LOCUS       sequence-331405-        9150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of dCas9 (aTc control) and sgRNA (arabinose 
            control).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9150)
  AUTHORS   Bradley RW
  TITLE     An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. 
            coli.
  JOURNAL   Biotechnol Rep (Amst). 2021 Oct 9;32:e00680. doi: 
            10.1016/j.btre.2021.e00680. eCollection 2021 Dec.
  PUBMED    34703773
REFERENCE   2  (bases 1 to 9150)
  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: "Biotechnol 
            Rep (Amst)."; date: "2021-10-9"; volume: "32:e00680. doi"; pages: " 
            10.1016/j.btre.2021.e00680. eCollection 2021 Dec"
COMMENT     Sequence Label: pdCas9-sgRNA-RFP
FEATURES             Location/Qualifiers
     source          1..9150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..22
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(89..191)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(221..847)
                     /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     752..771
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(860..894)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        895..950
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    931..949
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             968..979
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(986..1012,1013..1015,1016..3502,3503..3505,3506..5092)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDDIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(986..1012,1013..1015,1016..5092)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDDIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(5159..6037)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6064..6348
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     6271..6290
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    6456..6477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6492..6497,6498..6515,6516..6522)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6530..6546
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             6564..6575
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             6582..7259
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      7304..7375
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7391..7418
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        7432..7507
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      7529..7600
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7616..7643
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(7671..7688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7805..8350)
                     /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(7885..7904)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(8464..8558)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(8579..9150,1..88))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
       61 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
      121 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
      181 cgtgccgatc aacgtctcat tttcgccaga tatcgacgtc ttaagaccca ctttcacatt
      241 taagttgttt ttctaatccg catatgatca attcaaggcc gaataagaag gctggctctg
      301 caccttggtg atcaaataat tcgatagctt gtcgtaataa tggcggcata ctatcagtag
      361 taggtgtttc cctttcttct ttagcgactt gatgctcttg atcttccaat acgcaaccta
      421 aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt ctctagtgaa aaaccttgtt
      481 ggcataaaaa ggctaattga ttttcgagag tttcatactg tttttctgta ggccgtgtac
      541 ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc acatctaaaa cttttagcgt
      601 tattacgtaa aaaatcttgc cagctttccc cttctaaagg gcaaaagtga gtatggtgcc
      661 tatctaacat ctcaatggct aaggcgtcga gcaaagcccg cttatttttt acatgccaat
      721 acaatgtagg ctgctctaca cctagcttct gggcgagttt acgggttgtt aaaccttcga
      781 ttccgacctc attaagcagc tctaatgcgc tgttaatcac tttactttta tctaatctag
      841 acatcattaa ttcctaattg ctagcattgt acctaggact gagctagcca taaagttgac
      901 actctatcgt tgatagagtt attttaccac tccctatcag tgatagagaa aagaattcaa
      961 aagatctaaa gaggagaaag gatctatgga taagaaatac tcaataggct tagctatcgg
     1021 cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt ctaaaaagtt
     1081 caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag gggctctttt
     1141 atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc gtagaaggta
     1201 tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg agatggcgaa
     1261 agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag aagacaagaa
     1321 gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc atgagaaata
     1381 tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag cggatttgcg
     1441 cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt tgattgaggg
     1501 agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg tacaaaccta
     1561 caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta aagcgattct
     1621 ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc tccccggtga
     1681 gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga cccctaattt
     1741 taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag atacttacga
     1801 tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt tgtttttggc
     1861 agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa atactgaaat
     1921 aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc atcaagactt
     1981 gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag aaatcttttt
     2041 tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc aagaagaatt
     2101 ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat tattggtgaa
     2161 actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct ctattcccca
     2221 tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact tttatccatt
     2281 tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgtattc cttattatgt
     2341 tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt ctgaagaaac
     2401 aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc aatcatttat
     2461 tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac caaaacatag
     2521 tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat atgttactga
     2581 aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg ttgatttact
     2641 cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat
     2701 agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg cttcattagg
     2761 tacctaccat gatttgctaa aaattattaa agataaagat tttttggata atgaagaaaa
     2821 tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata gggagatgat
     2881 tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga aacagcttaa
     2941 acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg gtattaggga
     3001 taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg ccaatcgcaa
     3061 ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc aaaaagcaca
     3121 agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc
     3181 tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg
     3241 gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa
     3301 gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg
     3361 aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta
     3421 tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg
     3481 tttaagtgat tatgatgtcg atgacattgt tccacaaagt ttccttaaag acgattcaat
     3541 agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag
     3601 tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat
     3661 cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga
     3721 taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc
     3781 acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga
     3841 ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt
     3901 ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt
     3961 cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga
     4021 ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc
     4081 aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact
     4141 tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat
     4201 tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt
     4261 caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc
     4321 aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg
     4381 tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg
     4441 gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag
     4501 ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa
     4561 agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg
     4621 gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata
     4681 tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa
     4741 cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca
     4801 aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag
     4861 tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt
     4921 atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga
     4981 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat
     5041 cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact gataagaatt
     5101 gctcggtacc aaattccaga aaagagacgc tttcgagcgt cttttttcgt tttggtcctt
     5161 atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg
     5221 ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa
     5281 cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc
     5341 tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat
     5401 gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc
     5461 tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg
     5521 gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta
     5581 tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca
     5641 ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata
     5701 ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt
     5761 gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca
     5821 gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac
     5881 cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat
     5941 cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt
     6001 atcccggcag caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca
     6061 gagaagaaac caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc
     6121 tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga
     6181 ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca
     6241 ttgattattt gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag
     6301 cggatcctac ctgacgcttt ttatcgcaac tctctactgt ttctccattg agacccaata
     6361 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     6421 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     6481 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6541 taacaatttc acacatacta gagaaagagg agaaatacta gatggcttcc tccgaagacg
     6601 ttatcaaaga gttcatgcgt ttcaaagttc gtatggaagg ttccgttaac ggtcacgagt
     6661 tcgaaatcga aggtgaaggt gaaggtcgtc cgtacgaagg tacccagacc gctaaactga
     6721 aagttaccaa aggtggtccg ctgccgttcg cttgggacat cctgtccccg cagttccagt
     6781 acggttccaa agcttacgtt aaacacccgg ctgacatccc ggactacctg aaactgtcct
     6841 tcccggaagg tttcaaatgg gaacgtgtta tgaacttcga agacggtggt gttgttaccg
     6901 ttacccagga ctcctccctg caagacggtg agttcatcta caaagttaaa ctgcgtggta
     6961 ccaacttccc gtccgacggt ccggttatgc agaaaaaaac catgggttgg gaagcttcca
     7021 ccgaacgtat gtacccggaa gacggtgctc tgaaaggtga aatcaaaatg cgtctgaaac
     7081 tgaaagacgg tggtcactac gacgctgaag ttaaaaccac ctacatggct aaaaaaccgg
     7141 ttcagctgcc gggtgcttac aaaaccgaca tcaaactgga catcacctcc cacaacgaag
     7201 actacaccat cgttgaacag tacgaacgtg ctgaaggtcg tcactccacc ggtgcttaat
     7261 aacgctgata gtgctagtgt agatcgctac tagagccagg catcaaataa aacgaaaggc
     7321 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     7381 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataggtctc agttttagag
     7441 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     7501 tcggtgcttt ttttctcgag ccaggcatca aataaaacga aaggctcagt cgaaagactg
     7561 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     7621 accttcgggt gggcctttct gcgtttatac ctagggatat attccgcttc ctcgctcact
     7681 gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga
     7741 gatttcctgg aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc
     7801 cgtttttcca taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt
     7861 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggc ggctccctcg
     7921 tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt
     7981 tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt
     8041 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     8101 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
     8161 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
     8221 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
     8281 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
     8341 acgatctcaa gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt
     8401 tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt tactagtgct
     8461 tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg
     8521 gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt
     8581 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     8641 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     8701 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     8761 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     8821 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     8881 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     8941 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     9001 cgtctttcat tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa
     9061 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     9121 ccagctgaac ggtctggtta taggtacatt
//
