LOCUS       sequence-374760-       15796 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 15796)
  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 15796)
  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-EF1a-Zim3-dCas9-loxP-P2A-EGFP-loxP
FEATURES             Location/Qualifiers
     source          1..15796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..59)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        44..373
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      224..359
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1507..1572
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1702..1722
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2147..2281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2184..2203)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2238..2257
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2449..3082
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3129..3254
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3751..3984
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4169..4213
                     /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    4498..4615
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5347..6524
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5578..6516
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6473..6493
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(6881..6905)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6925..6945
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6994..7003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7000..7026,7027..7029,7030..9516,9517..9519,
                     9520..11103)
                     /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             7000..11103
                     /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             11107..11133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             11113..11139
                     /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             11152..11172
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11179..11199
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    11209..11242
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             11245..11301
                     /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             join(11302..11304,11305..12018)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(11605..11624)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     11952..11973
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    12018..12051
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    12108..12696
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12161..12181)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12579..12590)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12699..12715)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12700..12716)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12806..13039
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(13064..13082)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(13065..13082)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(13372..13391)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(13605..13624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13724..13746
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13784..13802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13870..13974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13975..14043,14044..14835)
                     /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(14193..14212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      15006..15594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15495..15514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     15748..15765
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctctccccgc gcgttggccg attcattaat gcagctgtgg aatgtgtgtc agttagggtg
       61 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      121 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
      181 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
      241 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
      301 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
      361 aggcttttgc aaaaagcttg gacacaagac aggcttgcga gatatgtttg agaataccac
      421 tttatcccgc gtcagggaga ggcagtgcgt aaaaagacgc ggactcatgt gaaatactgg
      481 tttttagtgc gccagatctc tataatctcg cgcaacctat tttcccctcg aacacttttt
      541 aagccgtaga taaacaggct gggacacttc acatgagcga aaaatacatc gtcacctggg
      601 acatgttgca gatccatgca cgtaaactcg caagccgact gatgccttct gaacaatgga
      661 aaggcattat tgccgtaagc cgtggcggtc tgtaccgggt gcgttactgg cgcgtgaact
      721 gggtattcgt catgtcgata ccgtttgtat ttccagctac gatcacgaca accagcgcga
      781 gcttaaagtg ctgaaacgcg cagaaggcga tggcgaaggc ttcatcgtta ttgatgacct
      841 ggtggatacc ggtggtactg cggttgcgat tcgtgaaatg tatccaaaag cgcactttgt
      901 caccatcttc gcaaaaccgg ctggtcgtcc gctggttgat gactatgttg ttgatatccc
      961 gcaagatacc tggattgaac agccgtggga tatgggcgtc gtattcgtcc cgccaatctc
     1021 cggtcgctaa tcttttcaac gcctggcact gccgggcgtt gttcttttta acttcaggcg
     1081 ggttacaata gtttccagta agtattctgg aggctgcatc catgacacag gcaaacctga
     1141 gcgaaaccct gttcaaaccc cgctttaaac atcctgaaac ctcgacgcta gtccgccgct
     1201 ttaatcacgg cgcacaaccg cctgtgcagt cggcccttga tggtaaaacc atccctcact
     1261 ggtatcgcat gattaaccgt ctgatgtgga tctggcgcgg cattgaccca cgcgaaatcc
     1321 tcgacgtcca ggcacgtatt gtgatgagcg atgccgaacg taccgacgat gatttatacg
     1381 atacggtgat tggctaccgt ggcggcaact ggatttatga gtgggccccg gatctttgtg
     1441 aaggaacctt acttctgtgg tgtgacataa ttggacaaac tacctacaga gatttaaagc
     1501 tctaaggtaa atataaaatt tttaagtgta taatgtgtta aactactgat tctaattgtt
     1561 tgtgtatttt agattccaac ctatggaact gatgaatggg agcagtggtg gaatgccttt
     1621 aatgaggaaa acctgttttg ctcagaagaa atgccatcta gtgatgatga ggctactgct
     1681 gactctcaac attctactcc tccaaaaaag aagagaaagg tagaagaccc caaggacttt
     1741 ccttcagaat tgctaagttt tttgagtcat gctgtgttta gtaatagaac tcttgcttgc
     1801 tttgctattt acaccacaaa ggaaaaagct gcactgctat acaagaaaat tatggaaaaa
     1861 tattctgtaa cctttataag taggcataac agttataatc ataacatact gttttttctt
     1921 actccacaca ggcatagagt gtctgctatt aataactatg ctcaaaaatt gtgtaccttt
     1981 agctttttaa tttgtaaagg ggttaataag gaatatttga tgtatagtgc cttgactaga
     2041 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     2101 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     2161 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2221 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     2281 caactggata actcaagcta accaaaatca tcccaaactt cccaccccat accctattac
     2341 cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg aattattttc
     2401 attttaaaga aattgtattt gttaaatatg tactacaaac ttagtagttg gaagggctaa
     2461 ttcactccca aagaagacaa gatatccttg atctgtggat ctaccacaca caaggctact
     2521 tccctgatta gcagaactac acaccagggc caggggtcag atatccactg acctttggat
     2581 ggtgctacaa gctagtacca gttgagccag ataaggtaga agaggccaat aaaggagaga
     2641 acaccagctt gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtgt
     2701 tagagtggag gtttgacagc cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg
     2761 agtacttcaa gaactgctga tatcgagctt gctacaaggg actttccgct ggggactttc
     2821 cagggaggcg tggcctgggc gggactgggg agtggcgagc cctcagatcc tgcatataag
     2881 cagctgcttt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     2941 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     3001 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     3061 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
     3121 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     3181 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     3241 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
     3301 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
     3361 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     3421 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     3481 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     3541 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc
     3601 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     3661 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     3721 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     3781 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     3841 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     3901 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     3961 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     4021 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     4081 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     4141 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     4201 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     4261 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     4321 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     4381 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     4441 cattcgatta gtgaacggat ctcgacggta tcgccaaatg gcagtattca tccacaattt
     4501 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     4561 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     4621 ttattacagg gacagcagag atccagtttg gatcgataag cttgatatcg aattcgggag
     4681 gtggtccctg cagttacgcc aatgataacc cccgccagaa aaatcttagt agccttccct
     4741 ttttgttttc cgtgccccaa ctcggcggat tgactcggcc ccttccggaa acacccgaat
     4801 caacttctag tcaaattatt gttcacgccg caatgaccca cccctggccc gcgtctgtgg
     4861 aactgacccc tggtgtacag gagagttcgc tgctgaaagt ggtcccaaag gggtactagt
     4921 ttttaagctc ccaactcccc ctcccccagc gtctggagga ttccacaccc tcgcaccgca
     4981 ggggcgagga agtgggcgga gtccggtttt ggcgccagcc gctgaggctg ccaagcagaa
     5041 aagccaccgc tgaggagact ccggtcactg tcctcgcccc gcctccccct tccctcccct
     5101 tggggaccac cgggcgccac gccgcgaacg gtaagtgccg cggtcgtcgg cgcctccgcc
     5161 ctccccctag ggccccaatt cccagcgggc gcggcgcgcg gcccctcccc ccgccgggcg
     5221 cgcgcccgct gccccgccct tcgtggccgc ccggcgtggg cggtgccacc cctccccccg
     5281 gcggccccgc gcgcagctcc cggctccctc ccccttcgga tgtggcttga gctgtaggcg
     5341 cggaggggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     5401 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     5461 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     5521 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
     5581 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
     5641 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     5701 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     5761 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     5821 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     5881 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     5941 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     6001 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     6061 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     6121 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     6181 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     6241 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     6301 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     6361 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     6421 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     6481 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtggaattc tcacgcgtct
     6541 gcaggatatc aagcttgcgg taccgcgggc ccggccacca tgaacaattc ccagggaaga
     6601 gtgaccttcg aggatgtcac tgtgaacttc acccaggggg agtggcagcg gctgaatccc
     6661 gaacagagaa acttgtacag ggatgtgatg ctggagaatt acagcaacct tgtctctgtg
     6721 ggacaagggg aaaccaccaa acccgatgtg atcttgaggt tggaacaagg aaaggagcca
     6781 tggttggagg aagaggaagt gctgggaagt ggccgtgcag aaaaaaatgg ggacattgga
     6841 gggcagattt ggaagccaaa ggatgtgaaa gagagtctct acccaacttt cttgtacaaa
     6901 gtggttggtg gatctggagg ttctcccaag aagaagcgca aggtgggacg cgtctgcagg
     6961 atatcaagct tgcggtaccg cgggcccggg atcgccacca tggacaagaa gtacagcatc
     7021 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     7081 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     7141 atcggcgccc tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc
     7201 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     7261 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     7321 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     7381 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     7441 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     7501 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     7561 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     7621 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     7681 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
     7741 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     7801 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     7861 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     7921 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     7981 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     8041 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc
     8101 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     8161 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     8221 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     8281 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     8341 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     8401 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
     8461 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     8521 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg
     8581 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc
     8641 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     8701 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     8761 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     8821 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     8881 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     8941 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     9001 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     9061 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     9121 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     9181 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     9241 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     9301 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     9361 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     9421 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     9481 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg
     9541 aaggacgact ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc
     9601 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg
     9661 aatgccaagc tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc
     9721 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     9781 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac
     9841 aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     9901 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     9961 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
    10021 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
    10081 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
    10141 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
    10201 gaaacaggcg agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg
    10261 tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
    10321 gagtctatcc tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac
    10381 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
    10441 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
    10501 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
    10561 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
    10621 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
    10681 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
    10741 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac
    10801 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
    10861 gacaaggtgc tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag
    10921 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
    10981 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
    11041 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
    11101 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
    11161 aaacgcaagg tggaagatcc taagaaaaag cggaaagtga gcggccgcat aacttcgtat
    11221 aaagtatcct atacgaagtt atccgctact aacttcagcc tgctgaagca ggctggagac
    11281 gtggaggaga accctggacc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
    11341 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc tggcgagggc
    11401 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
    11461 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
    11521 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
    11581 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
    11641 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
    11701 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
    11761 gccgacaagc agaagaacgg catcaaggcg aacttcaaga tccgccacaa catcgaggac
    11821 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
    11881 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
    11941 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
    12001 gacgagctgt acaagtaata acttcgtata aagtatccta tacgaagtta tccaagtagt
    12061 gagcgatcgc cctgcaggca tgcaagcttg atatcaagct tatcgataat caacctctgg
    12121 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
    12181 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
    12241 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
    12301 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
    12361 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
    12421 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
    12481 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
    12541 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
    12601 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
    12661 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgaggga
    12721 attaattcga gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
    12781 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
    12841 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
    12901 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
    12961 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
    13021 gtgtggaaaa tctctagcag catctagaat taattccgtg tattctatag tgtcacctaa
    13081 atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata
    13141 tgtacaagcc taattgtgta gcatctggct tactgaagca gaccctatca tctctctcgt
    13201 aaactgccgt cagagtcggt ttggttggac gaaccttctg agtttctggt aacgccgtcc
    13261 cgcacccgga aatggtcagc gaaccaatca gcagggtcat cgctagccag atcctctacg
    13321 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
    13381 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
    13441 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
    13501 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
    13561 tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc tcagtacaat ctgctctgat
    13621 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
    13681 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
    13741 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
    13801 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
    13861 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
    13921 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
    13981 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
    14041 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
    14101 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
    14161 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
    14221 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
    14281 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
    14341 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
    14401 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
    14461 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
    14521 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
    14581 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
    14641 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
    14701 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
    14761 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
    14821 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
    14881 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
    14941 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    15001 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    15061 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    15121 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
    15181 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    15241 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    15301 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    15361 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    15421 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    15481 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    15541 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    15601 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
    15661 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
    15721 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
    15781 ccaatacgca aaccgc
//
