LOCUS       sequence-496327-       12869 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Expresses nucleus-localized nCas9 (D10A) fused to PolI5M and mCherry
            using a CMV promoter. Includes a GFP cassette flanked by BsmbI cut 
            sites to knock in and coexpress user-defined gRNAs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12869)
  AUTHORS   Hurtado JE, Schieferecke AJ, Halperin SO, Guan J, Aidlen D, Schaffer
            DV, Dueber JE
  TITLE     Nickase fidelity drives EvolvR-mediated diversification in mammalian
            cells.
  JOURNAL   Nat Commun. 2025 Apr 19;16(1):3723. doi: 10.1038/s41467-025-58414-0.
  PUBMED    40253348
REFERENCE   2  (bases 1 to 12869)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-04-19"; volume: "16(1):3723. doi"; pages: " 
            10.1038/s41467-025-58414-0"
COMMENT     Sequence Label: pEvolvR-mCherry-nCas9-PolI5M
FEATURES             Location/Qualifiers
     source          1..12869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1153..1401
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1153..1173
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1324..1343
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     RBS             1557..1568
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1575..2288
                     /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"
     terminator      2300..2371
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2387..2414
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2428..2503
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2535..2914
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2915..3118
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3069..3089
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3137..3146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3149..3169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3194..3898
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3336..3354)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3604..3623
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(3908..3934,3935..3937,3938..8011)
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3908..8011
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8015..8041
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8021..8047
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             8060..8080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             8887..8895
                     /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)"
     primer_bind     complement(11069..11088)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11070..11088)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11095..11112)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11095..11111)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11104..11126)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11253..11708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11340..11359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11550..11571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11734..11838
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11839..11907,11908..12699)
                     /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     complement(12057..12076)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     1021 tcactaaagg gaacaaaagc tggagctcca ccgcggtggc ggccgctctt aaggggtgca
     1081 gcggcctccg cgccgggttt tggcgcctcg gtgagtgagt gtgtgcgtgt tggatccaag
     1141 gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     1201 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     1261 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     1321 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     1381 ttgtggaaag gacgaaacac ctgagacgga aagtgaaacg tgatttcatg cgtcattttg
     1441 aacattttgt aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt
     1501 tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag
     1561 aggagaaata ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg
     1621 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
     1681 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc
     1741 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
     1801 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
     1861 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
     1921 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata
     1981 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
     2041 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
     2101 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
     2161 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg
     2221 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     2281 tgtacaaatg accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     2341 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     2401 tgggcctttc tgcgtttata cgtctcagtt ttagagctag aaatagcaag ttaaaataag
     2461 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc cgcggcctct
     2521 agactcgagg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     2581 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     2641 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     2701 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     2761 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     2821 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     2881 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     2941 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     3001 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     3061 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     3121 ctggctaact accggtgcca ccatggcccc aaagaagaag cggaaggtcg gtatccacgg
     3181 agtcccagca gccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     3241 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     3301 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     3361 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     3421 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     3481 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     3541 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     3601 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     3661 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     3721 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     3781 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     3841 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg
     3901 atccggtatg gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
     3961 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     4021 cgaccggcac agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggagaaac
     4081 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     4141 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     4201 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     4261 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     4321 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     4381 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     4441 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     4501 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     4561 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     4621 cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     4681 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     4741 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     4801 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     4861 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     4921 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     4981 ctacgccggc tacatcgatg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     5041 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     5101 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     5161 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     5221 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     5281 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     5341 cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc atcgagcgga tgaccaactt
     5401 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     5461 caccgtgtac aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     5521 cttcctgagc ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa
     5581 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     5641 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     5701 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     5761 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     5821 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     5881 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     5941 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     6001 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     6061 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     6121 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     6181 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     6241 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     6301 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     6361 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     6421 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgataaca aagtgctgac
     6481 tcggagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     6541 gatgaagaac tactggcgcc agctgctgaa tgccaagctg attacccaga ggaagttcga
     6601 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     6661 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     6721 gatgaacact aagtacgacg agaacgacaa actgatccgg gaagtgaaag tgatcaccct
     6781 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     6841 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     6901 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     6961 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     7021 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     7081 gaagcggcct ctgatcgaga caaacggcga aacaggcgag atcgtgtggg ataagggccg
     7141 ggactttgcc accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac
     7201 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgacaa
     7261 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     7321 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     7381 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     7441 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     7501 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     7561 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     7621 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     7681 tgtggaacag cacaaacact acctggacga gatcatcgag cagatcagcg agttctccaa
     7741 gagagtgatc ctggccgacg ctaatctgga caaggtgctg agcgcctaca acaagcacag
     7801 agacaagcct atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     7861 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     7921 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     7981 acggatcgac ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc ccgattatgc
     8041 cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg
     8101 gaaaggttct agtgaaaccc cgggaacaag tgagtcggcc acccctgaag gtggatcagg
     8161 gggtagcgga tccgttcaga tcccgcagaa cccgctgatt ctggttgacg gatctagtta
     8221 cctgtaccgt gcttaccatg ctttcccgcc tttgaccaat tctgctggtg aacctacggg
     8281 agctatgtac ggagttctga atatgttgcg ttctttaatt atgcagtaca agcctaccca
     8341 cgctgctgtt gttttcgatg ctaaaggtaa gacgttccgc gacgagttat tcgagcacta
     8401 taagtctcac cgtcctccga tgcctgatga cttacgcgct cagattgagc cgctgcatgc
     8461 tatggtgaag gctatgggtt tacctctttt ggctgtcagc ggtgttgagg ctgatgatgt
     8521 cattggcacc ttagctcgtg aggctgagaa ggctggtcgc cctgttttga tttctaccgg
     8581 tgacaaggac atggctcaat tggttacccc gaacatcacc ctgatcaaca ccatgaccaa
     8641 cacgattctg ggtcctgagg aagttgttaa caaatatggt gttcctccgg agttgattat
     8701 tgactttctt gctcttatgg gcgattcttc agacaatatc ccgggtgttc caggtgttgg
     8761 agagaagact gctcaagctc tgcttcaggg tctgggtggt ttggacaccc tttacgctga
     8821 accggagaag atcgccggtc tgtcttttcg cggtgctaag accatggctg ctaaactgga
     8881 acagaataag gaggtcgcat acctgtctta tcaattggct accatcaaga cggatgtgga
     8941 gttagaactt acgtgcgagc agcttgaggt tcaacagcct gctgctgagg aactgctggg
     9001 tctttttaag aaatacgaat ttaagcgttg gaccgccgac gttgaggctg gtaagtggct
     9061 gcaagctaag ggtgctaagc cggctgctaa accgcaagaa acgagtgtcg ctgatgaggc
     9121 tccggaggtt accgctaccg ttatctctta cgataattat gttacgattc tggacgagga
     9181 aaccttaaag gcttggatcg ctaaattaga gaaggctcct gttttcgctt tcgacacgga
     9241 aacggattct ctggacaata ttagtgcgaa tcttgttggt ctgagtttcg caattgaacc
     9301 gggtgttgct gcttacatcc ctgtggcaca cgactacctg gacgctccgg accagatttc
     9361 acgtgaacgc gctctggaac tgctgaagcc tttattagag gacgagaaag ctttgaaagt
     9421 tggtcagaat ttgaagtatg ctcgtggaat cttagctaat tatggtatcg agttgcgcgg
     9481 tatcgctttc gacacgatgt tggaatctta tatcctgaac tctgtcgctg gtcgccatga
     9541 catggactct ctggctgagc gctggctgaa acataagacg attaccttcg aggaaatcgc
     9601 aggaaagggt aagaaccagc tcacgttcaa tcaaatcgct ctggaggaag ctggtcgcta
     9661 tgctgctgag gacgctgacg ttactctgca actgcacttg aagatgtggc ctgacttgca
     9721 gaagcataag ggtccactga atgtttttga aaacattgag atgcctttgg ttccagttct
     9781 gtctcgtatc gagcgcaatg gcgttaaaat tgacccaaag gttttacata accactcaga
     9841 ggaactgacg ctgcgcttag ccgaattgga gaaaaaggct cacgagatcg ctggcgaaga
     9901 gttcaatctg tcatctacga aacaactgca gactatcctg ttcgagaagc aaggtatcaa
     9961 gccattaaaa aagacccctg gcggtgctcc gtctacctct gaggaagttt tggaggagtt
    10021 agctttggat taccctctgc cgaaggttat cttggaatac cgcggtttgg ctaaattgaa
    10081 gtctacttat acggataaac ttcctttgat gattaatcca aagacgggtc gcgttcacac
    10141 gtcgtaccat caagctgtta ccgctaccgg tcgcctgtct tctacggatc cgaatttaca
    10201 gaatattcct gtgcgcaatg aggagggccg ccgcattcgt caagctttta tcgctccgga
    10261 agactacgtt atcgtttctg ctgattattc tcaaaatgaa ttacgtatca tggctcacct
    10321 gtctcgcgat aagggtctgt tgacggcctt tgctgagggt aaggacattc atcgtgctac
    10381 cgctgctgag gtttacggcc tgccgttgga aacggttacg tctgaacagc gtcgctctgc
    10441 taagcgtatt aatttcggct taatctacgg tatgtctgcg tttggcttag ctcgtcagct
    10501 gaatatcccg cgcaaggaag ctcaaaaata tatggatctg tattttgagc gttaccacgg
    10561 tgttttggaa tacatggagc gtacgcgcgc gcaagctaag gaacaaggtt atgtggaaac
    10621 cttggatggt cgtcgcttgt acttgcctga cattaagtct tctaacggcg cccgccgcgc
    10681 tgctgccgag cgcgctgcta tcaatgctcc gatgcaaggt actgctgctg atattattaa
    10741 gcgtgctatg atcgctgtgg acgcttggct gcaagctgaa cagcctcgcg ttcgcatgat
    10801 tatgcaagtt catgacgagt tggttttcga ggtgcataag gacgacgtgg acgctgttgc
    10861 taaacaaatc caccagttga tggagaattg cacgcgctta gacgttccgc tgctggttga
    10921 agttggttct ggtgaaaact gggaccaggc tcactaataa tggctgaaaa ctgggaccag
    10981 gctcactaac tcagatccta ctaggtttaa taaacatctt tattttcatt acatctgtgt
    11041 gttggttttt tgtgtggtac ccaattcgcc ctatagtgag tcgtattaca attcactggc
    11101 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
    11161 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
    11221 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
    11281 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
    11341 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
    11401 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
    11461 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
    11521 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
    11581 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
    11641 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
    11701 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
    11761 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
    11821 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
    11881 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
    11941 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
    12001 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
    12061 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
    12121 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
    12181 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
    12241 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
    12301 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
    12361 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
    12421 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
    12481 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
    12541 gccggtgagc gtggttctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
    12601 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
    12661 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
    12721 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
    12781 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
    12841 gaccccgtag aaaagatcaa aggatcttc
//
