LOCUS       sequence-350806-       14812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9 fused to mCherry and the KRAB domain of ZIM3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14812)
  AUTHORS   Alerasool N, Segal D, Lee H, Taipale M
  TITLE     An efficient KRAB domain for CRISPRi applications in human cells.
  JOURNAL   Nat Methods. 2020 Nov;17(11):1093-1096. doi: 
            10.1038/s41592-020-0966-x. Epub 2020 Oct 5.
  PUBMED    33020655
REFERENCE   2  (bases 1 to 14812)
  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"; doi: 
            "10.1038/s41592-020-0966-x"; journalName: "Nat Methods"; date: 
            "2020-11"; volume: "17"; issue: "11"; pages: "1093-1096"
COMMENT     Sequence Label: pHR-UCOE-SFFV-dCas9-mCherry-ZIM3-KRAB
FEATURES             Location/Qualifiers
     source          1..14812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     239..256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(326..346)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        331..660
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      511..646
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1794..1859
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1989..2009
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2434..2568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2471..2490)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2525..2544
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2736..3369
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3416..3541
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4038..4271
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4456..4500
                     /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    4785..4902
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5644..6051
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      6120..6129
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6126..6152,6153..6155,6156..8642,8643..8645,
                     8646..10229)
                     /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             6126..10229
                     /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             10233..10259
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10239..10265
                     /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             10278..10298
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10305..10325
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10365..11069
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(10510..10528)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     10778..10797
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    11411..11999
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11464..11484)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11882..11893)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12002..12018)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12003..12019)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12109..12342
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12367..12385)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12368..12385)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(12675..12694)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12908..12927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13027..13049
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13087..13105)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13173..13277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13278..13346,13347..14138)
                     /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(13496..13515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(14309..14812,1..85)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(14798..14812,1..5)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
       61 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      121 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
      181 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
      241 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
      301 ttggccgatt cattaatgca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
      361 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
      421 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
      481 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
      541 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
      601 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
      661 aagcttggac acaagacagg cttgcgagat atgtttgaga ataccacttt atcccgcgtc
      721 agggagaggc agtgcgtaaa aagacgcgga ctcatgtgaa atactggttt ttagtgcgcc
      781 agatctctat aatctcgcgc aacctatttt cccctcgaac actttttaag ccgtagataa
      841 acaggctggg acacttcaca tgagcgaaaa atacatcgtc acctgggaca tgttgcagat
      901 ccatgcacgt aaactcgcaa gccgactgat gccttctgaa caatggaaag gcattattgc
      961 cgtaagccgt ggcggtctgt accgggtgcg ttactggcgc gtgaactggg tattcgtcat
     1021 gtcgataccg tttgtatttc cagctacgat cacgacaacc agcgcgagct taaagtgctg
     1081 aaacgcgcag aaggcgatgg cgaaggcttc atcgttattg atgacctggt ggataccggt
     1141 ggtactgcgg ttgcgattcg tgaaatgtat ccaaaagcgc actttgtcac catcttcgca
     1201 aaaccggctg gtcgtccgct ggttgatgac tatgttgttg atatcccgca agatacctgg
     1261 attgaacagc cgtgggatat gggcgtcgta ttcgtcccgc caatctccgg tcgctaatct
     1321 tttcaacgcc tggcactgcc gggcgttgtt ctttttaact tcaggcgggt tacaatagtt
     1381 tccagtaagt attctggagg ctgcatccat gacacaggca aacctgagcg aaaccctgtt
     1441 caaaccccgc tttaaacatc ctgaaacctc gacgctagtc cgccgcttta atcacggcgc
     1501 acaaccgcct gtgcagtcgg cccttgatgg taaaaccatc cctcactggt atcgcatgat
     1561 taaccgtctg atgtggatct ggcgcggcat tgacccacgc gaaatcctcg acgtccaggc
     1621 acgtattgtg atgagcgatg ccgaacgtac cgacgatgat ttatacgata cggtgattgg
     1681 ctaccgtggc ggcaactgga tttatgagtg ggccccggat ctttgtgaag gaaccttact
     1741 tctgtggtgt gacataattg gacaaactac ctacagagat ttaaagctct aaggtaaata
     1801 taaaattttt aagtgtataa tgtgttaaac tactgattct aattgtttgt gtattttaga
     1861 ttccaaccta tggaactgat gaatgggagc agtggtggaa tgcctttaat gaggaaaacc
     1921 tgttttgctc agaagaaatg ccatctagtg atgatgaggc tactgctgac tctcaacatt
     1981 ctactcctcc aaaaaagaag agaaaggtag aagaccccaa ggactttcct tcagaattgc
     2041 taagtttttt gagtcatgct gtgtttagta atagaactct tgcttgcttt gctatttaca
     2101 ccacaaagga aaaagctgca ctgctataca agaaaattat ggaaaaatat tctgtaacct
     2161 ttataagtag gcataacagt tataatcata acatactgtt ttttcttact ccacacaggc
     2221 atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc tttttaattt
     2281 gtaaaggggt taataaggaa tatttgatgt atagtgcctt gactagagat cataatcagc
     2341 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2401 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2461 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2521 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa ctggataact
     2581 caagctaacc aaaatcatcc caaacttccc accccatacc ctattaccac tgccaattac
     2641 ctgtggtttc atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat
     2701 tgtatttgtt aaatatgtac tacaaactta gtagttggaa gggctaattc actcccaaag
     2761 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattagca
     2821 gaactacaca ccagggccag gggtcagata tccactgacc tttggatggt gctacaagct
     2881 agtaccagtt gagccagata aggtagaaga ggccaataaa ggagagaaca ccagcttgtt
     2941 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtgttag agtggaggtt
     3001 tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt acttcaagaa
     3061 ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag ggaggcgtgg
     3121 cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag ctgctttttg
     3181 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     3241 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     3301 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     3361 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     3421 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     3481 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     3541 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     3601 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     3661 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     3721 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     3781 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     3841 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct gatcttcaga
     3901 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     3961 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     4021 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     4081 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     4141 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     4201 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     4261 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     4321 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     4381 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     4441 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     4501 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     4561 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     4621 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     4681 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     4741 aacggatctc gacggtatcg ccaaatggca gtattcatcc acaattttaa aagaaaaggg
     4801 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     4861 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     4921 agcagagatc cagtttggat cgataagctt gatatcgaat tcgggaggtg gtccctgcag
     4981 ttacgccaat gataaccccc gccagaaaaa tcttagtagc cttccctttt tgttttccgt
     5041 gccccaactc ggcggattga ctcggcccct tccggaaaca cccgaatcaa cttctagtca
     5101 aattattgtt cacgccgcaa tgacccaccc ctggcccgcg tctgtggaac tgacccctgg
     5161 tgtacaggag agttcgctgc tgaaagtggt cccaaagggg tactagtttt taagctccca
     5221 actccccctc ccccagcgtc tggaggattc cacaccctcg caccgcaggg gcgaggaagt
     5281 gggcggagtc cggttttggc gccagccgct gaggctgcca agcagaaaag ccaccgctga
     5341 ggagactccg gtcactgtcc tcgccccgcc tcccccttcc ctccccttgg ggaccaccgg
     5401 gcgccacgcc gcgaacggta agtgccgcgg tcgtcggcgc ctccgccctc cccctagggc
     5461 cccaattccc agcgggcgcg gcgcgcggcc ccgcgcgcag ctcccggctc cctccccctt
     5521 cggatgtggc ttgagctgta ggcgcggagt cctgcagccc cgataaaata aaagatttta
     5581 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     5641 gcagtaacgc cattttgcaa ggcatggaaa aataccaaac caagaataga gaagttcaga
     5701 tcaagggcgg gtacatgaaa atagctaacg ttgggccaaa caggatatct gcggtgagca
     5761 gtttcggccc cggcccgggg ccaagaacag atggtcaccg cagtttcggc cccggcccga
     5821 ggccaagaac agatggtccc cagatatggc ccaaccctca gcagtttctt aagacccatc
     5881 agatgtttcc aggctccccc aaggacctga aatgaccctg cgccttattt gaattaacca
     5941 atcagcctgc ttctcgcttc tgttcgcgcg cttctgcttc ccgagctcta taaaagagct
     6001 cacaacccct cactcggcgc gccagtcctc cgacagactg agtcgcccgg gggggatctg
     6061 gagctctcga gaattctcac gcgtctgcag gatatcaagc ttgcggtacc gcgggcccgg
     6121 ccaccatgga caagaagtac agcatcggcc tggccatcgg caccaactct gtgggctggg
     6181 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     6241 accggcacag catcaagaag aacctgatcg gcgccctgct gttcgacagc ggagaaacag
     6301 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     6361 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     6421 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     6481 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     6541 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     6601 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     6661 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     6721 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     6781 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     6841 gcaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     6901 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     6961 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     7021 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     7081 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     7141 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     7201 acgccggcta catcgatggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     7261 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     7321 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     7381 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     7441 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     7501 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     7561 aggaagtggt ggacaagggc gccagcgccc agagcttcat cgagcggatg accaacttcg
     7621 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     7681 ccgtgtacaa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     7741 tcctgagcgg cgagcagaaa aaagccatcg tggacctgct gttcaagacc aaccggaaag
     7801 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     7861 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     7921 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     7981 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     8041 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     8101 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     8161 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     8221 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     8281 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     8341 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     8401 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     8461 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     8521 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     8581 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     8641 acgctatcgt gcctcagagc tttctgaagg acgactccat cgataacaaa gtgctgactc
     8701 ggagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     8761 tgaagaacta ctggcgccag ctgctgaatg ccaagctgat tacccagagg aagttcgaca
     8821 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     8881 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     8941 tgaacactaa gtacgacgag aacgacaaac tgatccggga agtgaaagtg atcaccctga
     9001 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     9061 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     9121 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     9181 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     9241 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     9301 agcggcctct gatcgagaca aacggcgaaa caggcgagat cgtgtgggat aagggccggg
     9361 actttgccac cgtgcggaaa gtgctgtcta tgccccaagt gaatatcgtg aaaaagaccg
     9421 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgacaagc
     9481 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     9541 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     9601 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     9661 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     9721 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     9781 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     9841 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     9901 tggaacagca caaacactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     9961 gagtgatcct ggccgacgct aatctggaca aggtgctgag cgcctacaac aagcacagag
    10021 acaagcctat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
    10081 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
    10141 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
    10201 ggatcgacct gtctcagctg ggaggcgacg cctatcccta tgacgtgccc gattatgcca
    10261 gcctgggcag cggctccccc aagaaaaaac gcaaggtgga agatcctaag aaaaagcgga
    10321 aagtggacgg cattggtagt gggagcaacg gcagcagcgg atccatggtg agcaagggcg
    10381 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
    10441 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
    10501 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
    10561 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacattcccg
    10621 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
    10681 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
    10741 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
    10801 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
    10861 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
    10921 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
    10981 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
    11041 actccaccgg cggcatggac gagctgtacg gcggtggcgg agggatggga agagtgacct
    11101 tcgaggatgt cactgtgaac ttcacccagg gggagtggca gcggctgaat cccgaacaga
    11161 gaaacttgta cagggatgtg atgctggaga attacagcaa ccttgtctct gtgggacaag
    11221 gggaaaccac caaacccgat gtgatcttga ggttggaaca aggaaaggag ccatggttgg
    11281 aggaagagga agtgctggga agtggccgtg cagaaaaaaa tggggacatt ggagggcaga
    11341 tttggaagcc aaaggatgtg aaagagagtc tctagtgcag gcatgcaagc ttgatatcaa
    11401 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
    11461 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
    11521 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
    11581 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
    11641 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
    11701 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
    11761 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
    11821 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
    11881 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
    11941 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
    12001 tcgataccgt cgacctcgag ggaattaatt cgagctcggt acctttaaga ccaatgactt
    12061 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
    12121 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
    12181 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
    12241 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
    12301 gatccctcag acccttttag tcagtgtgga aaatctctag cagcatctag aattaattcc
    12361 gtgtattcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt
    12421 gtagccgcgt tctaacgaca atatgtacaa gcctaattgt gtagcatctg gcttactgaa
    12481 gcagacccta tcatctctct cgtaaactgc cgtcagagtc ggtttggttg gacgaacctt
    12541 ctgagtttct ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa tcagcagggt
    12601 catcgctagc cagatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
    12661 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
    12721 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
    12781 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
    12841 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gaatggtgca
    12901 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
    12961 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
    13021 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
    13081 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
    13141 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
    13201 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
    13261 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
    13321 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    13381 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    13441 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    13501 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    13561 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    13621 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    13681 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    13741 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    13801 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    13861 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    13921 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    13981 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
    14041 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    14101 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    14161 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    14221 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    14281 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    14341 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    14401 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    14461 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    14521 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    14581 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    14641 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    14701 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    14761 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg ta
//
