaagtg gttactgtcg ctgaatccac agcgataggc gatgtcagta acgctggcct
     8521 cgctgtggcg tagcagatgt cgggctttca tcagtcgcag gcggttcagg tatcgctgag
     8581 gcgtcagtcc cgtttgctgc ttaagctgcc gatgtagcgt acgcagtgaa agagaaaatt
     8641 gatccgccac ggcatcccaa ttcacctcat cggcaaaatg gtcctccagc caggccagaa
     8701 gcaagttgag acgtgatgcg ctgttttcca ggttctcctg caaactgctt ttacgcagca
     8761 agagcagtaa ttgcataaac aagatctcgc gactggcggt cgagggtaaa tcattttccc
     8821 cttcctgctg ttccatctgt gcaaccagct gtcgcacctg ctgcaatacg ctgtggttaa
     8881 cgcgccagtg agacggatac tgcccatcca gctcttgtgg cagcaactga ttcagcccgg
     8941 cgagaaactg aaatcgatcc ggcgagcgat acagcacatt ggtcagacac agattatcgg
     9001 tatgttcata cagatgccga tcatgatcgc gtacgaaaca gaccgtgcca ccggtgatgg
     9061 tatagggctg cccattaaac acatgaatac ccgtgccatg ttcgacaatc acaatttcat
     9121 gaaaatcatg atgatgttca ggaaaatccg cctgcgggag ccggggttct atcgccacgg
     9181 acgcgttacc ggacggaaaa aaatccacac tatgtaatac ggtcatttgg gctccctcta
     9241 gtagctagca taatacctag gactgagcta gctgtaaact ctagtatcac actggctcac
     9301 cttcgggtgg gcctttctgc gtttatatac tagagagacc tttacgccgc tggagcagga
     9361 atgcggtgag catcacatca ccacaattca gcaaattgtg aacatcatca cgttcatctt
     9421 tccctggttg ccaatggccc attttcctgt cagtaacgag aaggtcgcga attgaggcgc
     9481 tttttagact ggtcgtaact agagtcacac aggaaagtac tagatggcag aatacaaaga
     9541 taatttactt ggtgaggcga acagctttct cgaagtgctg gaacaggttt cgcatctcgc
     9601 accgctggac aaaccggtgc tcatcatcgg cgaacgcggc accggtaaag agctgattgc
     9661 cagccgcctg cattatctct cctcccgttg gcaagggccg tttatttccc ttaactgcgc
     9721 ggcgttaaat gaaaatctgc tggattccga actgtttggt cacgaagcgg gggcgtttac
     9781 cggtgcgcaa aaacgtcatc cagggagatt tgaacgtgcc gacggcggta cgctatttct
     9841 tgatgaactc gctacggcac cgatgatggt gcaggagaaa ttattgcgcg tgattgagta
     9901 cggtgaactg gagcgcgttg gcggcagcca accattgcag gtgaatgtgc ggttggtatg
     9961 cgcgacgaat gccgatctcc cggcgatggt caatgaaggc acttttcgcg ctgacctgct
    10021 cgaccgactg gcttttgatg ttgtacaact gccaccactg cgcgagcgcg aaagcgacat
    10081 aatgttgatg gcagaatact ttgccatcca gatgtgtcgg gaaatcaagc tgcctctgtt
    10141 cccggggttt acggagcgcg ccagagaaac attgctgaat tatcgttggc cgggaaatat
    10201 tcgtgaattg aaaaacgtgg tggaacgttc agtgtatcgc cacggcacca gcgattatcc
    10261 gcttgatgac atcattattg atccctttaa acggcgtccg cctgaagacg ctatcgccgt
    10321 ttcagaaacc acctcgcttc caacactgcc gctggattta cgtgagtttc agatgcagca
    10381 ggaaaaagag ttgctgcaac tcagtttgca aatgaatgca cgcacacgcc gccgcgaacg
    10441 tcgcgcagag aaacaggctc aatggaaagc agcaaattaa tactagagcc aattattgaa
    10501 ggccgctaac gcggcctttt tttgtttctg gtctccctac tagaagttat ttccagtgat
    10561 cgcgcggcat agttcgtttc ccatcggtta gtgtaattcg ctaactcatc ctggcatgtt
    10621 gctgttgatt cttcaatcag atctttataa atcaaaaaga taaaaaattg gcacgcaaat
    10681 tgtattaaca gttcagcagg acaatcctga acgcaaatac tagagagctg tcaccggatg
    10741 tgctttccgg tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa
    10801 ctagagatta aagaggagaa ataccatatg cgtaaaggcg aagagctgtt cactggtgtc
    10861 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
    10921 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt
    10981 aaactgccgg taccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt
    11041 gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc
    11101 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa
    11161 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa
    11221 gaagacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac
    11281 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg
    11341 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt
    11401 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg
    11461 aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat
    11521 ggtatggatg aactgtacaa aaggcctgct gcaaacgacg aaaactacgc tgcatcagtt
    11581 taataatact agagccaatt attgaaggcc tccctaacgg ggggcctttt tttgtttctg
    11641 gtctccctac tagtagcggc cgctgca
//
