LOCUS       sequence-257361-       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"
     CDS             complement(166..1116)
                     /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      1164..1386
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(1878..1972)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2002..2793,2794..2862))
                     /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     2625..2644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2863..2967)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3032..3050
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3054..3097)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3100..3121
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(join(3185..4771,4772..4774,4775..7261,7262..7264,
                     7265..7291))
                     /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(3188..7291)
                     /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             7298..7309
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        7327..7382
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7328..7346
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7398..8024
                     /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(7474..7493)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             8039..8050
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(8194..9030)
                     /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(9048..9082)
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     terminator      9098..9125
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        9183..9301
                     /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             10299..10310
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_RNA        10532..10581
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             10614..10625
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             10632..11345
                     /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    11452..11472
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      11473..11530
                     /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(11554..11571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatgcaccaa aaactcgtaa aagctctgat gtatctatct tttttacacc gttttcatct
       61 gtgcatatgg acagttttcc ctttgatatg taacggtgaa cagttgttct acttttgttt
      121 gttagtcttg atgcttcact gatagataca agagccataa gaacctcaga tccttccgta
      181 tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc
      241 attgagatca tacttacttt gcatgtcact caaaaatttt gcctcaaaac tggtgagctg
      301 aatttttgca gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc
      361 tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt tctcaagttc
      421 ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg
      481 gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt tacttattgg
      541 tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa gcattaacat
      601 gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag
      661 ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag acttaacatg
      721 ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact
      781 aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact ggaaaatctc
      841 aaagccttta accaaaggat tcctgatttc cacagttctc gtcatcagct ctctggttgc
      901 tttagctaat acaccataag cattttccct actgatgttc atcatctgag cgtattggtt
      961 ataagtgaac gataccgtcc gttctttcct tgtagggttt tcaatcgtgg ggttgagtag
     1021 tgccacacag cataaaatta gcttggtttc atgctccgtt aagtcatagc gactaatcgc
     1081 tagttcattt gctttgaaaa caactaattc agacatacat ctcaattggt ctaggtgatt
     1141 ttaatcacta taccaattga gatgggctag tcaatgataa ttacatgtcc ttttcctttg
     1201 agttgtgggt atctgtaaat tctgctagac ctttgctgga aaacttgtaa attctgctag
     1261 accctctgta aattccgcta gacctttgtg tgtttttttt gtttatattc aagtggttat
     1321 aatttataga ataaagaaag aataaaaaaa gataaaaaga atagatccca gccctgtgta
     1381 taactcacta ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa cgctgtttgc
     1441 tcctctacaa aacagacctt aaaaccctaa aggcttaagt agcaccctcg caagctcggg
     1501 caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc tgtctttttc
     1561 gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa tggcactaca
     1621 ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag cccgtcacgg
     1681 gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt tttgctgttc
     1741 agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg tgacaggtca
     1801 ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacaggctt acccgtctta
     1861 ctgtccctag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg
     1921 aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagcgag
     1981 ctcgtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2041 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2101 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2161 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2221 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2281 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2341 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2401 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2461 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2521 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2581 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2641 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2701 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2761 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2821 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2881 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2941 gaaaaataaa caaatagggg ttccgcgcac atttcccatg gtgccacctg acgtctaaga
     3001 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag
     3061 ataaaaaaaa tccttagctt tcgctaagga tgatttctgg aattcgcggc cgcttctaga
     3121 ggggagacca gaaacaaaaa aaggggagcg gtttcccgct ccccttcaat aattggctct
     3181 agtattagtc acctcctagc tgactcaaat caatgcgtgt ttcataaaga ccagtgatgg
     3241 attgatggat aagagtggca tctaaaactt cttttgtaga cgtatatcgt ttacgatcaa
     3301 ttgttgtatc aaaatattta aaagcagcgg gagctccaag attcgtcaac gtaaataaat
     3361 gaataatatt ttctgcttgt tcacgtattg gtttgtctct atgtttgtta tatgcactaa
     3421 gaactttatc taaattggca tctgctaaaa taacacgctt agaaaattca ctgatttgct
     3481 caataatctc atctaaataa tgcttatgct gctccacaaa caattgtttt tgttcgttat
     3541 cttctggact acccttcaac ttttcataat gactagctaa atataaaaaa ttcacatatt
     3601 tgcttggcag agccagctca tttccttttt gtaaaactcc ggcactagcc agcatccgtt
     3661 tacgaccgtt ttctaactca aaaagactat atttaggtag tttaatgatt aagtcttttt
     3721 taacttcctt atatccttta gcttctaaaa agtcaatcgg atttttttca aaggaacttc
     3781 tttccataat tgtgatccct agtaactctt taacggattt taacttcttc gatttccctt
     3841 tttccacctt agcaaccact aggactgaat aagctaccgt tggactatca aaaccaccat
     3901 atttttttgg atcccagtct tttttacgag caataagctt gtccgaattt ctttttggta
     3961 aaattgactc cttggagaat ccgcctgtct gtacttctgt tttcttgaca atattgactt
     4021 ggggcatgga caatactttg cgcactgtgg caaaatctcg ccctttatcc cagacaattt
     4081 ctccagtttc cccattagtt tcgattagag ggcgtttgcg aatctctcca tttgcaagtg
     4141 taatttctgt tttgaagaag ttcatgatat tagagtaaaa gaaatatttt gcggttgctt
     4201 tgcctatttc ttgctcagac ttagcaatca ttttacgaac atcataaact ttataatcac
     4261 catagacaaa ctccgattca agttttggat atttcttaat caaagcagtt ccaacgacgg
     4321 catttagata cgcatcatgg gcatgatggt aattgttaat ctcacgtact ttatagaatt
     4381 ggaaatcttt tcggaagtca gaaactaatt tagattttaa ggtaatcact ttaacctctc
     4441 gaataagttt atcattttca tcgtatttag tattcatgcg actatccaaa atttgtgcca
     4501 catgcttagt gatttggcga gtttcaacca attggcgttt gataaaacca gctttatcaa
     4561 gttcactcaa acctccacgt tcagctttcg ttaaattatc aaacttacgt tgagtgatta
     4621 acttggcgtt tagaagttgt ctccaatagt ttttcatctt tttgactact tcttcacttg
     4681 gaacgttatc cgatttacca cgatttttat cagaacgcgt taagacctta ttgtctattg
     4741 aatcgtcttt aaggaaactt tgtggaacaa tggcatcgac atcataatca cttaaacgat
     4801 taatatctaa ttcttggtcc acatacatgt ctcttccatt ttggagataa tagagataga
     4861 gcttttcatt ttgcaattga gtattttcaa caggatgctc tttaagaatc tgacttccta
     4921 attctttgat accttcttcg attcgtttca tacgctctcg cgaatttttc tggccctttt
     4981 gagttgtctg attttcacgt gccatttcaa taacgatatt ttctggctta tgccgcccca
     5041 ttactttgac caattcatca acaactttta cagtctgtaa aatacctttt ttaatagcag
     5101 ggctaccagc taaatttgca atatgttcat gtaaactatc gccttgtcca gacacttgtg
     5161 ctttttgaat gtcttcttta aatgtcaaac tatcatcatg gatcagctga ataaaattgc
     5221 gattggcaaa accatctgat ttcaaaaaat ctaatattgt tttgccagat tgcttatccc
     5281 taataccatt aatcaatttt cgagacaaac gtccccaacc agtataacgg cgacgtttaa
     5341 gctgtttcat caccttatca tcaaagaggt gagcatatgt tttaagtctt tcctcaatca
     5401 tctccctatc ttcaaataag gtcaatgtta aaacaatatc ctctaagata tcttcatttt
     5461 cttcattatc caaaaaatct ttatctttaa taatttttag caaatcatgg taggtaccta
     5521 atgaagcatt aaatctatct tcaactcctg aaatttcaac actatcaaaa cattctattt
     5581 ttttgaaata atcttctttt aattgcttaa cggttacttt tcgatttgtt ttgaagagta
     5641 aatcaacaat ggctttcttc tgatcacctg aaagaaatgc tggttttcgc attccttcag
     5701 taacatattt gacctttgtc aattcgttat aaaccgtaaa atactcataa agcaaactat
     5761 gttttggtag tactttttca tttggaagat ttttatcaaa gtttgtcatg cgttcaataa
     5821 atgattgagc tgaagcacct ttatcgacaa ctttttcaaa attccatggg gtaattgttt
     5881 cttcagactt ccgagtcatc catgcaaaac gactattgcc acgcgccaat ggaccaacat
     5941 aataaggaat acgaaaagtc aagatttttt caatcttctc acgattgtct tttaaaaatg
     6001 gataaaagtc ttcttgtctt ctcaaaatag catgcagctc acccaagtga atttgatggg
     6061 gaataatgcc gttgtcaaag gtccgttgct tgcgcagcaa atcttcacga tttagtttca
     6121 ccaataattc ctcagtacca tccatttttt ctaaaattgg tttgataaat ttataaaatt
     6181 cttcttggct agctccccca tcaatataac ctgcatatcc gttttttgat tgatcaaaaa
     6241 agatttcttt atacttttct ggaagttgtt gtcgaactaa agcttttaaa agagtcaagt
     6301 cttgatgatg ttcatcgtat aatttaatca ttgaagctga taggggagcc ttagttattt
     6361 cagtatttac tcttaggata tctgaaagta aaatagcatc tgataaattc ttagctgcca
     6421 aaaacaaatc agcatattga tctccaattt gcgccaataa attatctaaa tcatcatcgt
     6481 aagtatcttt tgaaagctgt aatttggtat cttctgccaa atcaaaattt gatttaaaat
     6541 taggggtcaa acccaatgac aaagcaatga gattcccaaa taagccattt ttcttctcac
     6601 cggggagctg agcaatgaga ttttctaatc gtcttgattt actcaatcgt gcagaaagaa
     6661 tcgctttagc atctactcca cttgcgttaa tagggttttc ttcaaataat tgattgtagg
     6721 tttgtaccaa ctggataaat agtttgtcca catcactatt atcaggattt aaatctccct
     6781 caatcaaaaa atgaccacga aacttaatca tatgcgctaa ggccaaatag attaagcgca
     6841 aatccgcttt atcagtagaa tctaccaatt tttttcgcag atgatagata gttggatatt
     6901 tctcatgata agcaacttca tctactatat ttccaaaaat aggatgacgt tcatgcttct
     6961 tgtcttcttc caccaaaaaa gactcttcaa gtcgatgaaa gaaactatca tctactttcg
     7021 ccatctcatt tgaaaaaatc tcctgtagat aacaaatacg attcttccga cgtgtatacc
     7081 ttctacgagc tgtccgtttg aggcgagtcg cttccgctgt ctctccactg tcaaataaaa
     7141 gagcccctat aagatttttt ttgatactgt ggcggtctgt atttcccaga accttgaact
     7201 ttttagacgg aaccttatat tcatcagtga tcaccgccca tccgacgcta tttgtgccga
     7261 tagctaagcc tattgagtat ttcttatcca tagatccttt ctcctcttta gatcttttca
     7321 attcttttct ctatcactga tagggagtgg taaaataact ctatcaacga tagagtgtca
     7381 acaaaaatta ggaattaatg atgtcaagat tagataaaag taaagtgatt aacagcgcat
     7441 tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc
     7501 taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg
     7561 ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa
     7621 gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc
     7681 gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg
     7741 aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac
     7801 tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg
     7861 ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta
     7921 tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca
     7981 tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ttaatactag agccaattat
     8041 tgaaggccgc taacgcggcc tttttttgtt tctggtctcc ctactagagc ctagttgtct
     8101 tcatgcatga agacaaaatt aatactagag ggaccaaaac gaaaaaagac gcttttcagc
     8161 gtcttattgt tcgtctttgg taccgagcta gtattattgc agaaagccat cccgtccctg
     8221 gcgaatatca cgcggtgacc agttaaactc tcggcgaaaa agcgtcgaaa agtggttact
     8281 gtcgctgaat ccacagcgat aggcgatgtc agtaacgctg gcctcgctgt ggcgtagcag
     8341 atgtcgggct ttcatcagtc gcaggcggtt caggtatcgc tgaggcgtca gtcccgtttg
     8401 ctgcttaagc tgccgatgta gcgtacgcag tgaaagagaa aattgatccg ccacggcatc
     8461 ccaattcacc tcatcggcaa aatggtcctc cagccaggcc agaagcaagt tgagacgtga
     8521 tgcgctgttt tccaggttct cctgcaaact gcttttacgc agcaagagca gtaattgcat
     8581 aaacaagatc tcgcgactgg cggtcgaggg taaatcattt tccccttcct gctgttccat
     8641 ctgtgcaacc agctgtcgca cctgctgcaa tacgctgtgg ttaacgcgcc agtgagacgg
     8701 atactgccca tccagctctt gtggcagcaa ctgattcagc ccggcgagaa actgaaatcg
     8761 atccggcgag cgatacagca cattggtcag acacagatta tcggtatgtt catacagatg
     8821 ccgatcatga tcgcgtacga aacagaccgt gccaccggtg atggtatagg gctgcccatt
     8881 aaacacatga atacccgtgc catgttcgac aatcacaatt tcatgaaaat catgatgatg
     8941 ttcaggaaaa tccgcctgcg ggagccgggg ttctatcgcc acggacgcgt taccggacgg
     9001 aaaaaaatcc acactatgta atacggtcat ttgggctccc tctagtagct agcataatac
     9061 ctaggactga gctagctgta aactctagta tcacactggc tcaccttcgg gtgggccttt
     9121 ctgcgtttat atactagaga gacctttacg ccgctggagc aggaatgcgg tgagcatcac
     9181 atcaccacaa ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaatg
     9241 gcccattttc ctgtcagtaa cgagaaggtc gcgaattgag gcgcttttta gactggtcgt
     9301 aactagagtc acacaggaaa gtactagatg gcagaataca aagataattt acttggtgag
     9361 gcgaacagct ttctcgaagt gctggaacag gtttcgcatc tcgcaccgct ggacaaaccg
     9421 gtgctcatca tcggcgaacg cggcaccggt aaagagctga ttgccagccg cctgcattat
     9481 ctctcctccc gttggcaagg gccgtttatt tcccttaact gcgcggcgtt aaatgaaaat
     9541 ctgctggatt ccgaactgtt tggtcacgaa gcgggggcgt ttaccggtgc gcaaaaacgt
     9601 catccaggga gatttgaacg tgccgacggc ggtacgctat ttcttgatga actcgctacg
     9661 gcaccgatga tggtgcagga gaaattattg cgcgtgattg agtacggtga actggagcgc
     9721 gttggcggca gccaaccatt gcaggtgaat gtgcggttgg tatgcgcgac gaatgccgat
     9781 ctcccggcga tggtcaatga aggcactttt cgcgctgacc tgctcgaccg actggctttt
     9841 gatgttgtac aactgccacc actgcgcgag cgcgaaagcg acataatgtt gatggcagaa
     9901 tactttgcca tccagatgtg tcgggaaatc aagctgcctc tgttcccggg gtttacggag
     9961 cgcgccagag aaacattgct gaattatcgt tggccgggaa atattcgtga attgaaaaac
    10021 gtggtggaac gttcagtgta tcgccacggc accagcgatt atccgcttga tgacatcatt
    10081 attgatccct ttaaacggcg tccgcctgaa gacgctatcg ccgtttcaga aaccacctcg
    10141 cttccaacac tgccgctgga tttacgtgag tttcagatgc agcaggaaaa agagttgctg
    10201 caactcagtt tgcaaatgaa tgcacgcaca cgccgccgcg aacgtcgcgc agagaaacag
    10261 gctcaatgga aagcagcaaa ttaatactag agccaattat tgaaggccgc taacgcggcc
    10321 tttttttgtt tctggtctcc ctactagaag ttatttccag tgatcgcgcg gcatagttcg
    10381 tttcccatcg gttagtgtaa ttcgctaact catcctggca tgttgctgtt gattcttcaa
    10441 tcagatcttt ataaatcaaa aagataaaaa attggcacgc aaattgtatt aacagttcag
    10501 caggacaatc ctgaacgcaa atactagaga gctgtcaccg gatgtgcttt ccggtctgat
    10561 gagtccgtga ggacgaaaca gcctctacaa ataattttgt ttaactagag attaaagagg
    10621 agaaatacca tatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct attctggtgg
    10681 aactggatgg tgatgtcaac ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg
    10741 caactaatgg taaactgacg ctgaagttca tctgtactac tggtaaactg ccggtacctt
    10801 ggccgactct ggtaacgacg ctgacttatg gtgttcagtg ctttgctcgt tatccggacc
    10861 atatgaagca gcatgacttc ttcaagtccg ccatgccgga aggctatgtg caggaacgca
    10921 cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg
    10981 ataccctggt aaaccgcatt gagctgaaag gcattgactt taaagaagac ggcaatatcc
    11041 tgggccataa gctggaatac aattttaaca gccacaatgt ttacatcacc gccgataaac
    11101 aaaaaaatgg cattaaagcg aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc
    11161 agctggctga tcactaccag caaaacactc caatcggtga tggtcctgtt ctgctgccag
    11221 acaatcacta tctgagcacg caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc
    11281 atatggttct gctggagttc gtaaccgcag cgggcatcac gcatggtatg gatgaactgt
    11341 acaaaaggcc tgctgcaaac gacgaaaact acgctgcatc agtttaataa tactagagcc
    11401 aattattgaa ggcctcccta acggggggcc tttttttgtt tctggtctcc ctactagtag
    11461 cggccgctgc agtccggcaa aaaagggcaa ggtgtcacca ccctgccctt tttctttaaa
    11521 accgaaaaga ttacttcgcg ttatgcaggc ttcctcgctc actgactcgc tgcgctcggt
    11581 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatctcga ggttacattg
    11641 tcgatctgtt catggtgaac agctttg
//
