LOCUS       sequence-476350-       15803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express VPH-dCas9-P2A-mCherry in mammalian cells. Lentiviral.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15803)
  AUTHORS   Southard KM, Ardy RC, Tang A, O'Sullivan DD, Metzner E, 
            Guruvayurappan K, Norman TM
  TITLE     Comprehensive transcription factor perturbations recapitulate 
            fibroblast transcriptional states.
  JOURNAL   Nat Genet. 2025 Aug 6. doi: 10.1038/s41588-025-02284-1.
  PUBMED    40770575
REFERENCE   2  (bases 1 to 15803)
  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: "Nat Genet. 
            2025 Aug 6. doi: 10.1038/s41588-025-02284-1."
COMMENT     Sequence Label: pRCA422 - pHR-UCOE-SFFV-VPH-dcas9-p2a-mCherry
FEATURES             Location/Qualifiers
     source          1..15803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          1221..1286
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1416..1436
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1861..1995
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1898..1917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1952..1971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2163..2796
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    2843..2968
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3465..3698
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3883..3927
                     /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    4212..4329
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5071..5478
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      5553..5562
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5589..5738
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5745..6287
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             6312..6683
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     protein_bind    complement(6691..6715)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6735..6755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      6804..6813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6810..6836,6837..6839,6840..9326,9327..9329,
                     9330..10913)
                     /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             6810..10913
                     /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             10917..10943
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10923..10949
                     /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             10962..10982
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10989..11009
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11019..11075
                     /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             11076..11783
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(11218..11236)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     11486..11505
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    11829..12417
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11882..11902)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12300..12311)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12420..12436)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12421..12437)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12527..12760
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(12785..12803)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(12786..12803)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(13093..13112)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(13326..13345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13445..13467
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13505..13523)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13591..13695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13696..13764,13765..14556)
                     /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(13914..13933)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14727..15315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15216..15235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     15469..15486
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(15556..15576)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(15561..15803,1..87)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(15741..15803,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag cttggacaca agacaggctt gcgagatatg
      121 tttgagaata ccactttatc ccgcgtcagg gagaggcagt gcgtaaaaag acgcggactc
      181 atgtgaaata ctggttttta gtgcgccaga tctctataat ctcgcgcaac ctattttccc
      241 ctcgaacact ttttaagccg tagataaaca ggctgggaca cttcacatga gcgaaaaata
      301 catcgtcacc tgggacatgt tgcagatcca tgcacgtaaa ctcgcaagcc gactgatgcc
      361 ttctgaacaa tggaaaggca ttattgccgt aagccgtggc ggtctgtacc gggtgcgtta
      421 ctggcgcgtg aactgggtat tcgtcatgtc gataccgttt gtatttccag ctacgatcac
      481 gacaaccagc gcgagcttaa agtgctgaaa cgcgcagaag gcgatggcga aggcttcatc
      541 gttattgatg acctggtgga taccggtggt actgcggttg cgattcgtga aatgtatcca
      601 aaagcgcact ttgtcaccat cttcgcaaaa ccggctggtc gtccgctggt tgatgactat
      661 gttgttgata tcccgcaaga tacctggatt gaacagccgt gggatatggg cgtcgtattc
      721 gtcccgccaa tctccggtcg ctaatctttt caacgcctgg cactgccggg cgttgttctt
      781 tttaacttca ggcgggttac aatagtttcc agtaagtatt ctggaggctg catccatgac
      841 acaggcaaac ctgagcgaaa ccctgttcaa accccgcttt aaacatcctg aaacctcgac
      901 gctagtccgc cgctttaatc acggcgcaca accgcctgtg cagtcggccc ttgatggtaa
      961 aaccatccct cactggtatc gcatgattaa ccgtctgatg tggatctggc gcggcattga
     1021 cccacgcgaa atcctcgacg tccaggcacg tattgtgatg agcgatgccg aacgtaccga
     1081 cgatgattta tacgatacgg tgattggcta ccgtggcggc aactggattt atgagtgggc
     1141 cccggatctt tgtgaaggaa ccttacttct gtggtgtgac ataattggac aaactaccta
     1201 cagagattta aagctctaag gtaaatataa aatttttaag tgtataatgt gttaaactac
     1261 tgattctaat tgtttgtgta ttttagattc caacctatgg aactgatgaa tgggagcagt
     1321 ggtggaatgc ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg
     1381 atgaggctac tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag
     1441 accccaagga ctttccttca gaattgctaa gttttttgag tcatgctgtg tttagtaata
     1501 gaactcttgc ttgctttgct atttacacca caaaggaaaa agctgcactg ctatacaaga
     1561 aaattatgga aaaatattct gtaaccttta taagtaggca taacagttat aatcataaca
     1621 tactgttttt tcttactcca cacaggcata gagtgtctgc tattaataac tatgctcaaa
     1681 aattgtgtac ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata
     1741 gtgccttgac tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1801 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     1861 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1921 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1981 tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc
     2041 ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc
     2101 tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta
     2161 gttggaaggg ctaattcact cccaaagaag acaagatatc cttgatctgt ggatctacca
     2221 cacacaaggc tacttccctg attagcagaa ctacacacca gggccagggg tcagatatcc
     2281 actgaccttt ggatggtgct acaagctagt accagttgag ccagataagg tagaagaggc
     2341 caataaagga gagaacacca gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     2401 ggagagagaa gtgttagagt ggaggtttga cagccgccta gcatttcatc acgtggcccg
     2461 agagctgcat ccggagtact tcaagaactg ctgatatcga gcttgctaca agggactttc
     2521 cgctggggac tttccaggga ggcgtggcct gggcgggact ggggagtggc gagccctcag
     2581 atcctgcata taagcagctg ctttttgcct gtactgggtc tctctggtta gaccagatct
     2641 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     2701 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     2761 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
     2821 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
     2881 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
     2941 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
     3001 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
     3061 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
     3121 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
     3181 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
     3241 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
     3301 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
     3361 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
     3421 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     3481 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     3541 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     3601 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     3661 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     3721 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     3781 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     3841 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     3901 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     3961 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     4021 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     4081 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     4141 gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcca aatggcagta
     4201 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     4261 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     4321 caaaattttc gggtttatta cagggacagc agagatccag tttggatcga taagcttgat
     4381 atcgaattcg ggaggtggtc cctgcagtta cgccaatgat accccccgcc agaaaaatct
     4441 tagtagcctt ccctttttgt tttccgtgcc ccaactcggc ggattgactc ggccccttcc
     4501 ggaaacaccc gaatcaactt ctagtcaaat tattgttcac gccgcaatga cccacccctg
     4561 gcccgcgtct gtggaactga cccctggtgt acaggagagt tcgctgctga aagtggtccc
     4621 aaaggggtac tagtttttaa gctcccaact ccccctcccc cagcgtctgg aggattccac
     4681 accctcgcac cgcaggggcg aggaagtggg cggagtccgg ttttggcgcc agccgctgag
     4741 gctgccaagc agaaaagcca ccgctgagga gactccggtc actgtcctcg ccccgcctcc
     4801 cccttccctc cccttgggga ccaccgggcg ccacgccgcg aacggtaagt gccgcggtcg
     4861 tcggcgcctc cgccctcccc ctagggcccc aattcccagc gggcgcggcg cgcggccccg
     4921 cgcgcagctc ccggctccct cccccttcgg atgtggcttg agctgtaggc gcggagtcct
     4981 gcagccccga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     5041 cccacctgta ggtttggcaa gctagctgca gtaacgccat tttgcaaggc atggaaaaat
     5101 accaaaccaa gaatagagaa gttcagatca agggcgggta catgaaaata gctaacgttg
     5161 ggccaaacag gatatctgcg gtgagcagtt tcggccccgg cccggggcca agaacagatg
     5221 gtcaccgcag tttcggcccc ggcccgaggc caagaacaga tggtccccag atatggccca
     5281 accctcagca gtttcttaag acccatcaga tgtttccagg ctcccccaag gacctgaaat
     5341 gaccctgcgc cttatttgaa ttaaccaatc agcctgcttc tcgcttctgt tcgcgcgctt
     5401 ctgcttcccg agctctataa aagagctcac aacccctcac tcggcgcgcc agtcctccga
     5461 cagactgagt cgcccggggg ggatctggag ctctcgagaa ttctcacgcg tctgcaggat
     5521 atcaagcttg cggtaccgcg ggcccgcata tggccaccat gggttccgga ggaggaggta
     5581 gcggtagtga tgctttagac gattttgact tagatatgct tggttcagac gcgttagacg
     5641 acttcgacct agacatgtta ggctcagatg cattggacga cttcgattta gatatgttgg
     5701 gctccgatgc cctagatgac tttgatttag atatgctagg tagtccttca gggcagatca
     5761 gcaaccaggc cctggctctg gcccctagct ccgctccagt gctggcccag actatggtgc
     5821 cctctagtgc tatggtgcct ctggcccagc cacctgctcc agcccctgtg ctgaccccag
     5881 gaccacccca gtcactgagc gctccagtgc ccaagtctac acaggccggc gaggggactc
     5941 tgagtgaagc tctgctgcac ctgcagttcg acgctgatga ggacctggga gctctgctgg
     6001 ggaacagcac cgatcccgga gtgttcacag atctggcctc cgtggacaac tctgagtttc
     6061 agcagctgct gaatcagggc gtgtccatgt ctcatagtac agccgaacca atgctgatgg
     6121 agtaccccga agccattacc cggctggtga ccggcagcca gcggcccccc gaccccgctc
     6181 caactcccct gggaaccagc ggcctgccta atgggctgtc cggagatgaa gacttctcaa
     6241 gcatcgctga tatggacttt agtgccctgc tgtcacagat ttcctctagt gggcagggag
     6301 gaggtggaag cggcttcagc gtggacacca gtgccctgct ggacctgttc agcccctcgg
     6361 tgaccgtgcc cgacatgagc ctgcctgacc ttgacagcag cctggccagt atccaagagc
     6421 tcctgtctcc ccaggagccc cccaggcctc ccgaggcaga gaacagcagc ccggattcag
     6481 ggaagcagct ggtgcactac acagcgcagc cgctgttcct gctggacccc ggctccgtgg
     6541 acaccgggag caacgacctg ccggtgctgt ttgagctggg agagggctcc tacttctccg
     6601 aaggggacgg cttcgccgag gaccccacca tctccctgct gacaggctcg gagcctccca
     6661 aagccaagga ccccactgtc tccggatcct acccaacttt cttgtacaaa gtggttggtg
     6721 gatctggagg ttctcccaag aagaagcgca aggtgggacg cgtctgcagg atatcaagct
     6781 tgcggtaccg cgggcccggg atcgccacca tggacaagaa gtacagcatc ggcctggcca
     6841 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     6901 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggcgccc
     6961 tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     7021 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     7081 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     7141 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     7201 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     7261 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     7321 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     7381 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     7441 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     7501 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     7561 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     7621 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     7681 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     7741 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     7801 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     7861 tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc agccaggaag
     7921 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     7981 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     8041 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     8101 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     8161 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     8221 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc gcccagagct
     8281 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     8341 acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg aaatacgtga
     8401 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc atcgtggacc
     8461 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     8521 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     8581 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     8641 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     8701 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     8761 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     8821 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     8881 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     8941 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     9001 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     9061 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     9121 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     9181 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     9241 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     9301 accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg aaggacgact
     9361 ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc gacaacgtgc
     9421 cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg aatgccaagc
     9481 tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     9541 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     9601 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac aaactgatcc
     9661 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     9721 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     9781 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     9841 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     9901 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     9961 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaacaggcg
    10021 agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg tctatgcccc
    10081 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
    10141 tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac cctaagaagt
    10201 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
    10261 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
    10321 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
    10381 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
    10441 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
    10501 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
    10561 ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac gagatcatcg
    10621 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaggtgc
    10681 tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag aatatcatcc
    10741 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
    10801 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
    10861 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacgcctatc
    10921 cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa aaacgcaagg
    10981 tggaagatcc taagaaaaag cggaaagtga gcggccgcgc tactaacttc agcctgctga
    11041 agcaggctgg agacgtggag gagaaccctg gacctgtgag caagggcgag gaggataaca
    11101 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
    11161 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
    11221 agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
    11281 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
    11341 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
    11401 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
    11461 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
    11521 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
    11581 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
    11641 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
    11701 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
    11761 gcatggacga gctgtacaag taagcgatcg ccctgcaggc atgcaagctt gatatcaagc
    11821 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
    11881 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
    11941 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
    12001 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
    12061 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
    12121 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
    12181 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
    12241 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
    12301 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
    12361 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
    12421 gataccgtcg acctcgaggg aattaattcg agctcggtac ctttaagacc aatgacttac
    12481 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
    12541 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
    12601 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    12661 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    12721 tccctcagac ccttttagtc agtgtggaaa atctctagca gcatctagaa ttaattccgt
    12781 gtattctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg tattaattgt
    12841 agccgcgttc taacgacaat atgtacaagc ctaattgtgt agcatctggc ttactgaagc
    12901 agaccctatc atctctctcg taaactgccg tcagagtcgg tttggttgga cgaaccttct
    12961 gagtttctgg taacgccgtc ccgcacccgg aaatggtcag cgaaccaatc agcagggtca
    13021 tcgctagcca gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
    13081 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
    13141 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
    13201 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
    13261 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga atggtgcact
    13321 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
    13381 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
    13441 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
    13501 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
    13561 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
    13621 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
    13681 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
    13741 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
    13801 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
    13861 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
    13921 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
    13981 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
    14041 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
    14101 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
    14161 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
    14221 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
    14281 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
    14341 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
    14401 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
    14461 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
    14521 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
    14581 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
    14641 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
    14701 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
    14761 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
    14821 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
    14881 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
    14941 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
    15001 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
    15061 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
    15121 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
    15181 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
    15241 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
    15301 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
    15361 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
    15421 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
    15481 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
    15541 cattaatgca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
    15601 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
    15661 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
    15721 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
    15781 cccatggctg actaattttt ttt
//
