LOCUS       sequence-495988-       12834 bp    DNA     circular SYN 23-DEC-2025
DEFINITION  Expresses nucleus-localized enCas9 (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 12834)
  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 12834)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 23, 2025 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-enCas9(D10A)-PolI5M
FEATURES             Location/Qualifiers
     source          1..12834
                     /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..6445,6446..6448,6449..6910,6911..6913,6914..7081,
                     7082..7084,7085..8008)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8012..8038
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8018..8044
                     /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             8057..8077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8084..8104
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     RBS             8884..8892
                     /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(11034..11053)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11035..11053)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11060..11077)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11060..11076)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11069..11091)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11218..11673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11305..11324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11515..11536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11699..11803
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11804..11872,11873..12664)
                     /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(12022..12041)
                     /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 gttatccctt 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 atccggagac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
     3961 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     4021 ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg gagaaacagc
     4081 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     4141 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     4201 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     4261 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     4321 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     4381 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     4441 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     4501 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     4561 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     4621 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     4681 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     4741 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     4801 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     4861 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     4921 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     4981 cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     5041 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     5101 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     5161 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     5221 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     5281 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     5341 ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga ccaacttcga
     5401 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     5461 cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     5521 cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca accggaaagt
     5581 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     5641 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     5701 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     5761 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     5821 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     5881 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     5941 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     6001 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     6061 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     6121 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     6181 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     6241 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     6301 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     6361 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     6421 ccatatcgtg cctcagagct ttctggcaga cgactccatc gataacaaag tgctgactcg
     6481 gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     6541 gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga agttcgacaa
     6601 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     6661 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     6721 gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga tcaccctgaa
     6781 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     6841 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     6901 aaagtaccct gcgctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     6961 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     7021 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     7081 ggcgcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata agggccggga
     7141 ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga aaaagaccga
     7201 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgacaagct
     7261 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     7321 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     7381 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     7441 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     7501 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     7561 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     7621 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     7681 ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     7741 agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca agcacagaga
     7801 caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     7861 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     7921 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     7981 gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg attatgccag
     8041 cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa
     8101 agtggacagt gaaaccccgg gaacaagtga gtcggccacc cctgaaggtg gatcaggggg
     8161 tagcggatcc gttcagatcc cgcagaaccc gctgattctg gttgacggat ctagttacct
     8221 gtaccgtgct taccatgctt tcccgccttt gaccaattct gctggtgaac ctacgggagc
     8281 tatgtacgga gttctgaata tgttgcgttc tttaattatg cagtacaagc ctacccacgc
     8341 tgctgttgtt ttcgatgcta aaggtaagac gttccgcgac gagttattcg agcactataa
     8401 gtctcaccgt cctccgatgc ctgatgactt acgcgctcag attgagccgc tgcatgctat
     8461 ggtgaaggct atgggtttac ctcttttggc tgtcagcggt gttgaggctg atgatgtcat
     8521 tggcacctta gctcgtgagg ctgagaaggc tggtcgccct gttttgattt ctaccggtga
     8581 caaggacatg gctcaattgg ttaccccgaa catcaccctg atcaacacca tgaccaacac
     8641 gattctgggt cctgaggaag ttgttaacaa atatggtgtt cctccggagt tgattattga
     8701 ctttcttgct cttatgggcg attcttcaga caatatcccg ggtgttccag gtgttggaga
     8761 gaagactgct caagctctgc ttcagggtct gggtggtttg gacacccttt acgctgaacc
     8821 ggagaagatc gccggtctgt cttttcgcgg tgctaagacc atggctgcta aactggaaca
     8881 gaataaggag gtcgcatacc tgtcttatca attggctacc atcaagacgg atgtggagtt
     8941 agaacttacg tgcgagcagc ttgaggttca acagcctgct gctgaggaac tgctgggtct
     9001 ttttaagaaa tacgaattta agcgttggac cgccgacgtt gaggctggta agtggctgca
     9061 agctaagggt gctaagccgg ctgctaaacc gcaagaaacg agtgtcgctg atgaggctcc
     9121 ggaggttacc gctaccgtta tctcttacga taattatgtt acgattctgg acgaggaaac
     9181 cttaaaggct tggatcgcta aattagagaa ggctcctgtt ttcgctttcg acacggaaac
     9241 ggattctctg gacaatatta gtgcgaatct tgttggtctg agtttcgcaa ttgaaccggg
     9301 tgttgctgct tacatccctg tggcacacga ctacctggac gctccggacc agatttcacg
     9361 tgaacgcgct ctggaactgc tgaagccttt attagaggac gagaaagctt tgaaagttgg
     9421 tcagaatttg aagtatgctc gtggaatctt agctaattat ggtatcgagt tgcgcggtat
     9481 cgctttcgac acgatgttgg aatcttatat cctgaactct gtcgctggtc gccatgacat
     9541 ggactctctg gctgagcgct ggctgaaaca taagacgatt accttcgagg aaatcgcagg
     9601 aaagggtaag aaccagctca cgttcaatca aatcgctctg gaggaagctg gtcgctatgc
     9661 tgctgaggac gctgacgtta ctctgcaact gcacttgaag atgtggcctg acttgcagaa
     9721 gcataagggt ccactgaatg tttttgaaaa cattgagatg cctttggttc cagttctgtc
     9781 tcgtatcgag cgcaatggcg ttaaaattga cccaaaggtt ttacataacc actcagagga
     9841 actgacgctg cgcttagccg aattggagaa aaaggctcac gagatcgctg gcgaagagtt
     9901 caatctgtca tctacgaaac aactgcagac tatcctgttc gagaagcaag gtatcaagcc
     9961 attaaaaaag acccctggcg gtgctccgtc tacctctgag gaagttttgg aggagttagc
    10021 tttggattac cctctgccga aggttatctt ggaataccgc ggtttggcta aattgaagtc
    10081 tacttatacg gataaacttc ctttgatgat taatccaaag acgggtcgcg ttcacacgtc
    10141 gtaccatcaa gctgttaccg ctaccggtcg cctgtcttct acggatccga atttacagaa
    10201 tattcctgtg cgcaatgagg agggccgccg cattcgtcaa gcttttatcg ctccggaaga
    10261 ctacgttatc gtttctgctg attattctca aaatgaatta cgtatcatgg ctcacctgtc
    10321 tcgcgataag ggtctgttga cggcctttgc tgagggtaag gacattcatc gtgctaccgc
    10381 tgctgaggtt tacggcctgc cgttggaaac ggttacgtct gaacagcgtc gctctgctaa
    10441 gcgtattaat ttcggcttaa tctacggtat gtctgcgttt ggcttagctc gtcagctgaa
    10501 tatcccgcgc aaggaagctc aaaaatatat ggatctgtat tttgagcgtt accacggtgt
    10561 tttggaatac atggagcgta cgcgcgcgca agctaaggaa caaggttatg tggaaacctt
    10621 ggatggtcgt cgcttgtact tgcctgacat taagtcttct aacggcgccc gccgcgctgc
    10681 tgccgagcgc gctgctatca atgctccgat gcaaggtact gctgctgata ttattaagcg
    10741 tgctatgatc gctgtggacg cttggctgca agctgaacag cctcgcgttc gcatgattat
    10801 gcaagttcat gacgagttgg ttttcgaggt gcataaggac gacgtggacg ctgttgctaa
    10861 acaaatccac cagttgatgg agaattgcac gcgcttagac gttccgctgc tggttgaagt
    10921 tggttctggt gaaaactggg accaggctca ctaactcaga tcctactagg tttaataaac
    10981 atctttattt tcattacatc tgtgtgttgg ttttttgtgt ggtacccaat tcgccctata
    11041 gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
    11101 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
    11161 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
    11221 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
    11281 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
    11341 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
    11401 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
    11461 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
    11521 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
    11581 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
    11641 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
    11701 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
    11761 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
    11821 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
    11881 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
    11941 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
    12001 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
    12061 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
    12121 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
    12181 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
    12241 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
    12301 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
    12361 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
    12421 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
    12481 ctggtttatt gctgataaat ctggagccgg tgagcgtggt tctcgcggta tcattgcagc
    12541 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
    12601 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
    12661 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
    12721 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
    12781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
