LOCUS       sequence-257360-       10491 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A CRISPR activation device with the necessary genes (dxcas9 3.7 
            controlled by Ptet, pspF-Delta-HTH::lambda-N22plus controlled by 
            promoter J23106), and the reporter part (PpspA-LEA1B1 with 
            sfgfp::ASV)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10491)
  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 10491)
  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: pLY61
FEATURES             Location/Qualifiers
     source          1..10491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(228..1178)
                     /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      1226..1448
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(1940..2034)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2064..2855,2856..2924))
                     /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     2687..2706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2925..3029)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3094..3112
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3116..3159)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3162..3183
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(join(3247..4833,4834..4836,4837..7323,7324..7326,
                     7327..7353))
                     /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(3250..7353)
                     /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             7360..7371
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        7389..7444
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7390..7408
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7460..8086
                     /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(7536..7555)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             8101..8112
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8152..8186
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             9185..9196
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_RNA        9418..9467
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             9500..9511
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             9518..10231
                     /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    10338..10358
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      10359..10416
                     /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(10440..10457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gctcactcaa aggcggtaat ctcgaggtta cattgtcgat ctgttcatgg tgaacagctt
       61 tgaatgcacc aaaaactcgt aaaagctctg atgtatctat cttttttaca ccgttttcat
      121 ctgtgcatat ggacagtttt ccctttgata tgtaacggtg aacagttgtt ctacttttgt
      181 ttgttagtct tgatgcttca ctgatagata caagagccat aagaacctca gatccttccg
      241 tatttagcca gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa
      301 ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc
      361 tgaatttttg cagttaaagc atcgtgtagt gtttttctta gtccgttatg taggtaggaa
      421 tctgatgtaa tggttgttgg tattttgtca ccattcattt ttatctggtt gttctcaagt
      481 tcggttacga gatccatttg tctatctagt tcaacttgga aaatcaacgt atcagtcggg
      541 cggcctcgct tatcaaccac caatttcata ttgctgtaag tgtttaaatc tttacttatt
      601 ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc aagcattaac
      661 atgaacttaa attcatcaag gctaatctct atatttgcct tgtgagtttt cttttgtgtt
      721 agttctttta ataaccactc ataaatcctc atagagtatt tgttttcaaa agacttaaca
      781 tgttccagat tatattttat gaattttttt aactggaaaa gataaggcaa tatctcttca
      841 ctaaaaacta attctaattt ttcgcttgag aacttggcat agtttgtcca ctggaaaatc
      901 tcaaagcctt taaccaaagg attcctgatt tccacagttc tcgtcatcag ctctctggtt
      961 gctttagcta atacaccata agcattttcc ctactgatgt tcatcatctg agcgtattgg
     1021 ttataagtga acgataccgt ccgttctttc cttgtagggt tttcaatcgt ggggttgagt
     1081 agtgccacac agcataaaat tagcttggtt tcatgctccg ttaagtcata gcgactaatc
     1141 gctagttcat ttgctttgaa aacaactaat tcagacatac atctcaattg gtctaggtga
     1201 ttttaatcac tataccaatt gagatgggct agtcaatgat aattacatgt ccttttcctt
     1261 tgagttgtgg gtatctgtaa attctgctag acctttgctg gaaaacttgt aaattctgct
     1321 agaccctctg taaattccgc tagacctttg tgtgtttttt ttgtttatat tcaagtggtt
     1381 ataatttata gaataaagaa agaataaaaa aagataaaaa gaatagatcc cagccctgtg
     1441 tataactcac tactttagtc agttccgcag tattacaaaa ggatgtcgca aacgctgttt
     1501 gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccct cgcaagctcg
     1561 ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg cacctgagtc gctgtctttt
     1621 tcgtgacatt cagttcgctg cgctcacggc tctggcagtg aatgggggta aatggcacta
     1681 caggcgcctt ttatggattc atgcaaggaa actacccata atacaagaaa agcccgtcac
     1741 gggcttctca gggcgtttta tggcgggtct gctatgtggt gctatctgac tttttgctgt
     1801 tcagcagttc ctgccctctg attttccagt ctgaccactt cggattatcc cgtgacaggt
     1861 cattcagact ggctaatgca cccagtaagg cagcggtatc atcaacaggc ttacccgtct
     1921 tactgtccct agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     1981 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
     2041 agctcgtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2101 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2161 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2221 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2281 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2341 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2401 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2461 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2521 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2581 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2641 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     2701 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2761 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2821 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2881 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2941 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3001 tagaaaaata aacaaatagg ggttccgcgc acatttccca tggtgccacc tgacgtctaa
     3061 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gcagaatttc
     3121 agataaaaaa aatccttagc tttcgctaag gatgatttct ggaattcgcg gccgcttcta
     3181 gaggggagac cagaaacaaa aaaaggggag cggtttcccg ctccccttca ataattggct
     3241 ctagtattag tcacctccta gctgactcaa atcaatgcgt gtttcataaa gaccagtgat
     3301 ggattgatgg ataagagtgg catctaaaac ttcttttgta gacgtatatc gtttacgatc
     3361 aattgttgta tcaaaatatt taaaagcagc gggagctcca agattcgtca acgtaaataa
     3421 atgaataata ttttctgctt gttcacgtat tggtttgtct ctatgtttgt tatatgcact
     3481 aagaacttta tctaaattgg catctgctaa aataacacgc ttagaaaatt cactgatttg
     3541 ctcaataatc tcatctaaat aatgcttatg ctgctccaca aacaattgtt tttgttcgtt
     3601 atcttctgga ctacccttca acttttcata atgactagct aaatataaaa aattcacata
     3661 tttgcttggc agagccagct catttccttt ttgtaaaact ccggcactag ccagcatccg
     3721 tttacgaccg ttttctaact caaaaagact atatttaggt agtttaatga ttaagtcttt
     3781 tttaacttcc ttatatcctt tagcttctaa aaagtcaatc ggattttttt caaaggaact
     3841 tctttccata attgtgatcc ctagtaactc tttaacggat tttaacttct tcgatttccc
     3901 tttttccacc ttagcaacca ctaggactga ataagctacc gttggactat caaaaccacc
     3961 atattttttt ggatcccagt cttttttacg agcaataagc ttgtccgaat ttctttttgg
     4021 taaaattgac tccttggaga atccgcctgt ctgtacttct gttttcttga caatattgac
     4081 ttggggcatg gacaatactt tgcgcactgt ggcaaaatct cgccctttat cccagacaat
     4141 ttctccagtt tccccattag tttcgattag agggcgtttg cgaatctctc catttgcaag
     4201 tgtaatttct gttttgaaga agttcatgat attagagtaa aagaaatatt ttgcggttgc
     4261 tttgcctatt tcttgctcag acttagcaat cattttacga acatcataaa ctttataatc
     4321 accatagaca aactccgatt caagttttgg atatttctta atcaaagcag ttccaacgac
     4381 ggcatttaga tacgcatcat gggcatgatg gtaattgtta atctcacgta ctttatagaa
     4441 ttggaaatct tttcggaagt cagaaactaa tttagatttt aaggtaatca ctttaacctc
     4501 tcgaataagt ttatcatttt catcgtattt agtattcatg cgactatcca aaatttgtgc
     4561 cacatgctta gtgatttggc gagtttcaac caattggcgt ttgataaaac cagctttatc
     4621 aagttcactc aaacctccac gttcagcttt cgttaaatta tcaaacttac gttgagtgat
     4681 taacttggcg tttagaagtt gtctccaata gtttttcatc tttttgacta cttcttcact
     4741 tggaacgtta tccgatttac cacgattttt atcagaacgc gttaagacct tattgtctat
     4801 tgaatcgtct ttaaggaaac tttgtggaac aatggcatcg acatcataat cacttaaacg
     4861 attaatatct aattcttggt ccacatacat gtctcttcca ttttggagat aatagagata
     4921 gagcttttca ttttgcaatt gagtattttc aacaggatgc tctttaagaa tctgacttcc
     4981 taattctttg ataccttctt cgattcgttt catacgctct cgcgaatttt tctggccctt
     5041 ttgagttgtc tgattttcac gtgccatttc aataacgata ttttctggct tatgccgccc
     5101 cattactttg accaattcat caacaacttt tacagtctgt aaaatacctt ttttaatagc
     5161 agggctacca gctaaatttg caatatgttc atgtaaacta tcgccttgtc cagacacttg
     5221 tgctttttga atgtcttctt taaatgtcaa actatcatca tggatcagct gaataaaatt
     5281 gcgattggca aaaccatctg atttcaaaaa atctaatatt gttttgccag attgcttatc
     5341 cctaatacca ttaatcaatt ttcgagacaa acgtccccaa ccagtataac ggcgacgttt
     5401 aagctgtttc atcaccttat catcaaagag gtgagcatat gttttaagtc tttcctcaat
     5461 catctcccta tcttcaaata aggtcaatgt taaaacaata tcctctaaga tatcttcatt
     5521 ttcttcatta tccaaaaaat ctttatcttt aataattttt agcaaatcat ggtaggtacc
     5581 taatgaagca ttaaatctat cttcaactcc tgaaatttca acactatcaa aacattctat
     5641 ttttttgaaa taatcttctt ttaattgctt aacggttact tttcgatttg ttttgaagag
     5701 taaatcaaca atggctttct tctgatcacc tgaaagaaat gctggttttc gcattccttc
     5761 agtaacatat ttgacctttg tcaattcgtt ataaaccgta aaatactcat aaagcaaact
     5821 atgttttggt agtacttttt catttggaag atttttatca aagtttgtca tgcgttcaat
     5881 aaatgattga gctgaagcac ctttatcgac aactttttca aaattccatg gggtaattgt
     5941 ttcttcagac ttccgagtca tccatgcaaa acgactattg ccacgcgcca atggaccaac
     6001 ataataagga atacgaaaag tcaagatttt ttcaatcttc tcacgattgt cttttaaaaa
     6061 tggataaaag tcttcttgtc ttctcaaaat agcatgcagc tcacccaagt gaatttgatg
     6121 gggaataatg ccgttgtcaa aggtccgttg cttgcgcagc aaatcttcac gatttagttt
     6181 caccaataat tcctcagtac catccatttt ttctaaaatt ggtttgataa atttataaaa
     6241 ttcttcttgg ctagctcccc catcaatata acctgcatat ccgttttttg attgatcaaa
     6301 aaagatttct ttatactttt ctggaagttg ttgtcgaact aaagctttta aaagagtcaa
     6361 gtcttgatga tgttcatcgt ataatttaat cattgaagct gataggggag ccttagttat
     6421 ttcagtattt actcttagga tatctgaaag taaaatagca tctgataaat tcttagctgc
     6481 caaaaacaaa tcagcatatt gatctccaat ttgcgccaat aaattatcta aatcatcatc
     6541 gtaagtatct tttgaaagct gtaatttggt atcttctgcc aaatcaaaat ttgatttaaa
     6601 attaggggtc aaacccaatg acaaagcaat gagattccca aataagccat ttttcttctc
     6661 accggggagc tgagcaatga gattttctaa tcgtcttgat ttactcaatc gtgcagaaag
     6721 aatcgcttta gcatctactc cacttgcgtt aatagggttt tcttcaaata attgattgta
     6781 ggtttgtacc aactggataa atagtttgtc cacatcacta ttatcaggat ttaaatctcc
     6841 ctcaatcaaa aaatgaccac gaaacttaat catatgcgct aaggccaaat agattaagcg
     6901 caaatccgct ttatcagtag aatctaccaa tttttttcgc agatgataga tagttggata
     6961 tttctcatga taagcaactt catctactat atttccaaaa ataggatgac gttcatgctt
     7021 cttgtcttct tccaccaaaa aagactcttc aagtcgatga aagaaactat catctacttt
     7081 cgccatctca tttgaaaaaa tctcctgtag ataacaaata cgattcttcc gacgtgtata
     7141 ccttctacga gctgtccgtt tgaggcgagt cgcttccgct gtctctccac tgtcaaataa
     7201 aagagcccct ataagatttt ttttgatact gtggcggtct gtatttccca gaaccttgaa
     7261 ctttttagac ggaaccttat attcatcagt gatcaccgcc catccgacgc tatttgtgcc
     7321 gatagctaag cctattgagt atttcttatc catagatcct ttctcctctt tagatctttt
     7381 caattctttt ctctatcact gatagggagt ggtaaaataa ctctatcaac gatagagtgt
     7441 caacaaaaat taggaattaa tgatgtcaag attagataaa agtaaagtga ttaacagcgc
     7501 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa
     7561 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga
     7621 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga
     7681 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca
     7741 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct
     7801 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc
     7861 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt
     7921 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc
     7981 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat
     8041 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcttaatact agagccaatt
     8101 attgaaggcc gctaacgcgg cctttttttg tttctggtct ccctactaga gtttacggct
     8161 agctcagtcc taggtatagt gctagctact agagtcacac aggaaagtac tagatggcag
     8221 aatacaaaga taatttactt ggtgaggcga acagctttct cgaagtgctg gaacaggttt
     8281 cgcatctcgc accgctggac aaaccggtgc tcatcatcgg cgaacgcggc accggtaaag
     8341 agctgattgc cagccgcctg cattatctct cctcccgttg gcaagggccg tttatttccc
     8401 ttaactgcgc ggcgttaaat gaaaatctgc tggattccga actgtttggt cacgaagcgg
     8461 gggcgtttac cggtgcgcaa aaacgtcatc cagggagatt tgaacgtgcc gacggcggta
     8521 cgctatttct tgatgaactc gctacggcac cgatgatggt gcaggagaaa ttattgcgcg
     8581 tgattgagta cggtgaactg gagcgcgttg gcggcagcca accattgcag gtgaatgtgc
     8641 ggttggtatg cgcgacgaat gccgatctcc cggcgatggt caatgaaggc acttttcgcg
     8701 ctgacctgct cgaccgactg gcttttgatg ttgtacaact gccaccactg cgcgagcgcg
     8761 aaagcgacat aatgttgatg gcagaatact ttgccatcca gatgtgtcgg gaaatcaagc
     8821 tgcctctgtt cccggggttt acggagcgcg ccagagaaac attgctgaat tatcgttggc
     8881 cgggaaatat tcgtgaattg aaaaacgtgg tggaacgttc agtgtatcgc cacggcacca
     8941 gcgattatcc gcttgatgac atcattattg atccctttaa acggcgtccg cctgaagacg
     9001 ctatcgccgt ttcagaaacc acctcgcttc caacactgcc gctggattta cgtgagtttc
     9061 agatgcagca ggaaaaagag ttgctgcaac tcagtttgca aatgaatgca cgcacacgcc
     9121 gccgcgaacg tcgcgcagag aaacaggctc aatggaaagc agcaaattaa tactagagcc
     9181 aattattgaa ggccgctaac gcggcctttt tttgtttctg gtctccctac tagaagttat
     9241 cccgacgcgc cgtccggtct gaggtctgga agatcggtta gtgtaattcg ctaactcatc
     9301 ctggcatgtt gctgttgatt cttcaatcag atctttataa atcaaaaaga taaaaaattg
     9361 gcacgcaaat tgtattaaca gttcagcagg acaatcctga acgcaaatac tagagagctg
     9421 tcaccggatg tgctttccgg tctgatgagt ccgtgaggac gaaacagcct ctacaaataa
     9481 ttttgtttaa ctagagatta aagaggagaa ataccatatg cgtaaaggcg aagagctgtt
     9541 cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc
     9601 cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg
     9661 tactactggt aaactgccgg taccttggcc gactctggta acgacgctga cttatggtgt
     9721 tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat
     9781 gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac
     9841 gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat
     9901 tgactttaaa gaagacggca atatcctggg ccataagctg gaatacaatt ttaacagcca
     9961 caatgtttac atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg
    10021 ccacaacgtg gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat
    10081 cggtgatggt cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc
    10141 taaagatccg aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg
    10201 catcacgcat ggtatggatg aactgtacaa aaggcctgct gcaaacgacg aaaactacgc
    10261 tgcatcagtt taataatact agagccaatt attgaaggcc tccctaacgg ggggcctttt
    10321 tttgtttctg gtctccctac tagtagcggc cgctgcagtc cggcaaaaaa gggcaaggtg
    10381 tcaccaccct gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc
    10441 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc a
//
