LOCUS       sequence-374756-       14963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with an N-term Zim3 KRAB-NLS fusion, a C-term HA-2xNLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14963)
  AUTHORS   Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, 
            Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, 
            Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M
  TITLE     Maximizing CRISPRi efficacy and accessibility with dual-sgRNA 
            libraries and optimal effectors.
  JOURNAL   eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.
  PUBMED    36576240
REFERENCE   2  (bases 1 to 14963)
  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: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pHR-UCOE-SFFV-Zim3-dCas9-BFP
FEATURES             Location/Qualifiers
     source          1..14963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..55
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(125..145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        130..459
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      310..445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1593..1658
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1788..1808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2233..2367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2270..2289)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2324..2343
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2535..3168
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3215..3340
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3837..4070
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4255..4299
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    4584..4701
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5443..5850
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(6226..6250)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6270..6290
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6339..6348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6345..6371,6372..6374,6375..8861,8862..8864,
                     8865..10448)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6345..10448
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             10452..10478
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10458..10484
                     /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             10497..10517
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10524..10544
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10602..11309
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     misc_feature    11361..11949
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11414..11434)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11832..11843)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11952..11968)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11953..11969)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12059..12292
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12317..12335)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12318..12335)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(12625..12644)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12858..12877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12977..12999
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13037..13055)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13123..13227
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13228..13296,13297..14088)
                     /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(13446..13465)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14259..14847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14748..14767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
       61 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      121 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
      181 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
      241 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
      301 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
      361 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
      421 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttggaca caagacaggc
      481 ttgcgagata tgtttgagaa taccacttta tcccgcgtca gggagaggca gtgcgtaaaa
      541 agacgcggac tcatgtgaaa tactggtttt tagtgcgcca gatctctata atctcgcgca
      601 acctattttc ccctcgaaca ctttttaagc cgtagataaa caggctggga cacttcacat
      661 gagcgaaaaa tacatcgtca cctgggacat gttgcagatc catgcacgta aactcgcaag
      721 ccgactgatg ccttctgaac aatggaaagg cattattgcc gtaagccgtg gcggtctgta
      781 ccgggtgcgt tactggcgcg tgaactgggt attcgtcatg tcgataccgt ttgtatttcc
      841 agctacgatc acgacaacca gcgcgagctt aaagtgctga aacgcgcaga aggcgatggc
      901 gaaggcttca tcgttattga tgacctggtg gataccggtg gtactgcggt tgcgattcgt
      961 gaaatgtatc caaaagcgca ctttgtcacc atcttcgcaa aaccggctgg tcgtccgctg
     1021 gttgatgact atgttgttga tatcccgcaa gatacctgga ttgaacagcc gtgggatatg
     1081 ggcgtcgtat tcgtcccgcc aatctccggt cgctaatctt ttcaacgcct ggcactgccg
     1141 ggcgttgttc tttttaactt caggcgggtt acaatagttt ccagtaagta ttctggaggc
     1201 tgcatccatg acacaggcaa acctgagcga aaccctgttc aaaccccgct ttaaacatcc
     1261 tgaaacctcg acgctagtcc gccgctttaa tcacggcgca caaccgcctg tgcagtcggc
     1321 ccttgatggt aaaaccatcc ctcactggta tcgcatgatt aaccgtctga tgtggatctg
     1381 gcgcggcatt gacccacgcg aaatcctcga cgtccaggca cgtattgtga tgagcgatgc
     1441 cgaacgtacc gacgatgatt tatacgatac ggtgattggc taccgtggcg gcaactggat
     1501 ttatgagtgg gccccggatc tttgtgaagg aaccttactt ctgtggtgtg acataattgg
     1561 acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta agtgtataat
     1621 gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat ggaactgatg
     1681 aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca gaagaaatgc
     1741 catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca aaaaagaaga
     1801 gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg agtcatgctg
     1861 tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa aaagctgcac
     1921 tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt tataagtagg cataacagtt
     1981 ataatcataa catactgttt tttcttactc cacacaggca tagagtgtct gctattaata
     2041 actatgctca aaaattgtgt acctttagct ttttaatttg taaaggggtt aataaggaat
     2101 atttgatgta tagtgccttg actagagatc ataatcagcc ataccacatt tgtagaggtt
     2161 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     2221 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2281 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2341 atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc
     2401 aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa
     2461 acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact
     2521 acaaacttag tagttggaag ggctaattca ctcccaaaga agacaagata tccttgatct
     2581 gtggatctac cacacacaag gctacttccc tgattagcag aactacacac cagggccagg
     2641 ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agccagataa
     2701 ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga gcctgcatgg
     2761 gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc tagcatttca
     2821 tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc gagcttgcta
     2881 caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga ctggggagtg
     2941 gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg tctctctggt
     3001 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     3061 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     3121 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     3181 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     3241 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     3301 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     3361 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     3421 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     3481 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     3541 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     3601 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     3661 gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg
     3721 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     3781 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     3841 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     3901 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     3961 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     4021 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     4081 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     4141 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     4201 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     4261 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     4321 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     4381 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     4441 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     4501 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcgc
     4561 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     4621 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     4681 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggatc
     4741 gataagcttg atatcgaatt cgggaggtgg tccctgcagt tacgccaatg ataacccccg
     4801 ccagaaaaat cttagtagcc ttcccttttt gttttccgtg ccccaactcg gcggattgac
     4861 tcggcccctt ccggaaacac ccgaatcaac ttctagtcaa attattgttc acgccgcaat
     4921 gacccacccc tggcccgcgt ctgtggaact gacccctggt gtacaggaga gttcgctgct
     4981 gaaagtggtc ccaaaggggt actagttttt aagctcccaa ctccccctcc cccagcgtct
     5041 ggaggattcc acaccctcgc accgcagggg cgaggaagtg ggcggagtcc ggttttggcg
     5101 ccagccgctg aggctgccaa gcagaaaagc caccgctgag gagactccgg tcactgtcct
     5161 cgccccgcct cccccttccc tccccttggg gaccaccggg cgccacgccg cgaacggtaa
     5221 gtgccgcggt cgtcggcgcc tccgccctcc ccctagggcc ccaattccca gcgggcgcgg
     5281 cgcgcggccc cgcgcgcagc tcccggctcc ctcccccttc ggatgtggct tgagctgtag
     5341 gcgcggagtc ctgcagcccc gataaaataa aagattttat ttagtctcca gaaaaagggg
     5401 ggaatgaaag accccacctg taggtttggc aagctagctg cagtaacgcc attttgcaag
     5461 gcatggaaaa ataccaaacc aagaatagag aagttcagat caagggcggg tacatgaaaa
     5521 tagctaacgt tgggccaaac aggatatctg cggtgagcag tttcggcccc ggcccggggc
     5581 caagaacaga tggtcaccgc agtttcggcc ccggcccgag gccaagaaca gatggtcccc
     5641 agatatggcc caaccctcag cagtttctta agacccatca gatgtttcca ggctccccca
     5701 aggacctgaa atgaccctgc gccttatttg aattaaccaa tcagcctgct tctcgcttct
     5761 gttcgcgcgc ttctgcttcc cgagctctat aaaagagctc acaacccctc actcggcgcg
     5821 ccagtcctcc gacagactga gtcgcccggg ggggatctgg agctctcgag aattctcacg
     5881 cgtctgcagg atatcaagct tgcggtaccg cgggcccggc caccatgaac aattcccagg
     5941 gaagagtgac cttcgaggat gtcactgtga acttcaccca gggggagtgg cagcggctga
     6001 atcccgaaca gagaaacttg tacagggatg tgatgctgga gaattacagc aaccttgtct
     6061 ctgtgggaca aggggaaacc accaaacccg atgtgatctt gaggttggaa caaggaaagg
     6121 agccatggtt ggaggaagag gaagtgctgg gaagtggccg tgcagaaaaa aatggggaca
     6181 ttggagggca gatttggaag ccaaaggatg tgaaagagag tctctaccca actttcttgt
     6241 acaaagtggt tggtggatct ggaggttctc ccaagaagaa gcgcaaggtg ggacgcgtct
     6301 gcaggatatc aagcttgcgg taccgcgggc ccgggatcgc caccatggac aagaagtaca
     6361 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     6421 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     6481 acctgatcgg cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga
     6541 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     6601 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     6661 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     6721 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     6781 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     6841 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     6901 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     6961 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     7021 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc
     7081 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     7141 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     7201 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     7261 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     7321 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     7381 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg
     7441 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     7501 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     7561 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     7621 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     7681 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     7741 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     7801 ccagcgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     7861 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca
     7921 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     7981 aagccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     8041 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     8101 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     8161 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     8221 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     8281 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     8341 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     8401 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     8461 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     8521 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     8581 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     8641 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     8701 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     8761 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     8821 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct
     8881 ttctgaagga cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca
     8941 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc
     9001 tgctgaatgc caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag
     9061 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     9121 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     9181 acgacaaact gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     9241 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     9301 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     9361 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     9421 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     9481 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     9541 acggcgaaac aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag
     9601 tgctgtctat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     9661 gcaaagagtc tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact
     9721 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     9781 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     9841 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     9901 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     9961 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
    10021 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
    10081 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc
    10141 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
    10201 atctggacaa ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg
    10261 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
    10321 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
    10381 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
    10441 gaggcgacgc ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca
    10501 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtgagcggc cgcagcggaa
    10561 gtgagacccc aggtacatcc gaatcagcaa cgcctgaaag cgtgtctaag ggcgaagagc
    10621 tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact
    10681 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca
    10741 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct
    10801 acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct
    10861 tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg
    10921 ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg
    10981 tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca
    11041 ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc
    11101 tcgtgggcgg gagccatctg atcgcaaacg ccaagaccac atatagatcc aagaaacccg
    11161 ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg
    11221 aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc
    11281 tccctagcaa actggggcac aagcttaatt aataagcgat cgccctgcag gcatgcaagc
    11341 ttgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
    11401 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
    11461 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
    11521 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
    11581 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
    11641 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
    11701 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
    11761 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
    11821 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
    11881 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
    11941 cctccccgca tcgataccgt cgacctcgag ggaattaatt cgagctcggt acctttaaga
    12001 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
    12061 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
    12121 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
    12181 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
    12241 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagcatctag
    12301 aattaattcc gtgtattcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta
    12361 tgtattaatt gtagccgcgt tctaacgaca atatgtacaa gcctaattgt gtagcatctg
    12421 gcttactgaa gcagacccta tcatctctct cgtaaactgc cgtcagagtc ggtttggttg
    12481 gacgaacctt ctgagtttct ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa
    12541 tcagcagggt catcgctagc cagatcctct acgccggacg catcgtggcc ggcatcaccg
    12601 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
    12661 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
    12721 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
    12781 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
    12841 gaatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
    12901 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
    12961 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
    13021 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
    13081 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
    13141 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
    13201 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
    13261 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
    13321 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
    13381 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
    13441 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
    13501 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
    13561 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
    13621 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
    13681 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
    13741 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
    13801 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
    13861 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
    13921 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
    13981 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
    14041 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
    14101 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
    14161 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
    14221 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
    14281 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
    14341 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
    14401 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
    14461 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
    14521 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
    14581 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
    14641 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
    14701 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
    14761 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
    14821 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
    14881 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
    14941 aaccgtatta ccgcctttga gtg
//
