LOCUS       sequence-249638-       10050 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCas9 fused to wild-type AcrIIA4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10050)
  AUTHORS   Aschenbrenner S, Kallenberger SM, Hoffmann MD, Huck A, Eils R, 
            Niopek D
  TITLE     Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 
            target specificity.
  JOURNAL   Sci Adv. 2020 Feb 5;6(6):eaay0187. doi: 10.1126/sciadv.aay0187. 
            eCollection 2020 Feb.
  PUBMED    32076642
REFERENCE   2  (bases 1 to 10050)
  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: "Sci Adv."; 
            date: "2020-02-5"; volume: "6(6):eaay0187. doi"; pages: " 
            10.1126/sciadv.aay0187. eCollection 2020 Feb"
COMMENT     Sequence Label: Cas-Acr (wtAcrIIA4)
FEATURES             Location/Qualifiers
     source          1..10050
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     771..791
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     865..884
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        865..883
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      933..942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             942..1007
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1014..1034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1059..5159
                     /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             1059..5159
                     /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             5160..5207
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5643..5660)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5649..5873
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5919..6347
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6006..6025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6216..6237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6356..6376)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6361..6690
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6541..6676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6603..6622
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6757..7551
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6811..6830)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7421..7440
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7725..7846
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7762..7781)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7816..7835
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7895..7911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7895..7911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7908..7930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7919..7935
                     /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(7943..7949,7950..7967,7968..7973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7988..8009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8126..8143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8297..8885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8377..8396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9056..9847,9848..9916))
                     /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     9679..9698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9917..10021)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
       61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      781 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      841 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
      901 gcgtttaaac gggccctcta gactcgagcg gtgccaccat ggactataag gaccacgacg
      961 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
     1021 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc
     1081 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     1141 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     1201 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     1261 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     1321 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     1381 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     1441 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     1501 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     1561 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     1621 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     1681 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     1741 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga
     1801 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     1861 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     1921 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     1981 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     2041 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     2101 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     2161 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     2221 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     2281 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     2341 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     2401 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     2461 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     2521 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     2581 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     2641 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
     2701 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     2761 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     2821 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     2881 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     2941 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     3001 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     3061 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     3121 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     3181 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     3241 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     3301 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     3361 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     3421 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     3481 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     3541 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg
     3601 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
     3661 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
     3721 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     3781 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     3841 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
     3901 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     3961 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     4021 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     4081 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     4141 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     4201 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     4261 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
     4321 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     4381 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
     4441 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     4501 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     4561 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     4621 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     4681 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     4741 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     4801 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
     4861 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     4921 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
     4981 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     5041 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     5101 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca
     5161 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggaa ttcgggggat
     5221 cagggggagg gtctggtggt ggatctggtg gaggatctgg tgggggttca gggggcggtt
     5281 ccggaggagg ctcaggaggc ggctccggcg gtggctcagg aggtggatcc ggtatgaaca
     5341 ttaacgacct gattcgggaa ataaagaaca aggactacac ggtcaagctg tcaggcaccg
     5401 actccaactc tataacacag ctcattataa gggttaataa tgatggcaac gaatatgtca
     5461 tctctgaatc cgagaatgaa tcaatcgtgg agaaattcat cagtgcattt aaaaacggct
     5521 ggaaccagga gtacgaagat gaggaagagt tctacaatga tatgcaaact atcacactga
     5581 aatctgagct gaactagctg aagttcatct gcaccaaagc ttaagtttaa accgctgatc
     5641 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5701 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     5761 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5821 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     5881 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     5941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     6361 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     6421 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     6481 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     6541 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     6601 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     6661 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     6721 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     6781 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     6841 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     6901 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     6961 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     7021 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     7081 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     7141 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     7201 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     7261 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     7321 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     7381 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     7441 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     7501 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     7561 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     7621 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     7681 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     7741 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     7801 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     7861 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     7921 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     7981 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     8041 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     8101 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     8161 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     8221 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     8281 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     8341 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     8401 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     8461 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     8521 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     8581 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     8641 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     8701 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     8761 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     8821 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     8881 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     8941 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     9001 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     9061 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     9121 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     9181 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     9241 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     9301 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     9361 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     9421 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     9481 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     9541 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     9601 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     9661 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     9721 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     9781 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     9841 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     9901 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     9961 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
    10021 gcacatttcc ccgaaaagtg ccacctgacg
//
