LOCUS       sequence-324058-       14555 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 14555)
  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 14555)
  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..14555
                     /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    1369..1387
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1405..1423
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1441..1459
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1477..1495
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1513..1531
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1549..1567
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1585..1603
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1648..1672
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1777..1786
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1783..1809,1810..1812,1813..4296,4297..4302,4303..4368,
                     4369..4371,4372..5886)
                     /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             5899..5919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5956..5980
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6010..6159
                     /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             6184..6204
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6634..6990
                     /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             7009..7578
                     /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(7583..7607)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7740..7759)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7784..7832
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8020..8268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8290..8365
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8379..8627
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8649..8724
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8738..8986
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        9008..9083
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        9158..9369
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             9382..9650
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             9684..10427
                     /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             10428..10481
                     /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             10488..11087
                     /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(10488..10507)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     10984..11004
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    11126..11247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11163..11182)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11217..11236
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   12009..12043
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12534..12550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12534..12550)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12547..12569)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12558..12574
                     /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(12582..12588,12589..12606,12607..12612))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12627..12648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12765..12782)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12936..13524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13016..13035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13695..14486,14487..14555))
                     /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     14318..14337
                     /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 ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca
     1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     1321 acacctgggg ggatacgggg aaaaggcctc cacggccact agttttactc cctatcagtg
     1381 atagagaacg tatgaagagt ttactcccta tcagtgatag agaacgtatg cagactttac
     1441 tccctatcag tgatagagaa cgtataagga gtttactccc tatcagtgat agagaacgta
     1501 tgaccagttt actccctatc agtgatagag aacgtatcta cagtttactc cctatcagtg
     1561 atagagaacg tatatccagt ttactcccta tcagtgatag agaacgtata agctttaggc
     1621 gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     1681 caattccaca acacttttgt cttataccaa ctttccgtac cacttcctac cctcgtaaag
     1741 gtctagagct agcgaattcg aatttaaatc tacgtagcca ccatggacaa gaagtactcc
     1801 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag
     1861 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac
     1921 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga
     1981 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt
     2041 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg
     2101 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg
     2161 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact
     2221 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga
     2281 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc
     2341 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt
     2401 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc
     2461 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc
     2521 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg
     2581 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac
     2641 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg
     2701 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat
     2761 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag
     2821 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga
     2881 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc
     2941 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac
     3001 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa
     3061 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt
     3121 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact
     3181 cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc
     3241 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag
     3301 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag
     3361 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa
     3421 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa
     3481 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc
     3541 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc
     3601 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt
     3661 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa
     3721 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg
     3781 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat
     3841 ggatttgcca accggaactt catgcagttg atccatgatg actctctcac ctttaaggag
     3901 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat
     3961 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa
     4021 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag
     4081 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag
     4141 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt
     4201 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag
     4261 gaactggaca tcaatcggct ctccgactac gacgtggctg ctatcgtgcc ccagtctttt
     4321 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaagc tagagggaag
     4381 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg
     4441 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt
     4501 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag
     4561 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat
     4621 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc
     4681 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat
     4741 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct
     4801 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag
     4861 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc
     4921 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac
     4981 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc
     5041 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc
     5101 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg
     5161 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg
     5221 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc
     5281 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga
     5341 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt
     5401 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg
     5461 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa
     5521 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt
     5581 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac
     5641 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca
     5701 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac
     5761 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca
     5821 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt
     5881 ggagacagca gggctgaccc caagaagaag aggaaggtgt cgccagggat ccgtcgactt
     5941 gacgcgttga tatcaacaag tttgtacaaa aaagcaggct acaaagaggc cagcggttcc
     6001 ggacgggctg acgcattgga cgattttgat ctggatatgc tgggaagtga cgccctcgat
     6061 gattttgacc ttgacatgct tggttcggat gcccttgatg actttgacct cgacatgctc
     6121 ggcagtgacg cccttgatga tttcgacctg gacatgctga ttaactctag aagttccgga
     6181 tctccgaaaa agaaacgcaa agttggtagc cagtacctgc ccgacaccga cgaccggcac
     6241 cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat gaagaagtcc
     6301 cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt gcccagcaga
     6361 tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag cagcctgagc
     6421 accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat ctctcaggcc
     6481 tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc tgcaccagct
     6541 ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct ggctcctgga
     6601 cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga gggcacactg
     6661 tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct gctgggaaac
     6721 agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga gttccagcag
     6781 ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct gatggaatac
     6841 cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc agctcctgcc
     6901 cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga cttcagctct
     6961 atcgccgata tggatttctc agccttgctg ggctctggca gcggcagccg ggattccagg
     7021 gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt gtttgagggc
     7081 cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag tccatgggcc
     7141 aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca tgagccagtc
     7201 gggtcactga ccccggcacc agtccctcag ccactggatc cagcgcccgc agtgactccc
     7261 gaggccagtc acctgttgga ggatcccgat gaagagacga gccaggctgt caaagccctt
     7321 cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg tggccaaatg
     7381 gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac acttgagtcc
     7441 atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga gattctggat
     7501 accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg actgtccatc
     7561 ttcgacacat ctctgttttg aaacccagct ttcttgtaca aagtggttga tatcatctag
     7621 agcggccgca ggtacctgaa taactaaggc cgcttccctt tagtgagggt taatgcttcg
     7681 agcagacaaa gggttcgatc cctaccggtt agtaatgagt ttaaacgggg gaggctaact
     7741 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     7801 aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     7861 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     7921 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     7981 ggcaggccct gccatagcct aggggcgcgc cgtttaaacg agggcctatt tcccatgatt
     8041 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     8101 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     8161 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     8221 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt aggcgaccgg
     8281 ctttgagaag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     8341 tgaaaaagtg gcaccgagtc ggtgcttttt ttgtatacga gggcctattt cccatgattc
     8401 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     8461 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     8521 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     8581 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgct gtggaagtct
     8641 ccaaacgcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     8701 gaaaaagtgg caccgagtcg gtgctttttt tgttaacgag ggcctatttc ccatgattcc
     8761 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     8821 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     8881 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     8941 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtct tctctctcgg
     9001 cgctatggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     9061 aaaaagtggc accgagtcgg tgcttttttt atttaaatag ctagcttaat taaagctgaa
     9121 tctaagtgga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     9181 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
     9241 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     9301 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     9361 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
     9421 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     9481 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     9541 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     9601 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     9661 ccggcgccta ctctagagcc gccatgtcta gactggacaa gagcaaagtc ataaactctg
     9721 ctctggaatt actcaatgga gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa
     9781 agctgggagt tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg
     9841 atgccctgcc aatcgagatg ctggacaggc atcataccca ctcctgcccc ctggaaggcg
     9901 agtcatggca agactttctg cggaacaacg ccaagtcata ccgctgtgct ctcctctcac
     9961 atcgcgacgg ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc
    10021 tggaaaatca gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg
    10081 ctctgtccgc cgtgggccac tttacactgg gctgcgtatt ggaggaacag gagcatcaag
    10141 tagcaaaaga ggaaagagag acacctacca ccgattctat gcccccactt ctgaaacaag
    10201 caattgagct gttcgaccgg cagggagccg aacctgcctt ccttttcggc ctggaactaa
    10261 tcatatgtgg cctggagaaa cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg
    10321 acgattttga cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc
    10381 tgcctgctga cgctcttgac gattttgacc ttgacatgct ccccggggag ggcagaggaa
    10441 gtcttctaac atgcggtgac gtggaggaga atcccggccc tgaattcatg accgagtaca
    10501 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
    10561 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
    10621 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
    10681 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
    10741 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
    10801 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
    10861 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
    10921 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
    10981 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
    11041 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc gacaatcaac
    11101 ctctggatta caaaatttgt gggttaactt gtttattgca gcttataatg gttacaaata
    11161 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
    11221 tttgtccaaa ctcatcaatg tatcttatca tgtctggaat tgactcaaat gatgtcaatt
    11281 agtctatcag aagctatctg gtctcccttc cgggggacaa gacatccctg tttaatattt
    11341 aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac caggttgcag
    11401 ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca ggtttagccc
    11461 cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc cgtatccccc
    11521 caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca
    11581 ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc
    11641 ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt
    11701 acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga aaatatatat
    11761 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
    11821 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
    11881 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
    11941 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
    12001 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct agctgcatca
    12061 ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc gctatctggg
    12121 catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt
    12181 tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat
    12241 tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg ccacctggga
    12301 taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga agcgcatcag
    12361 caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag
    12421 cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga tgccgcagaa
    12481 atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc atggtcatag
    12541 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
    12601 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
    12661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
    12721 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
    12781 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
    12841 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
    12901 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    12961 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    13021 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    13081 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    13141 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    13201 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    13261 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    13321 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
    13381 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    13441 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    13501 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    13561 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
    13621 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
    13681 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
    13741 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
    13801 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
    13861 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
    13921 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
    13981 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
    14041 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
    14101 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
    14161 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
    14221 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
    14281 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
    14341 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
    14401 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
    14461 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
    14521 ggaataaggg cgacacggaa atgttgaata ctcat
//
