LOCUS       sequence-181341-        7953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified from pEM-Cas9HF1 (Addgene ID: 89961) to include 
            constitutive recA56 to block recA-mediated double-strand break 
            repair..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7953)
  AUTHORS   Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar
            R, Lynch MD
  TITLE     Managing the SOS Response for Enhanced CRISPR-Cas-Based 
            Recombineering in E. coli through Transient Inhibition of Host RecA 
            Activity.
  JOURNAL   ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174.
  PUBMED    28915012
REFERENCE   2  (bases 1 to 7953)
  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. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174."
COMMENT     Sequence Label: pEM-Cas9HF1-recA56
FEATURES             Location/Qualifiers
     source          1..7953
                     /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        complement(649..683)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        684..739
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(775..2262,2263..2265,2266..2754,2755..2757,2758..2856,
                     2857..2859,2860..3549,3550..3552,3553..4878)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             775..4878
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5030..6091
                     /codon_start=1
                     /gene="recA"
                     /product="DNA repair protein from E. coli"
                     /label=RecA
                     /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS
                     LSLDIALGAGGLPMGCIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY
                     ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS
                     HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV
                     RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK
                     EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV
                     DDSEGVAETNEDF"
     terminator      6121..6192
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6208..6235
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6263..6280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6397..6942)
                     /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(6477..6496)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(7056..7150)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7171..7830)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7745..7764
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7831..7933)
                     /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 tcctaattgc tagcattgta
      661 cctaggactg agctagccat aaagttgaca ctctatcgtt gatagagtta ttttaccact
      721 ccctatcagt gatagagaaa agaattcaaa agatctaaag aggacttcgg atctatggat
      781 aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc ggtgatcact
      841 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
      901 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
      961 cgcctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
     1021 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
     1081 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
     1141 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
     1201 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
     1261 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
     1321 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
     1381 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
     1441 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
     1501 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
     1561 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
     1621 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
     1681 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
     1741 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
     1801 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
     1861 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
     1921 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
     1981 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
     2041 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
     2101 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
     2161 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
     2221 gataaaggtg cttcagctca atcatttatt gaacgcatga cagcgtttga taaaaatctt
     2281 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
     2341 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
     2401 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
     2461 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
     2521 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
     2581 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
     2641 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
     2701 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggagcgttg
     2761 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
     2821 ttgaaatcag atggttttgc caatcgcaat tttatggcgc tgatccatga tgatagtttg
     2881 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
     2941 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
     3001 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
     3061 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
     3121 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
     3181 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
     3241 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tcacattgtt
     3301 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
     3361 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
     3421 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
     3481 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
     3541 gaaactcgcg cgatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
     3601 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
     3661 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
     3721 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
     3781 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
     3841 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
     3901 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
     3961 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
     4021 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
     4081 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
     4141 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
     4201 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
     4261 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
     4321 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
     4381 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
     4441 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
     4501 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
     4561 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
     4621 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
     4681 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
     4741 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
     4801 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
     4861 agtcagctag gaggtgacgc tgctaacgac gaaaactacg ctctggccgc ctaatattac
     4921 ccggcatgac atttacgggc atgcataagg ctcgtataat atattcaggg agaccacaac
     4981 ggtttccctc tacaaataat tttgtttaac tttcatgaca ggagtaaaaa tggctatcga
     5041 cgaaaacaaa cagaaagcgt tggcggcagc actgggccag attgagaaac aatttggtaa
     5101 aggctccatc atgcgcctgg gtgaagaccg ttccatggat gtggaaacca tctctaccgg
     5161 ttcgctttca ctggatatcg cgcttggggc aggtggtctg ccgatgggct gcatcgtcga
     5221 aatctacgga ccggaatctt ccggtaaaac cacgctgacg ctgcaggtga tcgccgcagc
     5281 gcagcgtgaa ggtaaaacct gtgcgtttat cgatgctgaa cacgcgctgg acccaatcta
     5341 cgcacgtaaa ctgggcgtcg atatcgacaa cctgctgtgc tcccagccgg acaccggcga
     5401 gcaggcactg gaaatctgtg acgccctggc gcgttctggc gcagtagacg ttatcgtcgt
     5461 tgactccgtg gcggcactga cgccgaaagc ggaaatcgaa ggcgaaatcg gcgactctca
     5521 catgggcctt gcggcacgta tgatgagcca ggcgatgcgt aagctggcgg gtaacctgaa
     5581 gcagtccaac acgctgctga tcttcatcaa ccagatccgt atgaaaattg gtgtgatgtt
     5641 cggtaacccg gaaaccacta ccggtggtaa cgcgctgaaa ttctacgcct ctgttcgtct
     5701 ggacatccgt cgtatcggcg cggtgaaaga gggcgaaaac gtggtgggta gcgaaacccg
     5761 cgtgaaagtg gtgaagaaca aaatcgctgc gccgtttaaa caggctgaat tccagatcct
     5821 ctacggcgaa ggtatcaact tctacggcga actggttgac ctgggcgtaa aagagaagct
     5881 gatcgagaaa gcaggcgcgt ggtacagcta caaaggtgag aagatcggtc agggtaaagc
     5941 gaatgcgact gcctggctga aagataaccc ggaaaccgcg aaagagatcg agaagaaagt
     6001 acgtgagttg ctgctgagca acccgaactc aacgccggat ttctctgtag atgatagcga
     6061 aggcgtagca gaaactaacg aagattttta agcttaactc gagtaaggat ctccaggcat
     6121 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     6181 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
     6241 acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     6301 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     6361 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     6421 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     6481 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     6541 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     6601 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     6661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     6721 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     6781 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     6841 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     6901 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     6961 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     7021 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg
     7081 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     7141 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
     7201 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     7261 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     7321 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     7381 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     7441 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     7501 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     7561 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     7621 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     7681 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     7741 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     7801 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     7861 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     7921 tacgtgccga tcaacgtctc attttcgcca gat
//
