LOCUS       sequence-374831-       14978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with an N-term Zim3 KRAB-NLS fusion, a C-term HA-2xNLS, and 
            GFP linked by a P2A site from a SFFV promoter with an upstream 
            ubiquitous chromatin opening element.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14978)
  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 14978)
  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-EGFP
FEATURES             Location/Qualifiers
     source          1..14978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(52..70)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(53..70)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(360..379)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(593..612)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     712..734
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(772..790)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        858..962
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(963..1031,1032..1823)
                     /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(1181..1200)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1994..2582
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2483..2502
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2736..2753
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2823..2843)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2828..3157
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3008..3143
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4291..4356
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4486..4506
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4931..5065
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4968..4987)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5022..5041
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5233..5866
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5913..6038
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6535..6768
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6953..6997
                     /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    7282..7399
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8141..8548
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(8924..8948)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8968..8988
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      9037..9046
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9043..9069,9070..9072,9073..11559,11560..11562,
                     11563..13146)
                     /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             9043..13146
                     /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             13150..13176
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             13156..13182
                     /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             13195..13215
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13222..13242
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13252..13308
                     /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(13309..13311,13312..14025)
                     /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(13612..13631)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     13959..13980
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    14074..14662
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14127..14147)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14545..14556)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14665..14681)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14666..14682)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(14772..14978,1..27)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 tttagtcagt gtggaaaatc tctagcagca tctagaatta attccgtgta ttctatagtg
       61 tcacctaaat cgtatgtgta tgatacataa ggttatgtat taattgtagc cgcgttctaa
      121 cgacaatatg tacaagccta attgtgtagc atctggctta ctgaagcaga ccctatcatc
      181 tctctcgtaa actgccgtca gagtcggttt ggttggacga accttctgag tttctggtaa
      241 cgccgtcccg cacccggaaa tggtcagcga accaatcagc agggtcatcg ctagccagat
      301 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
      361 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
      421 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
      481 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
      541 cttcctaatg caggagtcgc ataagggaga gcgtcgaatg gtgcactctc agtacaatct
      601 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
      661 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      721 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
      781 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
      841 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      901 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
      961 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1021 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1081 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1141 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1201 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1261 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1321 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1381 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1441 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     1501 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     1561 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     1621 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     1681 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     1741 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     1801 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     1861 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1921 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1981 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2041 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2101 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     2161 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2221 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2281 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2341 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2401 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2461 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2521 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2581 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2641 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2701 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2761 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     2821 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2881 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2941 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3001 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3061 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3121 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttggacaca agacaggctt
     3181 gcgagatatg tttgagaata ccactttatc ccgcgtcagg gagaggcagt gcgtaaaaag
     3241 acgcggactc atgtgaaata ctggttttta gtgcgccaga tctctataat ctcgcgcaac
     3301 ctattttccc ctcgaacact ttttaagccg tagataaaca ggctgggaca cttcacatga
     3361 gcgaaaaata catcgtcacc tgggacatgt tgcagatcca tgcacgtaaa ctcgcaagcc
     3421 gactgatgcc ttctgaacaa tggaaaggca ttattgccgt aagccgtggc ggtctgtacc
     3481 gggtgcgtta ctggcgcgtg aactgggtat tcgtcatgtc gataccgttt gtatttccag
     3541 ctacgatcac gacaaccagc gcgagcttaa agtgctgaaa cgcgcagaag gcgatggcga
     3601 aggcttcatc gttattgatg acctggtgga taccggtggt actgcggttg cgattcgtga
     3661 aatgtatcca aaagcgcact ttgtcaccat cttcgcaaaa ccggctggtc gtccgctggt
     3721 tgatgactat gttgttgata tcccgcaaga tacctggatt gaacagccgt gggatatggg
     3781 cgtcgtattc gtcccgccaa tctccggtcg ctaatctttt caacgcctgg cactgccggg
     3841 cgttgttctt tttaacttca ggcgggttac aatagtttcc agtaagtatt ctggaggctg
     3901 catccatgac acaggcaaac ctgagcgaaa ccctgttcaa accccgcttt aaacatcctg
     3961 aaacctcgac gctagtccgc cgctttaatc acggcgcaca accgcctgtg cagtcggccc
     4021 ttgatggtaa aaccatccct cactggtatc gcatgattaa ccgtctgatg tggatctggc
     4081 gcggcattga cccacgcgaa atcctcgacg tccaggcacg tattgtgatg agcgatgccg
     4141 aacgtaccga cgatgattta tacgatacgg tgattggcta ccgtggcggc aactggattt
     4201 atgagtgggc cccggatctt tgtgaaggaa ccttacttct gtggtgtgac ataattggac
     4261 aaactaccta cagagattta aagctctaag gtaaatataa aatttttaag tgtataatgt
     4321 gttaaactac tgattctaat tgtttgtgta ttttagattc caacctatgg aactgatgaa
     4381 tgggagcagt ggtggaatgc ctttaatgag gaaaacctgt tttgctcaga agaaatgcca
     4441 tctagtgatg atgaggctac tgctgactct caacattcta ctcctccaaa aaagaagaga
     4501 aaggtagaag accccaagga ctttccttca gaattgctaa gttttttgag tcatgctgtg
     4561 tttagtaata gaactcttgc ttgctttgct atttacacca caaaggaaaa agctgcactg
     4621 ctatacaaga aaattatgga aaaatattct gtaaccttta taagtaggca taacagttat
     4681 aatcataaca tactgttttt tcttactcca cacaggcata gagtgtctgc tattaataac
     4741 tatgctcaaa aattgtgtac ctttagcttt ttaatttgta aaggggttaa taaggaatat
     4801 ttgatgtata gtgccttgac tagagatcat aatcagccat accacatttg tagaggtttt
     4861 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     4921 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4981 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5041 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
     5101 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
     5161 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
     5221 aaacttagta gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt
     5281 ggatctacca cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg
     5341 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg
     5401 tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga
     5461 tggatgaccc ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc
     5521 acgtggcccg agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca
     5581 agggactttc cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc
     5641 gagccctcag atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta
     5701 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5761 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5821 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     5881 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     5941 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     6001 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     6061 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     6121 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     6181 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     6241 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     6301 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     6361 ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga
     6421 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     6481 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     6541 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     6601 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     6661 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     6721 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     6781 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     6841 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     6901 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     6961 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     7021 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     7081 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     7141 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     7201 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcca
     7261 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     7321 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     7381 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggatcga
     7441 taagcttgat atcgaattcg ggaggtggtc cctgcagtta cgccaatgat aacccccgcc
     7501 agaaaaatct tagtagcctt ccctttttgt tttccgtgcc ccaactcggc ggattgactc
     7561 ggccccttcc ggaaacaccc gaatcaactt ctagtcaaat tattgttcac gccgcaatga
     7621 cccacccctg gcccgcgtct gtggaactga cccctggtgt acaggagagt tcgctgctga
     7681 aagtggtccc aaaggggtac tagtttttaa gctcccaact ccccctcccc cagcgtctgg
     7741 aggattccac accctcgcac cgcaggggcg aggaagtggg cggagtccgg ttttggcgcc
     7801 agccgctgag gctgccaagc agaaaagcca ccgctgagga gactccggtc actgtcctcg
     7861 ccccgcctcc cccttccctc cccttgggga ccaccgggcg ccacgccgcg aacggtaagt
     7921 gccgcggtcg tcggcgcctc cgccctcccc ctagggcccc aattcccagc gggcgcggcg
     7981 cgcggccccg cgcgcagctc ccggctccct cccccttcgg atgtggcttg agctgtaggc
     8041 gcggagtcct gcagccccga taaaataaaa gattttattt agtctccaga aaaagggggg
     8101 aatgaaagac cccacctgta ggtttggcaa gctagctgca gtaacgccat tttgcaaggc
     8161 atggaaaaat accaaaccaa gaatagagaa gttcagatca agggcgggta catgaaaata
     8221 gctaacgttg ggccaaacag gatatctgcg gtgagcagtt tcggccccgg cccggggcca
     8281 agaacagatg gtcaccgcag tttcggcccc ggcccgaggc caagaacaga tggtccccag
     8341 atatggccca accctcagca gtttcttaag acccatcaga tgtttccagg ctcccccaag
     8401 gacctgaaat gaccctgcgc cttatttgaa ttaaccaatc agcctgcttc tcgcttctgt
     8461 tcgcgcgctt ctgcttcccg agctctataa aagagctcac aacccctcac tcggcgcgcc
     8521 agtcctccga cagactgagt cgcccggggg ggatctggag ctctcgagaa ttctcacgcg
     8581 tctgcaggat atcaagcttg cggtaccgcg ggcccggcca ccatgaacaa ttcccaggga
     8641 agagtgacct tcgaggatgt cactgtgaac ttcacccagg gggagtggca gcggctgaat
     8701 cccgaacaga gaaacttgta cagggatgtg atgctggaga attacagcaa ccttgtctct
     8761 gtgggacaag gggaaaccac caaacccgat gtgatcttga ggttggaaca aggaaaggag
     8821 ccatggttgg aggaagagga agtgctggga agtggccgtg cagaaaaaaa tggggacatt
     8881 ggagggcaga tttggaagcc aaaggatgtg aaagagagtc tctacccaac tttcttgtac
     8941 aaagtggttg gtggatctgg aggttctccc aagaagaagc gcaaggtggg acgcgtctgc
     9001 aggatatcaa gcttgcggta ccgcgggccc gggatcgcca ccatggacaa gaagtacagc
     9061 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     9121 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     9181 ctgatcggcg ccctgctgtt cgacagcgga gaaacagccg aggccacccg gctgaagaga
     9241 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     9301 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     9361 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     9421 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     9481 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     9541 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     9601 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     9661 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     9721 gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg
     9781 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     9841 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     9901 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     9961 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
    10021 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
    10081 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat cgatggcgga
    10141 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
    10201 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
    10261 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
    10321 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
    10381 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
    10441 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgcc
    10501 agcgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
    10561 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaaa
    10621 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaaa
    10681 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
    10741 gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
    10801 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
    10861 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
    10921 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
    10981 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
    11041 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
    11101 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
    11161 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
    11221 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
    11281 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
    11341 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
    11401 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
    11461 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
    11521 gaactggaca tcaaccggct gtccgactac gatgtggacg ctatcgtgcc tcagagcttt
    11581 ctgaaggacg actccatcga taacaaagtg ctgactcgga gcgacaagaa ccggggcaag
    11641 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcgccagctg
    11701 ctgaatgcca agctgattac ccagaggaag ttcgacaatc tgaccaaggc cgagagaggc
    11761 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
    11821 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaac
    11881 gacaaactga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
    11941 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
    12001 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
    12061 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
    12121 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
    12181 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
    12241 ggcgaaacag gcgagatcgt gtgggataag ggccgggact ttgccaccgt gcggaaagtg
    12301 ctgtctatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
    12361 aaagagtcta tcctgcccaa gaggaacagc gacaagctga tcgccagaaa gaaggactgg
    12421 gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg
    12481 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
    12541 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
    12601 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
    12661 gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg
    12721 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
    12781 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa acactacctg
    12841 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
    12901 ctggacaagg tgctgagcgc ctacaacaag cacagagaca agcctatcag agagcaggcc
    12961 gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac
    13021 tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc
    13081 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
    13141 ggcgacgcct atccctatga cgtgcccgat tatgccagcc tgggcagcgg ctcccccaag
    13201 aaaaaacgca aggtggaaga tcctaagaaa aagcggaaag tgagcggccg cgctactaac
    13261 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctgt gagcaagggc
    13321 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
    13381 cacaagttca gcgtgtctgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
    13441 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
    13501 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
    13561 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
    13621 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
    13681 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
    13741 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggcgaac
    13801 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
    13861 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
    13921 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
    13981 accgccgccg ggatcactct cggcatggac gagctgtaca agtaataagc gatcgccctg
    14041 caggcatgca agcttgatat caagcttatc gataatcaac ctctggatta caaaatttgt
    14101 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
    14161 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
    14221 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
    14281 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
    14341 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
    14401 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
    14461 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
    14521 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
    14581 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
    14641 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gagggaatta attcgagctc
    14701 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
    14761 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
    14821 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
    14881 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
    14941 gttgtgtgac tctggtaact agagatccct cagaccct
//
