LOCUS       sequence-329432-        7722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express miCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7722)
  AUTHORS   Ma L, Ruan J, Song J, Wen L, Yang D, Zhao J, Xia X, Chen YE, Zhang 
            J, Xu J
  TITLE     MiCas9 increases large size gene knock-in rates and reduces 
            undesirable on-target and off-target indel edits.
  JOURNAL   Nat Commun. 2020 Nov 27;11(1):6082. doi: 10.1038/s41467-020-19842-2.
  PUBMED    33247137
REFERENCE   2  (bases 1 to 7722)
  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: "Nat 
            Commun."; date: "2020-11-27"; volume: "11(1):6082. doi"; pages: " 
            10.1038/s41467-020-19842-2"
COMMENT     Sequence Label: pmiCas9
FEATURES             Location/Qualifiers
     source          1..7722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     623..643
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     714..733
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        714..732
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      740..749
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(746..4147,4148..4150,4151..4747,4748,4749..4751,
                     4752..4753,4754..4756,4757..4849)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             746..4849
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             746..4849
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4856..4876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4883..4948
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5074..5091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5114..5131)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5120..5344
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5415..5431)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5415..5431)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5428..5450)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5439..5455
                     /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)."
     promoter        complement(join(5463..5469,5470..5487,5488..5493))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5508..5529
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5646..5663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5817..6405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6576..7367,7368..7436))
                     /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     7199..7218
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7437..7541)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7620..7639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
       61 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      121 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      181 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      241 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      301 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      361 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      421 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      481 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      541 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      601 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      661 ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg
      721 actcactata gggagagccg ccaccatgga taaaaagtat tctattggtt tagatatcgg
      781 cactaattcc gttggatggg ctgtcataac cgatgaatac aaagtacctt caaagaaatt
      841 taaggtgttg gggaacacag accgtcattc gattaaaaag aatcttatcg gtgccctcct
      901 attcgatagt ggcgaaacgg cagaggcgac tcgcctgaaa cgaaccgctc ggagaaggta
      961 tacacgtcgc aagaaccgaa tatgttactt acaagaaatt tttagcaatg agatggccaa
     1021 agttgacgat tctttctttc accgtttgga agagtccttc cttgtcgaag aggacaagaa
     1081 acatgaacgg caccccatct ttggaaacat agtagatgag gtggcatatc atgaaaagta
     1141 cccaacgatt tatcacctca gaaaaaagct agttgactca actgataaag cggacctgag
     1201 gttaatctac ttggctcttg cccatatgat aaagttccgt gggcactttc tcattgaggg
     1261 tgatctaaat ccggacaact cggatgtcga caaactgttc atccagttag tacaaaccta
     1321 taatcagttg tttgaagaga accctataaa tgcaagtggc gtggatgcga aggctattct
     1381 tagcgcccgc ctctctaaat cccgacggct agaaaacctg atcgcacaat tacccggaga
     1441 gaagaaaaat gggttgttcg gtaaccttat agcgctctca ctaggcctga caccaaattt
     1501 taagtcgaac ttcgacttag ctgaagatgc caaattgcag cttagtaagg acacgtacga
     1561 tgacgatctc gacaatctac tggcacaaat tggagatcag tatgcggact tatttttggc
     1621 tgccaaaaac cttagcgatg caatcctcct atctgacata ctgagagtta atactgagat
     1681 taccaaggcg ccgttatccg cttcaatgat caaaaggtac gatgaacatc accaagactt
     1741 gacacttctc aaggccctag tccgtcagca actgcctgag aaatataagg aaatattctt
     1801 tgatcagtcg aaaaacgggt acgcaggtta tattgacggc ggagcgagtc aagaggaatt
     1861 ctacaagttt atcaaaccca tattagagaa gatggatggg acggaagagt tgcttgtaaa
     1921 actcaatcgc gaagatctac tgcgaaagca gcggactttc gacaacggta gcattccaca
     1981 tcaaatccac ttaggcgaat tgcatgctat acttagaagg caggaggatt tttatccgtt
     2041 cctcaaagac aatcgtgaaa agattgagaa aatcctaacc tttcgcatac cttactatgt
     2101 gggacccctg gcccgaggga actctcggtt cgcatggatg acaagaaagt ccgaagaaac
     2161 gattactcca tggaattttg aggaagttgt cgataaaggt gcgtcagctc aatcgttcat
     2221 cgagaggatg accaactttg acaagaattt accgaacgaa aaagtattgc ctaagcacag
     2281 tttactttac gagtatttca cagtgtacaa tgaactcacg aaagttaagt atgtcactga
     2341 gggcatgcgt aaacccgcct ttctaagcgg agaacagaag aaagcaatag tagatctgtt
     2401 attcaagacc aaccgcaaag tgacagttaa gcaattgaaa gaggactact ttaagaaaat
     2461 tgaatgcttc gattctgtcg agatctccgg ggtagaagat cgatttaatg cgtcacttgg
     2521 tacgtatcat gacctcctaa agataattaa agataaggac ttcctggata acgaagagaa
     2581 tgaagatatc ttagaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat
     2641 tgaggaaaga ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa
     2701 gaggcgtcgc tatacgggct ggggacgatt gtcgcggaaa cttatcaacg ggataagaga
     2761 caagcaaagt ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa
     2821 ctttatgcag ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca
     2881 ggtttccgga caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc
     2941 catcaaaaag ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg
     3001 acgtcacaaa ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa
     3061 ggggcaaaaa aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg
     3121 cagccagatc ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta
     3181 cctctattac ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg
     3241 tttatctgat tacgacgtcg atcatattgt accccaatcc tttttgaagg acgattcaat
     3301 cgacaataaa gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag
     3361 cgaggaagtc gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat
     3421 aacgcaaaga aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga
     3481 caaggccgga tttattaaac gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc
     3541 acagatacta gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga
     3601 agtcaaagta atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt
     3661 ctataaagtt agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt
     3721 cgtagggacc gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga
     3781 ttacaaagtt tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc
     3841 tacagccaaa tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct
     3901 ggcaaacgga gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat
     3961 cgtatgggat aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt
     4021 caacatagta aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc
     4081 aaaaaggaat agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg
     4141 tggcttcgat agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg
     4201 aaaatccaag aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc
     4261 gtcttttgaa aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa
     4321 ggatctcata attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg
     4381 gatgttggct agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata
     4441 cgtgaatttc ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa
     4501 cgaacagaag caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca
     4561 aatttcggaa ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag
     4621 cgcatacaac aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt
     4681 gtttactctt accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga
     4741 tcgcaaacga tacacttcta ccaaggaggt gctagacgcg acactgattc accaatccat
     4801 cacgggatta tatgaaactc ggatagattt gtcacagctt gggggtgacg gatcccccaa
     4861 gaagaagagg aaagtctcga gcgactacaa agaccatgac ggtgattata aagatcatga
     4921 catcgattac aaggatgacg atgacaaggc tgcaggagca ttggatttcc ttagtagact
     4981 ccccttgcca cctccggtgt cccccatctg cacgtttgtg agcccagcgg cacagaaggc
     5041 atttcaaccg ccacggagtt gcgggtaacc ggtcatcatc accatcacca ttgagtttaa
     5101 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5161 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5221 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5281 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     5341 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     5401 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5461 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     5521 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5581 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5641 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5701 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5761 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5821 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5881 aacccgacag gactataaag ataccaggca tttccccctg gaagctccct cgtgcgctct
     5941 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6001 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6061 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6121 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6181 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6241 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6301 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6361 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6421 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6481 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6541 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6601 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6661 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6721 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6781 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6841 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6901 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6961 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7021 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7081 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7141 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7201 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7261 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7321 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7381 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7441 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7501 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7561 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt
     7621 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     7681 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa gg
//
