LOCUS       sequence-417154-       16372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ngn2 mediated cortical neuron induction, along with SNCA(3K) 
            over-expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16372)
  AUTHORS   Lam I, Ndayisaba A, Lewis AJ, Fu Y, Sagredo GT, Zaccagnini L, Sandoe
            J, Sanz RL, Vahdatshoar A, Martin TD, Morshed N, Ichihashi T, 
            Tripathi A, Ramalingam N, Oettgen-Suazo C, Bartels T, Schabinger M, 
            Hallacli E, Jiang X, Verma A, Tea C, Wang Z, Hakozaki H, Yu X, Hyles
            K, Park C, Theunissen TW, Wang H, Jaenisch R, Lindquist S, Stevens 
            B, Stefanova N, Wenning G, Luk KC, Sanchez Pernaute R, Gomez-Esteban
            JC, Felsky D, Kiyota Y, Sahni N, Yi SS, Chung C, Stahlberg H, Ferrer
            I, Schoneberg J, Elledge SJ, Dettmer U, Halliday GM, Bartels T, 
            Khurana V
  TITLE     Rapid iPSC inclusionopathy models shed light on formation, 
            consequence and molecular subtype of alpha-synuclein inclusions
  JOURNAL   bioRxiv 2022.11.08.515615
REFERENCE   2  (bases 1 to 16372)
  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: "bioRxiv 
            2022.11.08.515615"
COMMENT     Sequence Label: piggyBac-rtTA
            (4th_Gen)-SNCA(3K)-sfGFP-IRES-NG...
FEATURES             Location/Qualifiers
     source          1..16372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..25)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    77..644
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    94..646
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(238..255)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     465..484
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             658..681
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1474..1527
                     /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             1534..2133
                     /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(1534..1553)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2030..2050
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2134..2722
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2187..2207)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2605..2616)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2737..3213
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2936..2955)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4972..5034
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5158..5206
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5231..5250
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5248..6378)
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN"
     promoter        complement(6385..6895)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7589..7609)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7589..7607)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7628..7644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7628..7644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7641..7663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7652..7668
                     /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(7676..7682,7683..7700,7701..7706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7721..7742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7859..7876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8030..8618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8110..8129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8789..9580,9581..9649))
                     /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     9412..9431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9650..9754)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9829..9848)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   10029..10063
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    10616..10840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     10829..10846
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(10869..11438)
                     /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"
     CDS             complement(11457..11813)
                     /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             complement(12243..12263)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(12288..12437)
                     /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             complement(13095..13148)
                     /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             complement(13158..13553)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(13563..14073)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     protein_bind    14785..14803
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14821..14839
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14895..14913
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14941..14959
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14988..15006
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    15024..15042
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    15163..15187
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      15190..15199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             15658..16371
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
ORIGIN
        1 acccagcttt cttgtacaaa gtggttgatc tgctggaaga tggcgattag agaggtacga
       61 agatggcgat tagagatccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
      121 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
      181 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
      241 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
      301 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
      361 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
      421 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
      481 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
      541 ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
      601 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaatggac
      661 tacaaggacg acgatgacaa gttcgtcaaa tctgagactc tggagttgaa ggaggaagag
      721 gaggtactga tgctgctggg ctcggcttcc ccggcctcgg cgaccctgac cccgatgtcc
      781 tccagcgcgg acgaggagga ggacgaggag ctgcgccggc cgggctccgc gcgtgggcag
      841 cgtggagcgg aagccgggca gggggtgcag ggcagtccgg cgtcgggtgc cgggggttgc
      901 cggccagggc ggctgctggg cctgatgcac gagtgcaagc gtcgcccgtc gcgctcacgg
      961 gccgtctccc gaggtgccaa gacggcggag acggtgcagc gcatcaagaa gacccgcagg
     1021 ctcaaggcca acaaccgcga gcgcaaccgc atgcacaacc taaacgccgc gctggacgcg
     1081 ctgcgcgagg tgctgcccac cttccccgag gatgccaagc tcacgaagat cgagacgctg
     1141 cgcttcgccc acaattacat ctgggcgctc accgagactc tgcgcctggc ggaccactgc
     1201 gccggcgccg gtggcctcca gggggcgctc ttcacggagg cggtgctcct gagcccggga
     1261 gctgcgctcg gcgccagcgg ggacagccct tctccacctt cctcctggag ctgcaccaac
     1321 agcccggcgt catcctccaa ctccacgtcc ccatacagct gcactttatc gcccgctagc
     1381 cccgggtcag acgtggacta ctggcagccc ccacctccgg agaagcatcg ttatgcgcct
     1441 cacctgcccc tcgccaggga ctgtatctct agagagggca ggggaagtct tctaacatgc
     1501 ggggacgtgg aggaaaatcc cggcccactc gagatgaccg agtacaagcc cacggtgcgc
     1561 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1621 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1681 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1741 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1801 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1861 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1921 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1981 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     2041 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2101 acctggtgca tgacccgcaa gcccggtgcc tgaaatcaac ctctggatta caaaatttgt
     2161 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     2221 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     2281 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     2341 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     2401 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     2461 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     2521 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     2581 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     2641 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     2701 tccctttggg ccgcctcccc gcctcgagag atctacgggt ggcatccctg tgacccctcc
     2761 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     2821 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     2881 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     2941 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     3001 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     3061 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     3121 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     3181 caggcgtgaa ccactgctcc cttccctgtc cttgtacctt aattaagtta acatgcatag
     3241 tactgagctc acggggacag cccccccccc aaagccccca gggatgtaat tacgtccctc
     3301 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     3361 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     3421 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     3481 aaaaagcttt aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgggttg
     3541 caaaggagca cagtgctcat ccagatccaa ccccctgcta tgtgcagggt catcaaccag
     3601 cagcccaggc tgcccagagc cacatccagc ctggccttga atgcctgcag ggatggggca
     3661 tccacagcct ccttgggcaa cctgttcagt gcgtcaccac cctctggggg aaaaactgcc
     3721 tcctcatatc caacccaaac ctcccctgtc tcagtgtaaa gccattcccc cttgtcctat
     3781 caagggggag tttgctgtga cattgttggt ctggggtgac acatgtttgc caattcagtg
     3841 catcacggag aggcagatct tggggataag gaagtgcagg acagcatgga cgtgggacat
     3901 gcaggtgttg agggctctgg gacactctcc aagtcacagc gttcagaaca gccttaagga
     3961 taagaagata ggatagaagg acaaagagca agttaaaacc cagcatggag aggagcacaa
     4021 aaaggccaca gacactgctg gtccctgtgt ctgagcctgc atgtttgatg gtgtctggat
     4081 gcaagcagaa ggggtggaag agcttgcctg gagagataca gctgggtcag taggactggg
     4141 acaggcagct ggagaattgc catgtagatg ttcatacaat cgtcaaatca tgaaggctgg
     4201 aaaagccctc caagatcccc aagaccaacc ccaacccacc caccgtgccc actggccatg
     4261 tccctcagtg ccacatcccc acagttcttc atcacctcca gggacggtga cccccccacc
     4321 tccgtgggca gctgtgccac tgcagcaccg ctctttggag aaggtaaatc ttgctaaatc
     4381 cagcccgacc ctcccctggc acaacgtaag gccattatct ctcatccaac tccaggacgg
     4441 agtcagtgag aatattgttt aaacattata ggcgcgccat tgctgggatt acaggcgtga
     4501 accactgctc ccttccctgt ccttttaaaa gttttgttac tttatakmms mamagttctt
     4561 catcacctcc agggacggtg acccccccac ctccgtgggc agctgtgcca ctgcagcacc
     4621 gctctttgga gaaggtaaat cttgctaaat ccagcccgac cctcccctgg cacaacgtaa
     4681 ggccattatc tctcatccaa ctccaggacg gagtcagtga gaatattgtt taaacattat
     4741 aggcgcgcca ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttttaaa
     4801 agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata aataaataaa
     4861 cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt
     4921 aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca
     4981 attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc
     5041 tagtatacgc gtatgcggcc gctggggaaa aggaagaaac gcgggcgtat tggccccaat
     5101 ggggtctcgg tggggtatcg acagagtgcc agccctggga ccgaaccccg cgtttatgaa
     5161 caaacgaccc aacacccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt
     5221 ccttccggta ttgtctcctt ccgtgtttca gttagcctcc cccatctccc gggcaaacgt
     5281 gcgcgccagg tcgcagatcg tcggtatgga gcctggggtg gtgacgtggg tctggatcat
     5341 cccggaggta agttgcagca gggcgtcccg gcagccggcg ggcgattggt cgtaatccag
     5401 gataaagacg tgcatgggac ggaggcgttt ggccaagacg tccaaggccc aggcaaacac
     5461 gttgtacagg tcgccgttgg gggccagcaa ctcgggggcc cgaaacaggg taaataacgt
     5521 gtccccgata tggggtcgtg ggcccgcgtt gctctggggc tcggcaccct ggggcggcac
     5581 ggccgtcccc gaaagctgtc cccaatcctc ccgccacgac ccgccgccct gcagataccg
     5641 caccgtattg gcaagcagcc cgtaaacgcg gcgaatcgcg gccagcatag ccaggtcaag
     5701 ccgctcgccg gggcgctggc gtttggccag gcggtcgatg tgtctgtcct ccggaagggc
     5761 ccccaacacg atgtttgtgc cgggcaaggt cggcgggatg agggccacga acgccagcac
     5821 ggcctggggg gtcatgctgc ccataaggta tcgcgcggcc gggtagcaca ggagggcggc
     5881 gatgggatgg cggtcgaaga tgagggtgag ggccgggggc ggggcatgtg agctcccagc
     5941 ctcccccccg atatgaggag ccagaacggc gtcggtcacg gcataaggca tgcccattgt
     6001 tatctgggcg cttgtcatta ccaccgccgc gtccccggcc gatatctcac cctggtcaag
     6061 gcggtgttgt gtggtgtaga tgttcgcgat tgtctcggaa gcccccagca cccgccagta
     6121 agtcatcggc tcgggtacgt agacgatatc gtcgcgcgaa cccagggcca ccagcagttg
     6181 cgtggtggtg gttttcccca tcccgtgggg accgtctata taaacccgca gtagcgtggg
     6241 cattttctgc tccgggcgga cttccgtggc ttcttgctgc cggcgagggc gcaacgccgt
     6301 acgtcggttg ctatggccgc gagaacgcgc agcctggtcg aacgcagacg cgtgctgatg
     6361 gccggggtac gaagccatgg cggtctgggg agagaggtcg gtgattcggt caacgaggga
     6421 gccgactgcc gacgtgcgct ccggaggctt gcagaatgcg gaacaccgcg cgggcaggaa
     6481 cagggcccac actaccgccc cacaccccgc ctcccgcacc gccccttccc ggccgctgct
     6541 ctcggcgcgc cctgctgagc agccgctatt ggccacagcc catcgcggtc ggcgcgctgc
     6601 cattgctccc tggcgctgtc cgtctgcgag ggtactagtg agacgtgcgg cttccgtttg
     6661 tcacgtccgg cacgccgcga accgcaagga accttcccga cttaggggcg gagcaggaag
     6721 cgtcgccggg gggcccacaa gggtagcggc gaagatccgg gtgacgctgc gaacggacgt
     6781 gaagaatgtg cgagacccag ggtcggcgcc gctgcgtttc ccggaaccac gcccagagca
     6841 gccgcgtccc tgcgcaaacc cagggctgcc ttggaaaagg cgcaacccca acccccctcc
     6901 gcgcctacag ctcaagccac atccgaaggg ggagggagcc gggagctgcg cgcggggccg
     6961 ccggggggag gggtggcacc gcccacgccg ggcggccacg aagggcgggg cagcgggcgc
     7021 gcgcccggcg gggggagggg ccgcgcgccg cgcccgctgg gaattggggc cctaggggga
     7081 gggcggaggc gccgacgacc gcggcactta ccgttcgcgg cgtggcgccc ggtggtcccc
     7141 aaggggaggg aagggggagg cggggcgagg acagtgaccg gagtctcctc agcggtggct
     7201 tttctgcttg gcagcctcag cggctggcgc caaaaccgga ctccgcccac ttcctcgccc
     7261 ctgcggtgcg agggtgtgga atcctccaga cgctggggga gggggagttg ggagcttaaa
     7321 aactagtacc cctttgggac cactttcagc agcgaactct cctgtacacc aggggtcagt
     7381 tccacagacg cgggccaggg gtgggtcatt gcggcgtgaa caataatttg actagaagtt
     7441 gattcgggtg tttccggaag gggccgagtc aatccgccga gttggggcac ggaaaacaaa
     7501 aagggaaggc tactaagatt tttctggcgg gggttatcat tggcgtaact gcagggacca
     7561 cctcccttaa ttaatccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     7621 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     7681 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     7741 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     7801 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     7861 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     7921 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     7981 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     8041 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     8101 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     8161 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     8221 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     8281 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     8341 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     8401 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     8461 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     8521 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     8581 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     8641 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     8701 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     8761 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     8821 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     8881 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     8941 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     9001 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     9061 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     9121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     9181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     9241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     9301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     9361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     9421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     9481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     9541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     9601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     9661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     9721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     9781 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     9841 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     9901 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     9961 tctgcttagg gttaggcgtt ttgcgctgct tcggggagat gggggaggct aactgagctc
    10021 tagattaacc ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc
    10081 tctctctttc taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct
    10141 tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac
    10201 gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca
    10261 tacgataatt atattgttat ttcatgttct acttacgtga taacttatta tatatatatt
    10321 ttcttgttat agatatcaca aatgcttgtc cttccttcct gtaacacagc cattaaacca
    10381 ggagcatcgc ccttccccgg ccctcaggta agaggaccaa ataccgtagc cgtttccaat
    10441 ttcagtcctt tagcgccacc tggtgctaac tactctatca cgcttttatc caataactac
    10501 ctttgtaaat ttcctttcaa aagttctggc cgggcgcggt ggctcacgct tgtaatccca
    10561 gcactttgtg aggggtcagg agttcgagac gatcaactag aatgaatgtc gagacccata
    10621 gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc
    10681 tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc
    10741 tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg
    10801 gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt
    10861 taaactcaaa acagagatgt gtcgaagatg gacagtcctg tgctgatatg catggcatgc
    10921 aagaggcact cgtcgttcag gaaggtatcc agaatctcgt tcaattccgg ggtcaggggt
    10981 gagtccaggt tcagatcctc ggtcatggac tcaagtgtgg ttgtcagctc atccagatgg
    11041 ccccttgggg gcggatggga aaggtccatt tggccacaga ttgcagcctc ttccttctgg
    11101 ggaatcacag tatcggccat ctcccgaagg gctttgacag cctggctcgt ctcttcatcg
    11161 ggatcctcca acaggtgact ggcctcggga gtcactgcgg gcgctggatc cagtggctga
    11221 gggactggtg ccggggtcag tgacccgact ggctcatgta ctggaccggt tggtgttggt
    11281 gcgaggctgg cggggagtgg gcggttggcc catggacttc ctggaggatg aaatggccgg
    11341 attcgttttg gctggcacac ctcgcggccc tcaaacacgt cactaatagc ggagccggcc
    11401 tcaggcttcg gcaaaaacat cccttccctg gaatcccggc tgccgctgcc agagcccagc
    11461 aaggctgaga aatccatatc ggcgatagag ctgaagtcct cgtcgccaga cagcagtcca
    11521 ttaggcaggc ctggtgctcc cagaggggca ggagctggat caggaggcct ctgagcgcct
    11581 gtcacgagcc gggtgatggc ctcggggtat tccatcagca tgggctcggt ggtgtgaggg
    11641 gccacaggga tgccctggtt cagcagctgc tggaactcgc tgttgtccac gctggccagg
    11701 tcggtgaaca cggcaggatc ggtgctgttt cccagcaggg ctcccagatc ctcgtcgtcg
    11761 aactgcagct gcagcagagc ttcagacagt gtgccctcgc cggcctgtgt aggtttaggg
    11821 gctggtggag ccacagcctg tggaggtcca ggagccagca caggcacggg tgctggtgcc
    11881 tgagccagtg cagacaccat ggctggagct ggtgcaggag caggagcctg aggcagcacc
    11941 tgaggagggg ctggagccag agcagaggcc tgagagatct ggccgctggg gaacaccatg
    12001 gtagggaact cgtcgtagtt gatggtgctc aggctgctgg tgaaggggta aggctggggg
    12061 gcaggttttg gcacgctggc gctggatctg ctgggcacgg cgattcttct aggtggaggt
    12121 ctagggtcgg tggggccgct gaagggggac ttcttcatga tgctcttgaa tgtctcgtag
    12181 gtccgcttcc gcttttcctc gatccggtgc cggtcgtcgg tgtcgggcag gtactggcta
    12241 ccaactttgc gtttcttttt cggagatccg gaacttctag agttaatcag catgtccagg
    12301 tcgaaatcat caagggcgtc actgccgagc atgtcgaggt caaagtcatc aagggcatcc
    12361 gaaccaagca tgtcaaggtc aaaatcatcg agggcgtcac ttcccagcat atccagatca
    12421 aaatcgtcca atgcgtcagc ccgtccggaa ccgctggcct ccggcccgcc gctttcgcac
    12481 tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg
    12541 gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg
    12601 gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc
    12661 agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc
    12721 tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg
    12781 ccgagatgca ctttagcccc gtcgcgatgt gagaggagag cacagcggta tgacttggcg
    12841 ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc
    12901 ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag
    12961 tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg
    13021 ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta
    13081 gacatagaac cacctggacc agggttttct tcaacatcac cacaagtgag gagagaacct
    13141 ctaccttcgc cggatccgcc ctcccacaca taaccagagg gcagcaattc acgaatccca
    13201 actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg
    13261 agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc
    13321 gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt
    13381 tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg
    13441 ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta
    13501 gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc catggtgaat
    13561 tgctggggag agaggtcggt gattcggtca acgagggagc cgactgccga cgtgcgctcc
    13621 ggaggcttgc agaatgcgga acaccgcgcg ggcaggaaca gggcccacac taccgcccca
    13681 caccccgcct cccgcaccgc cccttcccgg ccgctgctct cggcgcgccc tgctgagcag
    13741 ccgctattgg ccacagccca tcgcggtcgg cgcgctgcca ttgctccctg gcgctgtccg
    13801 tctgcgaggg tactagtgag acgtgcggct tccgtttgtc acgtccggca cgccgcgaac
    13861 cgcaaggaac cttcccgact taggggcgga gcaggaagcg tcgccggggg gcccacaagg
    13921 gtagcggcga agatccgggt gacgctgcga acggacgtga agaatgtgcg agacccaggg
    13981 tcggcgccgc tgcgtttccc ggaaccacgc ccagagcagc cgcgtccctg cgcaaaccca
    14041 gggctgcctt ggaaaaggcg caaccccaac cccggggagg tggtccctgc agttacgcca
    14101 atgataaccc ccgccagaaa aatcttagta gccttccctt tttgttttcc gtgccccaac
    14161 tcggcggatt gactcggccc cttccggaaa cacccgaatc aacttctagt caaattattg
    14221 ttcacgccgc aatgacccac ccctggcccg cgtctgtgga actgacccct ggtgtacagg
    14281 agagttcgct gctgaaagtg gtcccaaagg ggtactagtt tttaagctcc caactccccc
    14341 tcccccagcg tctggaggat tccacaccct cgcaccgcag gggcgaggaa gtgggcggag
    14401 tccggttttg gcgccagccg ctgaggctgc caagcagaaa agccaccgct gaggagactc
    14461 cggtcactgt cctcgccccg cctccccctt ccctcccctt ggggaccacc gggcgccacg
    14521 ccgcgaacgg taagtgccgc ggtcgtcggc gcctccgccc tccccctagg gccccaattc
    14581 ccagcgggcg cggcgcgcgg cccctccccc cgccgggcgc gcgcccgctg ccccgccctt
    14641 cgtggccgcc cggcgtgggc ggtgccaccc ctccccccgg cggccccgcg cgcagctccc
    14701 ggctccctcc cccttcggat gtggcttgag ctgtaggcgc ggaggctagc tggggatctg
    14761 gtgggctcta ttggtagact ttagtcccta tcagtgatag agaaggtatc tccagtttac
    14821 tccctatcag tgatagagac atccatggaa tcaacttcag ctttatgagt aagaagccct
    14881 ataggatgtg gctttcccta tcagtgatag agaactaatg ataagaccac tgcactttag
    14941 tccctatcag tgatagagat gagatcttct ggtgtacagg agttttctcc ctatcagtga
    15001 tagagagtct gtgtgccatt tgatccctat cagtgataga gactgtattt ccaggtaggc
    15061 ctgtacagtg ggaggcctat ataagcagag ctcctttagt gaaccctcag atggccagag
    15121 ataagagaga aaagaagagt aagaagaaat ataagagcca ccacaagttt gtacaaaaaa
    15181 gcaggcttcg ccgccatgga tgtattcatg aaaggacttt caaaggccaa ggagggagtt
    15241 gtggctgctg ctgagaaaac caaacagggt gtggcagaag cagcaggaaa gacaaaaaag
    15301 ggtgttctct atgtaggctc caaaaccaag aagggagtgg tgcatggtgt ggcaacagtg
    15361 gctgagaaga ccaaaaagca agtgacaaat gttggaggag cagtggtgac gggtgtgaca
    15421 gcagtagccc agaagacagt ggagggagca gggagcattg cagcagccac tggctttgtc
    15481 aaaaaggacc agttgggcaa gaatgaagaa ggagccccac aggaaggaat tctggaagat
    15541 atgcctgtgg atcctgacaa tgaggcttat gaaatgcctt ctgaggaagg gtatcaagac
    15601 tacgaacctg aagcccagag caccgtgccc agagccaggg acccacctgt ggccaccagc
    15661 aaaggcgagg aactctttac gggggtagta ccgattcttg tggagttgga cggtgatgtc
    15721 aatggtcaca agttttctgt ccgaggcgaa ggggagggcg atgcgaccaa tggtaagctt
    15781 acattgaagt tcatttgtac gacaggcaag ctgccagtac cttggccaac gttggtgacg
    15841 acacttacat acggagtaca gtgcttcagc cgctatcctg atcacatgaa acgacatgat
    15901 ttttttaaat ccgcaatgcc cgaagggtac gtgcaggaac ggacaatttc ctttaaagac
    15961 gacgggacat ataagacaag ggcggaggtg aagtttgaag gcgacactct tgtgaatagg
    16021 atagaactca aaggcattga ttttaaagaa gatgggaaca ttttgggtca taagcttgag
    16081 tataacttca attcccacaa cgtctacata acagccgaca aacaaaaaaa cggaataaaa
    16141 gcaaacttca aaattaggca taacgttgag gatggttccg tccagctcgc cgatcattat
    16201 cagcagaata cgcctattgg agatgggccg gttctcctcc ccgataatca ctatctgagt
    16261 acccagagtg tgctttctaa agatcccaac gaaaagcgcg atcatatggt tctcctcgaa
    16321 tttgtcactg cagcagggat tacccatggt atggacgaat tgtataaatg aa
//
