LOCUS       sequence-488014-       11501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of Spy-dCas9-AsiA and Sth1-dCas9, and 
            constitutive expression of MCP-SoxS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11501)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 11501)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pKO025
FEATURES             Location/Qualifiers
     source          1..11501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(10..636)
                     /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     541..560
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        652..707
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    688..706
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             725..736
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(743..769,770..772,773..3259,3260..3262,3263..4846)
                     /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"
     terminator      5179..5250
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5266..5293
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        5361..5416
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5397..5415
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             5434..5445
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             7439..7447
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      8841..8912
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      8928..8955
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        8988..9022
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             9032..9043
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(9811..9828)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9945..10490)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(10025..10044)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(10604..10698)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(10719..11378)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     11293..11312
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(11379..11481)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
       61 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
      121 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
      181 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
      241 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
      301 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
      361 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
      421 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
      481 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
      541 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
      601 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
      661 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
      721 atctaaagag gagaaaggat ctatggataa gaaatactca ataggcttag ctatcggcac
      781 aaatagcgtc ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa
      841 ggttctggga aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt
      901 tgacagtgga gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac
      961 acgtcggaag aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt
     1021 agatgatagt ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca
     1081 tgaacgtcat cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc
     1141 aactatctat catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt
     1201 aatctatttg gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga
     1261 tttaaatcct gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa
     1321 tcaattattt gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc
     1381 tgcacgattg agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa
     1441 gaaaaatggc ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa
     1501 atcaaatttt gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga
     1561 tgatttagat aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc
     1621 taagaattta tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac
     1681 taaggctccc ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac
     1741 tcttttaaaa gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga
     1801 tcaatcaaaa aacggatatg caggttatat tgatggggga gctagccaag aagaatttta
     1861 taaatttatc aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact
     1921 aaatcgtgaa gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca
     1981 aattcacttg ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt
     2041 aaaagacaat cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg
     2101 tccattggcg cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat
     2161 taccccatgg aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga
     2221 acgcatgaca aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt
     2281 gctttatgag tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg
     2341 aatgcgaaaa ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt
     2401 caaaacaaat cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga
     2461 atgttttgat agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac
     2521 ctaccatgat ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga
     2581 agatatctta gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga
     2641 ggaaagactt aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg
     2701 tcgccgttat actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa
     2761 gcaatctggc aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt
     2821 tatgcagctg atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt
     2881 gtctggacaa ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat
     2941 taaaaaaggt attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg
     3001 gcataagcca gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg
     3061 ccagaaaaat tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag
     3121 tcagattctt aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct
     3181 ctattatctc caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt
     3241 aagtgattat gatgtcgatg ccattgttcc acaaagtttc cttaaagacg attcaataga
     3301 caataaggtc ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga
     3361 agaagtagtc aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac
     3421 tcaacgtaag tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa
     3481 agctggtttt atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca
     3541 aattttggat agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt
     3601 taaagtgatt accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta
     3661 taaagtacgt gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt
     3721 tggaactgct ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta
     3781 taaagtttat gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac
     3841 cgcaaaatat ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc
     3901 aaatggagag attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt
     3961 ctgggataaa gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa
     4021 tattgtcaag aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa
     4081 aagaaattcg gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg
     4141 ttttgatagt ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa
     4201 atcgaagaag ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc
     4261 ctttgaaaaa aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga
     4321 cttaatcatt aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat
     4381 gctggctagt gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt
     4441 gaatttttta tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga
     4501 acaaaaacaa ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat
     4561 cagtgaattt tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc
     4621 atataacaaa catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt
     4681 tacgttgacg aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg
     4741 taaacgatat acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac
     4801 tggtctttat gaaacacgca ttgatttgag tcagctagga ggtgactgcg cgggtggcgg
     4861 aggaagtggg ggcggtggta gcatgaataa aaacattgat acagttcgtg aaattattac
     4921 tgttgcatct attttgatta aattttccag agaagatatt gttgagaatc gtgctaattt
     4981 tattgcattt ctaaatgaga ttggagtaac gcatgaaggt agaaagttaa atcgcaattc
     5041 attccgtaaa attatttcta aattaactca agaagataag aaaaccctca tcgacgaatt
     5101 caacgagggt tttgagggtg tatatcgata tctagagatg tatacgaaca aataactcga
     5161 gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     5221 tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt
     5281 gggcctttct gcgtttatac ctagagcgcc cggaagagag tcaattcagg gtggtgaatc
     5341 tgcagtaatt cctaattttt gttgacactc tatcgttgat agagttattt taccactccc
     5401 tatcagtgat agagaaaaga attcaaaaga tctaaagagg agaaaggatc tatgagtgac
     5461 ttagttttag gacttgctat cggtataggt tctgttggtg tgggtatcct taacaaagtg
     5521 acaggagaaa ttatccataa aaactcacgc atcttcccag cagctcaagc ggaaaataac
     5581 ctagtacgta gaacgaatcg tcaaggaaga cgcttggcac gacgtaaaaa acatcgtaga
     5641 gttcgtttaa atcgtctatt tgaggaaagt ggattaatca ctgattttac gaagatttca
     5701 attaatctta acccatatca attacgagtt aagggcttga ccgatgaatt gtctaatgaa
     5761 gaactgttta tcgctcttaa aaatatggtg aaacaccgtg ggattagtta cctcgatgat
     5821 gctagtgatg acggaaattc atcagtagga gactatgcac aaattgttaa ggaaaatagt
     5881 aaacaattag aaactaagac accgggacag atacagttgg aacgctacca aacatatggt
     5941 caattacgtg gtgattttac tgttgagaaa gatggcaaaa aacatcgctt gattaatgtc
     6001 tttccaacat cagcttatcg ttcagaagcc ttaaggatac tgcaaactca acaagaattt
     6061 aatccacaga ttacagatga atttattaat cgttatctcg aaattttaac tggaaaacgg
     6121 aaatattatc atggacccgg aaatgaaaag tcacggactg attatggtcg ttacagaacg
     6181 agtggagaaa ctttagacaa tatttttgga attctaattg ggaaatgtac attttatcca
     6241 gacgagttta gagcagcaaa agcttcctac acggctcaag aattcaattt gctaaatgat
     6301 ttgaacaatc taacagttcc tactgaaacc aaaaagttga gcaaagaaca gaagaatcaa
     6361 atcattaatt atgtcaaaaa tgaaaaggca atggggccag cgaaactttt taaatatatc
     6421 gctaagttac tttcttgtga tgttgcagat atcaagggat accgtatcga caaatcaggt
     6481 aaggctgaga ttcatacttt cgaagcctat cgaaaaatga aaacgcttga aaccttagat
     6541 attgagcaaa tggatagaga aacgcttgat aaattagcct atgtcttaac attaaacact
     6601 gagagggaag gtattcaaga agccttagaa catgaatttg ctgatggtag ctttagccag
     6661 aagcaagttg acgaattggt tcaattccgc aaagcaaata gttccatttt tggaaaagga
     6721 tggcataatt tttctgtcaa actgatgatg gagttaattc cagaattgta tgagacgtca
     6781 gaagagcaaa tgactatcct aacacgactt ggaaaacaaa aaacgacttc gtcttcaaat
     6841 aaaacaaaat atatagatga aaaactatta actgaagaaa tctataatcc tgttgttgct
     6901 aagtctgttc gccaggctat aaaaatcgta aatgcggcga ttaaagaata cggagacttt
     6961 gacaatattg tcatcgaaat ggctcgtgaa acaaatgaag atgatgaaaa gaaagctatt
     7021 caaaagattc aaaaagccaa caaagatgaa aaagatgcag caatgcttaa ggctgctaac
     7081 caatataatg gaaaggctga attaccacat agtgttttcc acggtcataa gcaattagcg
     7141 actaaaatcc gcctttggca tcagcaagga gaacgttgcc tttatactgg taagacaatc
     7201 tcaatccatg atttgataaa taattctaat cagtttgaag tagatgctat tttacctctt
     7261 tctatcacat tcgatgatag ccttgcaaat aaggttttgg tttatgcaac tgctaaccaa
     7321 gaaaaaggac aacgaacacc ttatcaggct ttagatagta tggatgatgc gtggtctttc
     7381 cgtgaattaa aagcttttgt acgtgagtca aaaacacttt cgaacaagaa aaaagaatac
     7441 ctccttacag aagaagatat ttcaaagttt gatgttcgaa agaaatttat tgaacgaaat
     7501 cttgtagata caagatacgc ttcaagagtt gtcctcaatg cccttcaaga acactttaga
     7561 gctcacaaga ttgatacaaa agtttccgtg gttcgtggcc aatttacatc tcaattgaga
     7621 cgccattggg gaattgagaa gactcgtgat acttatcatc accatgctgt cgatgcattg
     7681 attattgccg cctcaagtca gttgaatttg tggaaaaaac aaaagaatac ccttgtaagt
     7741 tattcagaag accaactcct tgatattgaa acaggtgaac ttattagtga tgatgagtac
     7801 aaggaatctg tgttcaaagc cccttatcaa cattttgttg atacattgaa gagtaaagaa
     7861 tttgaagaca gtatcttatt ctcttatcaa gtggattcta agtttaatcg taaaatatca
     7921 gatgccacta tttatgcgac aagacaggct aaagtaggaa aagataaggc ggatgaaact
     7981 tatgtcttag ggaaaatcaa agatatctat actcaggatg gttatgatgc ctttatgaag
     8041 atttataaga aggataagtc aaaattcctc atgtatcgtc acgacccaca aacctttgag
     8101 aaagttatcg agccaatttt agagaactat cctaataagc aaataaatga aaaagggaaa
     8161 gaggtaccat gtaatccttt cctaaaatat aaagaagaac atggctatat tcgtaaatat
     8221 agtaaaaaag gcaatggtcc tgaaatcaag agtcttaaat actatgatag taagctaggc
     8281 aatcatattg atattactcc gaaggatagt aacaataaag ttgtcttaca gtcagtttct
     8341 ccatggagag cggatgtcta tttcaataag actactggaa aatacgaaat cctcggattg
     8401 aaatatgctg atttacaatt tgaaaaaggg acaggaacat ataagatttc ccaggaaaaa
     8461 tacaatgaca ttaagaaaaa agagggtgta gattctgatt cagaattcaa gtttacactt
     8521 tataaaaatg atttgttact cgttaaagat acagaaacaa aagaacaaca gcttttccgt
     8581 tttctttctc gaactatgcc taaacaaaag cattatgttg aattaaaacc ttatgataaa
     8641 cagaaatttg aaggaggtga ggcgttaatt aaagtgttgg gtaacgttgc taatagtggt
     8701 caatgcaaaa aagggctagg aaaatcaaat atttctattt ataaggtaag aacagatgtc
     8761 ctaggaaatc agcatatcat caaaaatgag ggtgataagc ctaagctaga tttttaactc
     8821 gagtaaggat ctccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     8881 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
     8941 gtgggccttt ctgcgtttat agtcaattca gggtggtgaa tctgcagttt acggctagct
     9001 cagccctagg tattatgcta gcgaattcat taaagaggag aaaggtacca tggggcccgc
     9061 ttctaacttt actcagttcg ttctcgtcga caatggcgga actggcgacg tgactgtcgc
     9121 cccaagcaac ttcgctaacg ggatcgctga atggatcagc tctaactcgc gttcacaggc
     9181 ttacaaagta acctgtagcg ttcgtcagag ctctgcgcag aatcgcaaat acaccatcaa
     9241 agtcgaggtg cctaaaggcg cctggcgttc gtacttaaat atggaactaa ccattccaat
     9301 tttcgccacg aattccgact gcgagcttat tgttaaggca atgcaaggtc tcctaaaaga
     9361 tggaaacccg attccctcag caatcgcagc aaactccggc atctacggtg gcggaggtag
     9421 catgtcccat cagaaaatta ttcaggatct tatcgcatgg attgacgagc atattgacca
     9481 gccgcttaac attgatgtag tcgcaaaaaa atcaggctat tcaaagtggt acttgcaacg
     9541 aatgttccgc acggtgacgc atcagacgct tggcgattac attcgccaac gccgcctgtt
     9601 actggccgcc gttgagttgc gcaccaccga gcgtccgatt tttgatatcg caatggacct
     9661 gggttatgtc tcgcagcaga ccttctcccg cgttttcgcg cggcagtttg atcgcactcc
     9721 cgcggattat cgccaccgcc tgtaagcggc cgccacgcaa aaaaccccgc ttcggcgggg
     9781 ttttttcgcc ctagggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     9841 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     9901 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     9961 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
    10021 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
    10081 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
    10141 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
    10201 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
    10261 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
    10321 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
    10381 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
    10441 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
    10501 ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac
    10561 ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca ccaataaaaa
    10621 acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg
    10681 gatctatcaa caggagtcca agcgagctcg atatcaaatt acgccccgcc ctgccactca
    10741 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
    10801 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
    10861 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
    10921 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
    10981 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
    11041 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
    11101 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacga
    11161 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
    11221 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
    11281 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
    11341 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
    11401 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
    11461 gaacctctta cgtgccgatc aacgtctcat tttcgccaga t
//
