LOCUS       sequence-178235-       10815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG-driven ubiquitous expression of Cas9 with Histone2B-RFP reporter
            controlled by IRES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10815)
  AUTHORS   Williams RM, Senanayake U, Artibani M, Taylor G, Wells D, Ahmed AA, 
            Sauka-Spengler T
  TITLE     Genome and epigenome engineering CRISPR toolkit for in vivo 
            modulation of cis-regulatory interactions and gene expression in the
            chicken embryo.
  JOURNAL   Development. 2018 Feb 23;145(4). pii: dev.160333. doi: 
            10.1242/dev.160333.
  PUBMED    29386245
REFERENCE   2  (bases 1 to 10815)
  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: 
            "Development."; date: "2018-02-23"; volume: "145(4). pii: 
            dev.160333. doi"; pages: " 10.1242/dev.160333"
COMMENT     Sequence Label: pCI Cas9 H2B-RFP
FEATURES             Location/Qualifiers
     source          1..10815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1807..1816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1819..1839
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1864..5964
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1864..5964
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5965..6012
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    join(6050..6624,6625..6627,6628..6636)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6217..6234)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6444..6463
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6643..7020
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      7033..7042
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7039..7716
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             7039..7716
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(7169..7187)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7437..7456
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7631..7654
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(7807..7826)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7872..7927
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7926..7945)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(8318..8334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8318..8334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8331..8353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8342..8358
                     /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(8366..8372,8373..8390,8391..8396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8411..8432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8491..8686
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      8537..8672
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    8692..8826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8729..8748)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8783..8802
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8894..8911)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9065..9653)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9145..9164)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9824..10615,10616..10684))
                     /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     10447..10466
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10685..10789)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgct cgagagttcg
     1741 aagccttaag tacgcgtagg cgcgccttaa ttaacgttta aacgctagct catcgataag
     1801 cttgatgcca ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     1861 gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     1921 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     1981 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2041 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2101 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     2161 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     2221 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     2281 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     2341 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     2401 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     2461 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     2521 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     2581 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     2641 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     2701 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     2761 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     2821 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     2881 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     2941 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3001 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3061 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3121 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     3181 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     3241 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     3301 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     3361 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     3421 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     3481 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     3541 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     3601 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     3661 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     3721 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     3781 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     3841 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     3901 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     3961 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4021 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4081 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4141 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     4201 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     4261 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     4321 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     4381 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     4441 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     4501 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     4561 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     4621 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     4681 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     4741 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     4801 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     4861 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     4921 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     4981 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     5041 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     5101 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     5161 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     5221 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     5281 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     5341 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     5401 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     5461 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     5521 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     5581 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     5641 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     5701 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     5761 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     5821 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     5881 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     5941 gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca
     6001 aaaaagaaaa agtaatgata gatcgaattc ctgcagcccg ggggatccgc ccctctccct
     6061 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     6121 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     6181 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     6241 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     6301 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     6361 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     6421 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     6481 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta
     6541 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     6601 tcctttgaaa aacacgatga taatatggcc acaaccatgg ttatgccaga gccagcgaag
     6661 tctgctcccg ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaaggc
     6721 ggcaagaagc gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg
     6781 aagcaggtcc accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt
     6841 gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag
     6901 cgctcgacca tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag
     6961 ttggccaagc acgccgtgtc cgagggtact aaggccatca ccaagtacac cagcgctaag
     7021 gatccaccgg tcgccaccat ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc
     7081 aaggtgcgca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     7141 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     7201 cccttcgcct gggacatcct gtcccctcag ttccagtacg gctccaaggc ctacgtgaag
     7261 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     7321 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     7381 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     7441 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcggatgta ccccgaggac
     7501 ggcgccctga agggcgagat caagatgagg ctgaagctga aggacggcgg ccactacgac
     7561 gccgaggtca agaccaccta catggccaag aagcccgtgc agctgcccgg cgcctacaag
     7621 accgacatca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     7681 gagcgcgccg agggccgcca ctccaccggc gcctaagcgg ccgcaattca ctcctcaggt
     7741 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     7801 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     7861 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     7921 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     7981 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     8041 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     8101 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     8161 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     8221 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcaccgtc gaccagctgg
     8281 tcgacggtgc accgtcgacc agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     8341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     8401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     8461 agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc
     8521 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     8581 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     8641 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt
     8701 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     8761 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     8821 tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     8881 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     8941 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     9001 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     9061 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     9121 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     9181 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     9241 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
     9301 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     9361 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     9421 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     9481 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     9541 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     9601 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     9661 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     9721 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     9781 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     9841 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     9901 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     9961 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
    10021 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
    10081 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
    10141 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
    10201 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
    10261 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
    10321 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
    10381 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
    10441 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    10501 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    10561 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    10621 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    10681 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
    10741 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
    10801 gaaaagtgcc acctg
//
