LOCUS       sequence-374753-       15719 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 15719)
  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 15719)
  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-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..15719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..34)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        19..348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      199..334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1482..1547
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1677..1697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2122..2256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2159..2178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2213..2232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2424..3057
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3104..3229
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3726..3959
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4144..4188
                     /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    4473..4590
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5322..6499
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5553..6491
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6448..6468
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(6856..6880)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6900..6920
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6969..6978
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6975..7001,7002..7004,7005..9491,9492..9494,
                     9495..11078)
                     /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             6975..11078
                     /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             11082..11108
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             11088..11114
                     /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             11127..11147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11154..11174
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11184..11240
                     /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(11241..11243,11244..11957)
                     /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(11544..11563)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     11891..11912
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    12006..12594
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12059..12079)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12477..12488)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12597..12613)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12598..12614)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12704..12937
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12962..12980)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12963..12980)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(13270..13289)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(13503..13522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13622..13644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13682..13700)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13768..13872
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13873..13941,13942..14733)
                     /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(14091..14110)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14904..15492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15393..15412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     15646..15663
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttaatgcagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
       61 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
      121 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
      181 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
      241 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
      301 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttggacac
      361 aagacaggct tgcgagatat gtttgagaat accactttat cccgcgtcag ggagaggcag
      421 tgcgtaaaaa gacgcggact catgtgaaat actggttttt agtgcgccag atctctataa
      481 tctcgcgcaa cctattttcc cctcgaacac tttttaagcc gtagataaac aggctgggac
      541 acttcacatg agcgaaaaat acatcgtcac ctgggacatg ttgcagatcc atgcacgtaa
      601 actcgcaagc cgactgatgc cttctgaaca atggaaaggc attattgccg taagccgtgg
      661 cggtctgtac cgggtgcgtt actggcgcgt gaactgggta ttcgtcatgt cgataccgtt
      721 tgtatttcca gctacgatca cgacaaccag cgcgagctta aagtgctgaa acgcgcagaa
      781 ggcgatggcg aaggcttcat cgttattgat gacctggtgg ataccggtgg tactgcggtt
      841 gcgattcgtg aaatgtatcc aaaagcgcac tttgtcacca tcttcgcaaa accggctggt
      901 cgtccgctgg ttgatgacta tgttgttgat atcccgcaag atacctggat tgaacagccg
      961 tgggatatgg gcgtcgtatt cgtcccgcca atctccggtc gctaatcttt tcaacgcctg
     1021 gcactgccgg gcgttgttct ttttaacttc aggcgggtta caatagtttc cagtaagtat
     1081 tctggaggct gcatccatga cacaggcaaa cctgagcgaa accctgttca aaccccgctt
     1141 taaacatcct gaaacctcga cgctagtccg ccgctttaat cacggcgcac aaccgcctgt
     1201 gcagtcggcc cttgatggta aaaccatccc tcactggtat cgcatgatta accgtctgat
     1261 gtggatctgg cgcggcattg acccacgcga aatcctcgac gtccaggcac gtattgtgat
     1321 gagcgatgcc gaacgtaccg acgatgattt atacgatacg gtgattggct accgtggcgg
     1381 caactggatt tatgagtggg ccccggatct ttgtgaagga accttacttc tgtggtgtga
     1441 cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa
     1501 gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg
     1561 gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag
     1621 aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa
     1681 aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga
     1741 gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa
     1801 aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc
     1861 ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg
     1921 ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta
     1981 ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt
     2041 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2101 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2161 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2221 tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa
     2281 aatcatccca aacttcccac cccataccct attaccactg ccaattacct gtggtttcat
     2341 ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa
     2401 atatgtacta caaacttagt agttggaagg gctaattcac tcccaaagaa gacaagatat
     2461 ccttgatctg tggatctacc acacacaagg ctacttccct gattagcaga actacacacc
     2521 agggccaggg gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     2581 gccagataag gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag
     2641 cctgcatggg atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct
     2701 agcatttcat cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg
     2761 agcttgctac aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac
     2821 tggggagtgg cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt
     2881 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     2941 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     3001 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
     3061 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
     3121 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
     3181 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
     3241 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
     3301 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     3361 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     3421 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     3481 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     3541 caaaagtaag accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga
     3601 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     3661 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     3721 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     3781 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     3841 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     3901 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     3961 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     4021 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     4081 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     4141 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     4201 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     4261 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     4321 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     4381 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga
     4441 cggtatcgcc aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     4501 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     4561 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     4621 gtttggatcg ataagcttga tatcgaattc gggaggtggt ccctgcagtt acgccaatga
     4681 taacccccgc cagaaaaatc ttagtagcct tccctttttg ttttccgtgc cccaactcgg
     4741 cggattgact cggccccttc cggaaacacc cgaatcaact tctagtcaaa ttattgttca
     4801 cgccgcaatg acccacccct ggcccgcgtc tgtggaactg acccctggtg tacaggagag
     4861 ttcgctgctg aaagtggtcc caaaggggta ctagttttta agctcccaac tccccctccc
     4921 ccagcgtctg gaggattcca caccctcgca ccgcaggggc gaggaagtgg gcggagtccg
     4981 gttttggcgc cagccgctga ggctgccaag cagaaaagcc accgctgagg agactccggt
     5041 cactgtcctc gccccgcctc ccccttccct ccccttgggg accaccgggc gccacgccgc
     5101 gaacggtaag tgccgcggtc gtcggcgcct ccgccctccc cctagggccc caattcccag
     5161 cgggcgcggc gcgcggcccc tccccccgcc gggcgcgcgc ccgctgcccc gcccttcgtg
     5221 gccgcccggc gtgggcggtg ccacccctcc ccccggcggc cccgcgcgca gctcccggct
     5281 ccctccccct tcggatgtgg cttgagctgt aggcgcggag gggctccggt gcccgtcagt
     5341 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     5401 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     5461 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     5521 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     5581 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     5641 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     5701 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     5761 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     5821 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     5881 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     5941 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     6001 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     6061 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     6121 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     6181 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     6241 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     6301 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     6361 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     6421 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     6481 ttccatttca ggtgtcgtgg aattctcacg cgtctgcagg atatcaagct tgcggtaccg
     6541 cgggcccggc caccatgaac aattcccagg gaagagtgac cttcgaggat gtcactgtga
     6601 acttcaccca gggggagtgg cagcggctga atcccgaaca gagaaacttg tacagggatg
     6661 tgatgctgga gaattacagc aaccttgtct ctgtgggaca aggggaaacc accaaacccg
     6721 atgtgatctt gaggttggaa caaggaaagg agccatggtt ggaggaagag gaagtgctgg
     6781 gaagtggccg tgcagaaaaa aatggggaca ttggagggca gatttggaag ccaaaggatg
     6841 tgaaagagag tctctaccca actttcttgt acaaagtggt tggtggatct ggaggttctc
     6901 ccaagaagaa gcgcaaggtg ggacgcgtct gcaggatatc aagcttgcgg taccgcgggc
     6961 ccgggatcgc caccatggac aagaagtaca gcatcggcct ggccatcggc accaactctg
     7021 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     7081 gcaacaccga ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg
     7141 gagaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     7201 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     7261 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     7321 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     7381 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     7441 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     7501 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     7561 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     7621 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     7681 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     7741 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     7801 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     7861 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     7921 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     7981 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     8041 agaacggcta cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca
     8101 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     8161 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     8221 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     8281 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     8341 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     8401 ggaacttcga ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga
     8461 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     8521 agtacttcac cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     8581 agcccgcctt cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca
     8641 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     8701 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     8761 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     8821 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     8881 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     8941 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     9001 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     9061 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     9121 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     9181 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     9241 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     9301 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     9361 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     9421 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     9481 acgatgtgga cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag
     9541 tgctgactcg gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     9601 tgaagaagat gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga
     9661 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     9721 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     9781 actcccggat gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga
     9841 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     9901 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     9961 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
    10021 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
    10081 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
    10141 agatccggaa gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata
    10201 agggccggga ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga
    10261 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
    10321 gcgacaagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
    10381 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
    10441 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
    10501 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
    10561 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
    10621 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
    10681 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
    10741 agctgtttgt ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt
    10801 tctccaagag agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca
    10861 agcacagaga caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga
    10921 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
    10981 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
    11041 acgagacacg gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg
    11101 attatgccag cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga
    11161 aaaagcggaa agtgagcggc cgcgctacta acttcagcct gctgaagcag gctggagacg
    11221 tggaggagaa ccctggacct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
    11281 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtct ggcgagggcg
    11341 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
    11401 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
    11461 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
    11521 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
    11581 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
    11641 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
    11701 ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg
    11761 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
    11821 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
    11881 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
    11941 acgagctgta caagtaataa gcgatcgccc tgcaggcatg caagcttgat atcaagctta
    12001 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
    12061 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
    12121 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
    12181 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
    12241 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
    12301 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
    12361 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
    12421 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
    12481 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
    12541 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
    12601 accgtcgacc tcgagggaat taattcgagc tcggtacctt taagaccaat gacttacaag
    12661 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
    12721 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
    12781 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
    12841 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
    12901 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tctagaatta attccgtgta
    12961 ttctatagtg tcacctaaat cgtatgtgta tgatacataa ggttatgtat taattgtagc
    13021 cgcgttctaa cgacaatatg tacaagccta attgtgtagc atctggctta ctgaagcaga
    13081 ccctatcatc tctctcgtaa actgccgtca gagtcggttt ggttggacga accttctgag
    13141 tttctggtaa cgccgtcccg cacccggaaa tggtcagcga accaatcagc agggtcatcg
    13201 ctagccagat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
    13261 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
    13321 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
    13381 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
    13441 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaatg gtgcactctc
    13501 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
    13561 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
    13621 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
    13681 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
    13741 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    13801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    13861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    13921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    13981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    14041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    14101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    14161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    14221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    14281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    14341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    14401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    14461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    14521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    14581 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    14641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    14701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    14761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    14821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    14881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    14941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    15001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    15061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    15121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    15181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    15241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    15301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    15361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    15421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    15481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    15541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    15601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    15661 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattca
//
