LOCUS       sequence-331043-       14537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express dCas9-VPR in a doxycyclin-inducable system and 3 sgRNAs 
            targeting the murine Cnga1 promoter. Can be stably integrated into 
            the genome via the PiggyBac Transposon system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14537)
  AUTHORS   Bohm S, Splith V, Riedmayr LM, Rotzer RD, Gasparoni G, Nordstrom 
            KJV, Wagner JE, Hinrichsmeyer KS, Walter J, Wahl-Schott C, Fenske S,
            Biel M, Michalakis S, Becirovic E
  TITLE     A gene therapy for inherited blindness using dCas9-VPR-mediated 
            transcriptional activation.
  JOURNAL   Sci Adv. 2020 Aug 19;6(34):eaba5614. doi: 10.1126/sciadv.aba5614. 
            eCollection 2020 Aug.
  PUBMED    32875106
REFERENCE   2  (bases 1 to 14537)
  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: "Sci Adv."; 
            date: "2020-08-19"; volume: "6(34):eaba5614. doi"; pages: " 
            10.1126/sciadv.aba5614. eCollection 2020 Aug"
COMMENT     Sequence Label: 
            PB-TRE-dCas9-VPR-pA-3xsgRNA-EF1a-Tet.O-T2A-PuroR-polyA
FEATURES             Location/Qualifiers
     source          1..14537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1393..1411
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1429..1447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1465..1483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1501..1519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1537..1555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1573..1591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1636..1660
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1765..1774
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1771..1797,1798..1800,1801..4284,4285..4290,4291..4356,
                     4357..4359,4360..5874)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5887..5907
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5944..5968
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5998..6147
                     /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             6172..6192
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6622..6978
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             6997..7566
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     protein_bind    complement(7571..7595)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7728..7747)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7772..7820
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8008..8256
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8278..8353
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8367..8615
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8637..8712
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8726..8974
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8996..9071
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        9146..9357
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             9370..9638
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             9672..10415
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             10416..10469
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             10470..11069
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(10470..10489)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     10966..10986
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11108..11229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11145..11164)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11199..11218
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11991..12025
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12516..12532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12516..12532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12529..12551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12540..12556
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(12564..12570,12571..12588,12589..12594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12609..12630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12747..12764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12918..13506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12998..13017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13677..14468,14469..14537))
                     /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     14300..14319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtctttcgtc ttcaagaatt
     1381 cctcgagttt actccctatc agtgatagag aacgtatgaa gagtttactc cctatcagtg
     1441 atagagaacg tataaggagt ttactcccta tcagtgatag agaacgtatg accagtttac
     1501 tccctatcag tgatagagaa cgtatctaca gtttactccc tatcagtgat agagaacgta
     1561 tatccagttt actccctatc agtgatagag aacgtataag ctttaggcgt gtacggtggg
     1621 cgcctataaa agcagagctc gtttagtgaa ccgtcagatc gcctggagca attccacaac
     1681 acttttgtct tataccaact ttccgtacca cttcctaccc tcgtaaaggt ctagagctag
     1741 cgaattcgaa tttaaatcta cgtagccacc atggacaaga agtactccat tgggctcgct
     1801 atcggcacaa acagcgtcgg ctgggccgtc attacggacg agtacaaggt gccgagcaaa
     1861 aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc
     1921 ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc
     1981 agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg
     2041 gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat
     2101 aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa
     2161 aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac
     2221 ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc
     2281 gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag
     2341 acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca
     2401 atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct
     2461 ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc
     2521 aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc
     2581 tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt
     2641 ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg
     2701 gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa
     2761 gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt
     2821 ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag
     2881 gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg
     2941 gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc
     3001 ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac
     3061 ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac
     3121 tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa
     3181 gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc
     3241 ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa
     3301 cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc
     3361 acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac
     3421 ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa
     3481 aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc
     3541 ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag
     3601 gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag
     3661 atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag
     3721 ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc
     3781 cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac
     3841 cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa
     3901 gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct tgcaggtagc
     3961 ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta
     4021 atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc
     4081 cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa
     4141 ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag
     4201 ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc
     4261 aatcggctct ccgactacga cgtggctgct atcgtgcccc agtcttttct caaagatgat
     4321 tctattgata ataaagtgtt gacaagatcc gataaagcta gagggaagag tgataacgtc
     4381 ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa
     4441 ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag
     4501 ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac
     4561 gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt
     4621 cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag aaaggacttt
     4681 cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat
     4741 gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac
     4801 ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc
     4861 aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt
     4921 acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga
     4981 gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg
     5041 caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc
     5101 ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa
     5161 tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag
     5221 aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag
     5281 cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc
     5341 aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg
     5401 aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct
     5461 aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg gtctcccgaa
     5521 gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc
     5581 gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg
     5641 ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc
     5701 cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc
     5761 atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag
     5821 tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg
     5881 gctgacccca agaagaagag gaaggtgtcg ccagggatcc gtcgacttga cgcgttgata
     5941 tcaacaagtt tgtacaaaaa agcaggctac aaagaggcca gcggttccgg acgggctgac
     6001 gcattggacg attttgatct ggatatgctg ggaagtgacg ccctcgatga ttttgacctt
     6061 gacatgcttg gttcggatgc ccttgatgac tttgacctcg acatgctcgg cagtgacgcc
     6121 cttgatgatt tcgacctgga catgctgatt aactctagaa gttccggatc tccgaaaaag
     6181 aaacgcaaag ttggtagcca gtacctgccc gacaccgacg accggcaccg gatcgaggaa
     6241 aagcggaagc ggacctacga gacattcaag agcatcatga agaagtcccc cttcagcggc
     6301 cccaccgacc ctagacctcc acctagaaga atcgccgtgc ccagcagatc cagcgccagc
     6361 gtgccaaaac ctgcccccca gccttacccc ttcaccagca gcctgagcac catcaactac
     6421 gacgagttcc ctaccatggt gttccccagc ggccagatct ctcaggcctc tgctctggct
     6481 ccagcccctc ctcaggtgct gcctcaggct cctgctcctg caccagctcc agccatggtg
     6541 tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc tccacaggct
     6601 gtggctccac cagcccctaa acctacacag gccggcgagg gcacactgtc tgaagctctg
     6661 ctgcagctgc agttcgacga cgaggatctg ggagccctgc tgggaaacag caccgatcct
     6721 gccgtgttca ccgacctggc cagcgtggac aacagcgagt tccagcagct gctgaaccag
     6781 ggcatccctg tggcccctca caccaccgag cccatgctga tggaataccc cgaggccatc
     6841 acccggctcg tgacaggcgc tcagaggcct cctgatccag ctcctgcccc tctgggagca
     6901 ccaggcctgc ctaatggact gctgtctggc gacgaggact tcagctctat cgccgatatg
     6961 gatttctcag ccttgctggg ctctggcagc ggcagccggg attccaggga agggatgttt
     7021 ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt ttgagggccg cgaggtgtgc
     7081 cagccaaaac gaatccggcc atttcatcct ccaggaagtc catgggccaa ccgcccactc
     7141 cccgccagcc tcgcaccaac accaaccggt ccagtacatg agccagtcgg gtcactgacc
     7201 ccggcaccag tccctcagcc actggatcca gcgcccgcag tgactcccga ggccagtcac
     7261 ctgttggagg atcccgatga agagacgagc caggctgtca aagcccttcg ggagatggcc
     7321 gatactgtga ttccccagaa ggaagaggct gcaatctgtg gccaaatgga cctttcccat
     7381 ccgcccccaa ggggccatct ggatgagctg acaaccacac ttgagtccat gaccgaggat
     7441 ctgaacctgg actcacccct gaccccggaa ttgaacgaga ttctggatac cttcctgaac
     7501 gacgagtgcc tcttgcatgc catgcatatc agcacaggac tgtccatctt cgacacatct
     7561 ctgttttgaa acccagcttt cttgtacaaa gtggttgata tcatctagag cggccgcagg
     7621 tacctgaata actaaggccg cttcccttta gtgagggtta atgcttcgag cagacaaagg
     7681 gttcgatccc taccggttag taatgagttt aaacggggga ggctaactga aacacggaag
     7741 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     7801 gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     7861 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     7921 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     7981 catagcctag gggcgcgccg tttaaacgag ggcctatttc ccatgattcc ttcatatttg
     8041 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     8101 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     8161 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     8221 ttggctttat atatcttgtg gaaaggacga aacaccgtag gcgaccggct ttgagaagtt
     8281 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     8341 accgagtcgg tgcttttttt gtatacgagg gcctatttcc catgattcct tcatatttgc
     8401 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     8461 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     8521 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     8581 tggctttata tatcttgtgg aaaggacgaa acaccgctgt ggaagtctcc aaacgcgttt
     8641 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     8701 ccgagtcggt gctttttttg ttaacgaggg cctatttccc atgattcctt catatttgca
     8761 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     8821 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     8881 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     8941 ggctttatat atcttgtgga aaggacgaaa caccgtcttc tctctcggcg ctatggtttt
     9001 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     9061 cgagtcggtg ctttttttat ttaaatagct agcttaatta aagctgaatc taagtggatc
     9121 tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     9181 ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
     9241 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     9301 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagctg
     9361 aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
     9421 ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc
     9481 gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
     9541 agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc tcaactctac
     9601 gtctttgttt cgttttctgt tctgcgccgt tacagatcta agctgtgacc ggcgcctact
     9661 ctagagccgc catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac
     9721 tcaatggagt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg
     9781 agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa
     9841 tcgagatgct ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag
     9901 actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg
     9961 ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc
    10021 tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg
    10081 tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg
    10141 aaagagagac acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt
    10201 tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc
    10261 tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact
    10321 tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg
    10381 ctcttgacga ttttgacctt gacatgctcc ccggggaggg cagaggaagt cttctaacat
    10441 gcggtgacgt ggaggagaat cccggcccta tgaccgagta caagcccacg gtgcgcctcg
    10501 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
    10561 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
    10621 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
    10681 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
    10741 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
    10801 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
    10861 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
    10921 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
    10981 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
    11041 ggtgcatgac ccgcaagccc ggtgcctgag tcgacaatca acctctggat tacaaaattt
    11101 gtgggttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
    11161 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
    11221 tgtatcttat catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctatc
    11281 tggtctccct tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa
    11341 actgggttct tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag
    11401 gaacgaagtc cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc
    11461 ttttttaggg cccattggta tggctttttc cccgtatccc cccaggtgtc tgcaggctca
    11521 aagagcagcg agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc
    11581 tgtccccgca cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc
    11641 gggcggctcg ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg
    11701 ggggggggct gtccctgata tctataacaa gaaaatatat atataataag ttatcacgta
    11761 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
    11821 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
    11881 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
    11941 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
    12001 ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata tcgtcgggtc
    12061 ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg aggaagaagc
    12121 ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg atgactgctg
    12181 ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag gtgtggccat
    12241 gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt cgtcgcggct
    12301 tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga acaataccgg
    12361 cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg cgctgggata
    12421 ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat ggataccccg
    12481 tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
    12541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
    12601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
    12661 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
    12721 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
    12781 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
    12841 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
    12901 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
    12961 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
    13021 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
    13081 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
    13141 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
    13201 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
    13261 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
    13321 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
    13381 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
    13441 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
    13501 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
    13561 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
    13621 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
    13681 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
    13741 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
    13801 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
    13861 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
    13921 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
    13981 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
    14041 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
    14101 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
    14161 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
    14221 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
    14281 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
    14341 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
    14401 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
    14461 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
    14521 aaatgttgaa tactcat
//
