LOCUS       sequence-374752-       14969 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 14969)
  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 14969)
  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-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..14969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..66)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        51..380
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      231..366
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1514..1579
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1709..1729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2154..2288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2191..2210)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2245..2264
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2456..3089
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3136..3261
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3758..3991
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4176..4220
                     /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    4505..4622
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5364..5771
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(6147..6171)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6191..6211
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6260..6269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6266..6292,6293..6295,6296..8782,8783..8785,
                     8786..10369)
                     /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             6266..10369
                     /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             10373..10399
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10379..10405
                     /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             10418..10438
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10445..10465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10475..10531
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             10532..11239
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(10674..10692)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     10942..10961
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    11288..11876
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11341..11361)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11759..11770)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11879..11895)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11880..11896)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11986..12219
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12244..12262)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12245..12262)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(12552..12571)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12785..12804)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12904..12926
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(12964..12982)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13050..13154
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13155..13223,13224..14015)
                     /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(13373..13392)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14186..14774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     14675..14694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14928..14945
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctgtggaat gtgtgtcagt
       61 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
      121 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
      181 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
      241 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
      301 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
      361 gaggcctagg cttttgcaaa aagcttggac acaagacagg cttgcgagat atgtttgaga
      421 ataccacttt atcccgcgtc agggagaggc agtgcgtaaa aagacgcgga ctcatgtgaa
      481 atactggttt ttagtgcgcc agatctctat aatctcgcgc aacctatttt cccctcgaac
      541 actttttaag ccgtagataa acaggctggg acacttcaca tgagcgaaaa atacatcgtc
      601 acctgggaca tgttgcagat ccatgcacgt aaactcgcaa gccgactgat gccttctgaa
      661 caatggaaag gcattattgc cgtaagccgt ggcggtctgt accgggtgcg ttactggcgc
      721 gtgaactggg tattcgtcat gtcgataccg tttgtatttc cagctacgat cacgacaacc
      781 agcgcgagct taaagtgctg aaacgcgcag aaggcgatgg cgaaggcttc atcgttattg
      841 atgacctggt ggataccggt ggtactgcgg ttgcgattcg tgaaatgtat ccaaaagcgc
      901 actttgtcac catcttcgca aaaccggctg gtcgtccgct ggttgatgac tatgttgttg
      961 atatcccgca agatacctgg attgaacagc cgtgggatat gggcgtcgta ttcgtcccgc
     1021 caatctccgg tcgctaatct tttcaacgcc tggcactgcc gggcgttgtt ctttttaact
     1081 tcaggcgggt tacaatagtt tccagtaagt attctggagg ctgcatccat gacacaggca
     1141 aacctgagcg aaaccctgtt caaaccccgc tttaaacatc ctgaaacctc gacgctagtc
     1201 cgccgcttta atcacggcgc acaaccgcct gtgcagtcgg cccttgatgg taaaaccatc
     1261 cctcactggt atcgcatgat taaccgtctg atgtggatct ggcgcggcat tgacccacgc
     1321 gaaatcctcg acgtccaggc acgtattgtg atgagcgatg ccgaacgtac cgacgatgat
     1381 ttatacgata cggtgattgg ctaccgtggc ggcaactgga tttatgagtg ggccccggat
     1441 ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat
     1501 ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct
     1561 aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa
     1621 tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc
     1681 tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa
     1741 ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct
     1801 tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat
     1861 ggaaaaatat tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt
     1921 ttttcttact ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg
     1981 tacctttagc tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt
     2041 gactagagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2101 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2161 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2221 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     2281 tctggatcaa ctggataact caagctaacc aaaatcatcc caaacttccc accccatacc
     2341 ctattaccac tgccaattac ctgtggtttc atttactcta aacctgtgat tcctctgaat
     2401 tattttcatt ttaaagaaat tgtatttgtt aaatatgtac tacaaactta gtagttggaa
     2461 gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta ccacacacaa
     2521 ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc
     2581 tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa
     2641 ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
     2701 gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg
     2761 catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg
     2821 gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc
     2881 atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg
     2941 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     3001 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     3061 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
     3121 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
     3181 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
     3241 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
     3301 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     3361 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     3421 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     3481 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     3541 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     3601 gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     3661 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     3721 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     3781 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     3841 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     3901 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     3961 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     4021 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     4081 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     4141 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     4201 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     4261 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     4321 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     4381 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     4441 acagatccat tcgattagtg aacggatctc gacggtatcg ccaaatggca gtattcatcc
     4501 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     4561 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     4621 ttcgggttta ttacagggac agcagagatc cagtttggat cgataagctt gatatcgaat
     4681 tcgggaggtg gtccctgcag ttacgccaat gataaccccc gccagaaaaa tcttagtagc
     4741 cttccctttt tgttttccgt gccccaactc ggcggattga ctcggcccct tccggaaaca
     4801 cccgaatcaa cttctagtca aattattgtt cacgccgcaa tgacccaccc ctggcccgcg
     4861 tctgtggaac tgacccctgg tgtacaggag agttcgctgc tgaaagtggt cccaaagggg
     4921 tactagtttt taagctccca actccccctc ccccagcgtc tggaggattc cacaccctcg
     4981 caccgcaggg gcgaggaagt gggcggagtc cggttttggc gccagccgct gaggctgcca
     5041 agcagaaaag ccaccgctga ggagactccg gtcactgtcc tcgccccgcc tcccccttcc
     5101 ctccccttgg ggaccaccgg gcgccacgcc gcgaacggta agtgccgcgg tcgtcggcgc
     5161 ctccgccctc cccctagggc cccaattccc agcgggcgcg gcgcgcggcc ccgcgcgcag
     5221 ctcccggctc cctccccctt cggatgtggc ttgagctgta ggcgcggagt cctgcagccc
     5281 cgataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     5341 gtaggtttgg caagctagct gcagtaacgc cattttgcaa ggcatggaaa aataccaaac
     5401 caagaataga gaagttcaga tcaagggcgg gtacatgaaa atagctaacg ttgggccaaa
     5461 caggatatct gcggtgagca gtttcggccc cggcccgggg ccaagaacag atggtcaccg
     5521 cagtttcggc cccggcccga ggccaagaac agatggtccc cagatatggc ccaaccctca
     5581 gcagtttctt aagacccatc agatgtttcc aggctccccc aaggacctga aatgaccctg
     5641 cgccttattt gaattaacca atcagcctgc ttctcgcttc tgttcgcgcg cttctgcttc
     5701 ccgagctcta taaaagagct cacaacccct cactcggcgc gccagtcctc cgacagactg
     5761 agtcgcccgg gggggatctg gagctctcga gaattctcac gcgtctgcag gatatcaagc
     5821 ttgcggtacc gcgggcccgg ccaccatgaa caattcccag ggaagagtga ccttcgagga
     5881 tgtcactgtg aacttcaccc agggggagtg gcagcggctg aatcccgaac agagaaactt
     5941 gtacagggat gtgatgctgg agaattacag caaccttgtc tctgtgggac aaggggaaac
     6001 caccaaaccc gatgtgatct tgaggttgga acaaggaaag gagccatggt tggaggaaga
     6061 ggaagtgctg ggaagtggcc gtgcagaaaa aaatggggac attggagggc agatttggaa
     6121 gccaaaggat gtgaaagaga gtctctaccc aactttcttg tacaaagtgg ttggtggatc
     6181 tggaggttct cccaagaaga agcgcaaggt gggacgcgtc tgcaggatat caagcttgcg
     6241 gtaccgcggg cccgggatcg ccaccatgga caagaagtac agcatcggcc tggccatcgg
     6301 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     6361 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gcgccctgct
     6421 gttcgacagc ggagaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     6481 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     6541 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     6601 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     6661 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     6721 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     6781 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     6841 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     6901 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     6961 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     7021 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     7081 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     7141 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     7201 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     7261 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     7321 cgaccagagc aagaacggct acgccggcta catcgatggc ggagccagcc aggaagagtt
     7381 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     7441 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     7501 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     7561 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     7621 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     7681 catcaccccc tggaacttcg aggaagtggt ggacaagggc gccagcgccc agagcttcat
     7741 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     7801 cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaagtgaaat acgtgaccga
     7861 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaagccatcg tggacctgct
     7921 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     7981 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     8041 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     8101 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     8161 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     8221 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     8281 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     8341 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     8401 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     8461 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     8521 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     8581 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     8641 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     8701 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     8761 gctgtccgac tacgatgtgg acgctatcgt gcctcagagc tttctgaagg acgactccat
     8821 cgataacaaa gtgctgactc ggagcgacaa gaaccggggc aagagcgaca acgtgccctc
     8881 cgaagaggtc gtgaagaaga tgaagaacta ctggcgccag ctgctgaatg ccaagctgat
     8941 tacccagagg aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     9001 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     9061 acagatcctg gactcccgga tgaacactaa gtacgacgag aacgacaaac tgatccggga
     9121 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     9181 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     9241 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     9301 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     9361 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     9421 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa caggcgagat
     9481 cgtgtgggat aagggccggg actttgccac cgtgcggaaa gtgctgtcta tgccccaagt
     9541 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     9601 caagaggaac agcgacaagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     9661 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     9721 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     9781 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     9841 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     9901 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     9961 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
    10021 tgagcagaaa cagctgtttg tggaacagca caaacactac ctggacgaga tcatcgagca
    10081 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aggtgctgag
    10141 cgcctacaac aagcacagag acaagcctat cagagagcag gccgagaata tcatccacct
    10201 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
    10261 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
    10321 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacg cctatcccta
    10381 tgacgtgccc gattatgcca gcctgggcag cggctccccc aagaaaaaac gcaaggtgga
    10441 agatcctaag aaaaagcgga aagtgagcgg ccgcgctact aacttcagcc tgctgaagca
    10501 ggctggagac gtggaggaga accctggacc tgtgagcaag ggcgaggagg ataacatggc
    10561 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
    10621 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
    10681 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
    10741 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
    10801 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
    10861 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
    10921 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
    10981 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
    11041 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
    11101 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
    11161 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
    11221 ggacgagctg tacaagtaat aagcgatcgc cctgcaggca tgcaagcttg atatcaagct
    11281 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
    11341 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
    11401 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
    11461 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
    11521 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
    11581 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
    11641 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
    11701 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
    11761 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
    11821 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
    11881 ataccgtcga cctcgaggga attaattcga gctcggtacc tttaagacca atgacttaca
    11941 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
    12001 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
    12061 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
    12121 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
    12181 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catctagaat taattccgtg
    12241 tattctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta
    12301 gccgcgttct aacgacaata tgtacaagcc taattgtgta gcatctggct tactgaagca
    12361 gaccctatca tctctctcgt aaactgccgt cagagtcggt ttggttggac gaaccttctg
    12421 agtttctggt aacgccgtcc cgcacccgga aatggtcagc gaaccaatca gcagggtcat
    12481 cgctagccag atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
    12541 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
    12601 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
    12661 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
    12721 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc
    12781 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
    12841 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
    12901 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
    12961 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
    13021 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
    13081 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
    13141 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
    13201 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
    13261 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
    13321 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
    13381 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
    13441 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
    13501 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
    13561 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
    13621 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
    13681 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
    13741 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
    13801 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
    13861 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
    13921 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
    13981 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
    14041 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
    14101 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    14161 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    14221 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    14281 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    14341 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    14401 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    14461 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    14521 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    14581 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    14641 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    14701 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    14761 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    14821 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
    14881 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
    14941 gcgaggaagc ggaagagcgc ccaatacgc
//
