LOCUS       sequence-416548-       16374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ngn2 mediated cortical neuron induction, along with SNCA(A53T)-sfGFP
            over-expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16374)
  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 16374)
  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(A53T)-sfGFP-IRES-...
FEATURES             Location/Qualifiers
     source          1..16374
                     /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   4975..5037
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5161..5209
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5234..5253
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5251..6381)
                     /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(6388..6898)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7592..7612)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(7592..7610)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7631..7647)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7631..7647)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7644..7666)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7655..7671
                     /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(7679..7685,7686..7703,7704..7709))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7724..7745
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7862..7879)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8033..8621)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8113..8132)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8792..9583,9584..9652))
                     /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     9415..9434
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9653..9757)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9832..9851)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   10032..10066
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    10619..10843
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     10832..10849
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(10872..11441)
                     /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(11460..11816)
                     /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(12246..12266)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(12291..12440)
                     /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(13098..13151)
                     /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(13161..13556)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(13566..14076)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     protein_bind    14788..14806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14824..14842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14898..14916
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14944..14962
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    14991..15009
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    15027..15045
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    15166..15190
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      15193..15202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             15661..16374
                     /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 tttatagaag aaattttgag
     4561 tttttgtttt tttttaataa ataaataaac ataaataaat kgywkstkkr mywytgcagc
     4621 accgctcttt ggagaaggta aatcttgcta aatccagccc gaccctcccc tggcacaacg
     4681 taaggccatt atctctcatc caactccagg acggagtcag tgagaatatt gtttaaacat
     4741 tataggcgcg ccattgctgg gattacaggc gtgaaccact gctcccttcc ctgtcctttt
     4801 aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttta ataaataaat
     4861 aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa
     4921 cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg
     4981 tcaattttac gcatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta
     5041 atctagtata cgcgtatgcg gccgctgggg aaaaggaaga aacgcgggcg tattggcccc
     5101 aatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc ccgcgtttat
     5161 gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg
     5221 gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccgggcaaa
     5281 cgtgcgcgcc aggtcgcaga tcgtcggtat ggagcctggg gtggtgacgt gggtctggat
     5341 catcccggag gtaagttgca gcagggcgtc ccggcagccg gcgggcgatt ggtcgtaatc
     5401 caggataaag acgtgcatgg gacggaggcg tttggccaag acgtccaagg cccaggcaaa
     5461 cacgttgtac aggtcgccgt tgggggccag caactcgggg gcccgaaaca gggtaaataa
     5521 cgtgtccccg atatggggtc gtgggcccgc gttgctctgg ggctcggcac cctggggcgg
     5581 cacggccgtc cccgaaagct gtccccaatc ctcccgccac gacccgccgc cctgcagata
     5641 ccgcaccgta ttggcaagca gcccgtaaac gcggcgaatc gcggccagca tagccaggtc
     5701 aagccgctcg ccggggcgct ggcgtttggc caggcggtcg atgtgtctgt cctccggaag
     5761 ggcccccaac acgatgtttg tgccgggcaa ggtcggcggg atgagggcca cgaacgccag
     5821 cacggcctgg ggggtcatgc tgcccataag gtatcgcgcg gccgggtagc acaggagggc
     5881 ggcgatggga tggcggtcga agatgagggt gagggccggg ggcggggcat gtgagctccc
     5941 agcctccccc ccgatatgag gagccagaac ggcgtcggtc acggcataag gcatgcccat
     6001 tgttatctgg gcgcttgtca ttaccaccgc cgcgtccccg gccgatatct caccctggtc
     6061 aaggcggtgt tgtgtggtgt agatgttcgc gattgtctcg gaagccccca gcacccgcca
     6121 gtaagtcatc ggctcgggta cgtagacgat atcgtcgcgc gaacccaggg ccaccagcag
     6181 ttgcgtggtg gtggttttcc ccatcccgtg gggaccgtct atataaaccc gcagtagcgt
     6241 gggcattttc tgctccgggc ggacttccgt ggcttcttgc tgccggcgag ggcgcaacgc
     6301 cgtacgtcgg ttgctatggc cgcgagaacg cgcagcctgg tcgaacgcag acgcgtgctg
     6361 atggccgggg tacgaagcca tggcggtctg gggagagagg tcggtgattc ggtcaacgag
     6421 ggagccgact gccgacgtgc gctccggagg cttgcagaat gcggaacacc gcgcgggcag
     6481 gaacagggcc cacactaccg ccccacaccc cgcctcccgc accgcccctt cccggccgct
     6541 gctctcggcg cgccctgctg agcagccgct attggccaca gcccatcgcg gtcggcgcgc
     6601 tgccattgct ccctggcgct gtccgtctgc gagggtacta gtgagacgtg cggcttccgt
     6661 ttgtcacgtc cggcacgccg cgaaccgcaa ggaaccttcc cgacttaggg gcggagcagg
     6721 aagcgtcgcc ggggggccca caagggtagc ggcgaagatc cgggtgacgc tgcgaacgga
     6781 cgtgaagaat gtgcgagacc cagggtcggc gccgctgcgt ttcccggaac cacgcccaga
     6841 gcagccgcgt ccctgcgcaa acccagggct gccttggaaa aggcgcaacc ccaacccccc
     6901 tccgcgccta cagctcaagc cacatccgaa gggggaggga gccgggagct gcgcgcgggg
     6961 ccgccggggg gaggggtggc accgcccacg ccgggcggcc acgaagggcg gggcagcggg
     7021 cgcgcgcccg gcggggggag gggccgcgcg ccgcgcccgc tgggaattgg ggccctaggg
     7081 ggagggcgga ggcgccgacg accgcggcac ttaccgttcg cggcgtggcg cccggtggtc
     7141 cccaagggga gggaaggggg aggcggggcg aggacagtga ccggagtctc ctcagcggtg
     7201 gcttttctgc ttggcagcct cagcggctgg cgccaaaacc ggactccgcc cacttcctcg
     7261 cccctgcggt gcgagggtgt ggaatcctcc agacgctggg ggagggggag ttgggagctt
     7321 aaaaactagt acccctttgg gaccactttc agcagcgaac tctcctgtac accaggggtc
     7381 agttccacag acgcgggcca ggggtgggtc attgcggcgt gaacaataat ttgactagaa
     7441 gttgattcgg gtgtttccgg aaggggccga gtcaatccgc cgagttgggg cacggaaaac
     7501 aaaaagggaa ggctactaag atttttctgg cgggggttat cattggcgta actgcaggga
     7561 ccacctccct taattaatcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     7621 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     7681 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     7741 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7801 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     7861 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     7921 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     7981 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     8041 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     8101 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     8161 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     8221 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     8281 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     8341 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     8401 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     8461 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     8521 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     8581 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     8641 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     8701 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     8761 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     8821 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     8881 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     8941 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     9001 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     9061 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     9121 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     9181 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     9241 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     9301 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     9361 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     9421 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     9481 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     9541 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     9601 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     9661 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     9721 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     9781 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     9841 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     9901 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     9961 gaatctgctt agggttaggc gttttgcgct gcttcgggga gatgggggag gctaactgag
    10021 ctctagatta accctagaaa gatagtctgc gtaaaattga cgcatgcatt cttgaaatat
    10081 tgctctctct ttctaaatag cgcgaatccg tcgctgtgca tttaggacat ctcagtcgcc
    10141 gcttggagct cccgtgaggc gtgcttgtca atgcggtaag tgtcactgat tttgaactat
    10201 aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta cgtgactttt aagatttaac
    10261 tcatacgata attatattgt tatttcatgt tctacttacg tgataactta ttatatatat
    10321 attttcttgt tatagatatc acaaatgctt gtccttcctt cctgtaacac agccattaaa
    10381 ccaggagcat cgcccttccc cggccctcag gtaagaggac caaataccgt agccgtttcc
    10441 aatttcagtc ctttagcgcc acctggtgct aactactcta tcacgctttt atccaataac
    10501 tacctttgta aatttccttt caaaagttct ggccgggcgc ggtggctcac gcttgtaatc
    10561 ccagcacttt gtgaggggtc aggagttcga gacgatcaac tagaatgaat gtcgagaccc
    10621 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
    10681 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
    10741 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
    10801 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
    10861 gtttaaactc aaaacagaga tgtgtcgaag atggacagtc ctgtgctgat atgcatggca
    10921 tgcaagaggc actcgtcgtt caggaaggta tccagaatct cgttcaattc cggggtcagg
    10981 ggtgagtcca ggttcagatc ctcggtcatg gactcaagtg tggttgtcag ctcatccaga
    11041 tggccccttg ggggcggatg ggaaaggtcc atttggccac agattgcagc ctcttccttc
    11101 tggggaatca cagtatcggc catctcccga agggctttga cagcctggct cgtctcttca
    11161 tcgggatcct ccaacaggtg actggcctcg ggagtcactg cgggcgctgg atccagtggc
    11221 tgagggactg gtgccggggt cagtgacccg actggctcat gtactggacc ggttggtgtt
    11281 ggtgcgaggc tggcggggag tgggcggttg gcccatggac ttcctggagg atgaaatggc
    11341 cggattcgtt ttggctggca cacctcgcgg ccctcaaaca cgtcactaat agcggagccg
    11401 gcctcaggct tcggcaaaaa catcccttcc ctggaatccc ggctgccgct gccagagccc
    11461 agcaaggctg agaaatccat atcggcgata gagctgaagt cctcgtcgcc agacagcagt
    11521 ccattaggca ggcctggtgc tcccagaggg gcaggagctg gatcaggagg cctctgagcg
    11581 cctgtcacga gccgggtgat ggcctcgggg tattccatca gcatgggctc ggtggtgtga
    11641 ggggccacag ggatgccctg gttcagcagc tgctggaact cgctgttgtc cacgctggcc
    11701 aggtcggtga acacggcagg atcggtgctg tttcccagca gggctcccag atcctcgtcg
    11761 tcgaactgca gctgcagcag agcttcagac agtgtgccct cgccggcctg tgtaggttta
    11821 ggggctggtg gagccacagc ctgtggaggt ccaggagcca gcacaggcac gggtgctggt
    11881 gcctgagcca gtgcagacac catggctgga gctggtgcag gagcaggagc ctgaggcagc
    11941 acctgaggag gggctggagc cagagcagag gcctgagaga tctggccgct ggggaacacc
    12001 atggtaggga actcgtcgta gttgatggtg ctcaggctgc tggtgaaggg gtaaggctgg
    12061 ggggcaggtt ttggcacgct ggcgctggat ctgctgggca cggcgattct tctaggtgga
    12121 ggtctagggt cggtggggcc gctgaagggg gacttcttca tgatgctctt gaatgtctcg
    12181 taggtccgct tccgcttttc ctcgatccgg tgccggtcgt cggtgtcggg caggtactgg
    12241 ctaccaactt tgcgtttctt tttcggagat ccggaacttc tagagttaat cagcatgtcc
    12301 aggtcgaaat catcaagggc gtcactgccg agcatgtcga ggtcaaagtc atcaagggca
    12361 tccgaaccaa gcatgtcaag gtcaaaatca tcgagggcgt cacttcccag catatccaga
    12421 tcaaaatcgt ccaatgcgtc agcccgtccg gaaccgctgg cctccggccc gccgctttcg
    12481 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
    12541 tcggctccct gccggtcgaa cagctcaatt gcttgtttca gaagtggggg catagaatcg
    12601 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgttcctc caatacgcag
    12661 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
    12721 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
    12781 gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg gtatgacttg
    12841 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagga gtgggtatga
    12901 tgcctgtcca gcatctcgat tggcagggca tcgagcaggg cccgcttgtt cttcacgtgc
    12961 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
    13021 tcgataccga ctccattgag taattccaga gcagagttta tgactttgct cttgtccagt
    13081 ctagacatag aaccacctgg accagggttt tcttcaacat caccacaagt gaggagagaa
    13141 cctctacctt cgccggatcc gccctcccac acataaccag agggcagcaa ttcacgaatc
    13201 ccaactgccg tcggctgtcc atcactgtcc ttcactatgg ctttgatccc aggatgcaga
    13261 tcgagaagca cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg
    13321 atcgcgacga tacaagtcag gttgccagct gccgcagcag cagcagtgcc cagcaccacg
    13381 agttctgcac aaggtccccc agtaaaatga tatacattga caccagtgaa gatgcggccg
    13441 tcgctagaga gagctgcgct ggcgacgctg tagtcttcag agatggggat gctgttgatt
    13501 gtagccgttg ctctttcaat gagggtggat tcttcttgag acaaaggctt ggccatggtg
    13561 aattgctggg gagagaggtc ggtgattcgg tcaacgaggg agccgactgc cgacgtgcgc
    13621 tccggaggct tgcagaatgc ggaacaccgc gcgggcagga acagggccca cactaccgcc
    13681 ccacaccccg cctcccgcac cgccccttcc cggccgctgc tctcggcgcg ccctgctgag
    13741 cagccgctat tggccacagc ccatcgcggt cggcgcgctg ccattgctcc ctggcgctgt
    13801 ccgtctgcga gggtactagt gagacgtgcg gcttccgttt gtcacgtccg gcacgccgcg
    13861 aaccgcaagg aaccttcccg acttaggggc ggagcaggaa gcgtcgccgg ggggcccaca
    13921 agggtagcgg cgaagatccg ggtgacgctg cgaacggacg tgaagaatgt gcgagaccca
    13981 gggtcggcgc cgctgcgttt cccggaacca cgcccagagc agccgcgtcc ctgcgcaaac
    14041 ccagggctgc cttggaaaag gcgcaacccc aaccccgggg aggtggtccc tgcagttacg
    14101 ccaatgataa cccccgccag aaaaatctta gtagccttcc ctttttgttt tccgtgcccc
    14161 aactcggcgg attgactcgg ccccttccgg aaacacccga atcaacttct agtcaaatta
    14221 ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt ggaactgacc cctggtgtac
    14281 aggagagttc gctgctgaaa gtggtcccaa aggggtacta gtttttaagc tcccaactcc
    14341 ccctccccca gcgtctggag gattccacac cctcgcaccg caggggcgag gaagtgggcg
    14401 gagtccggtt ttggcgccag ccgctgaggc tgccaagcag aaaagccacc gctgaggaga
    14461 ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc cttggggacc accgggcgcc
    14521 acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg ccctccccct agggccccaa
    14581 ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg cgcgcgcccg ctgccccgcc
    14641 cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc cggcggcccc gcgcgcagct
    14701 cccggctccc tcccccttcg gatgtggctt gagctgtagg cgcggaggct agctggggat
    14761 ctggtgggct ctattggtag actttagtcc ctatcagtga tagagaaggt atctccagtt
    14821 tactccctat cagtgataga gacatccatg gaatcaactt cagctttatg agtaagaagc
    14881 cctataggat gtggctttcc ctatcagtga tagagaacta atgataagac cactgcactt
    14941 tagtccctat cagtgataga gatgagatct tctggtgtac aggagttttc tccctatcag
    15001 tgatagagag tctgtgtgcc atttgatccc tatcagtgat agagactgta tttccaggta
    15061 ggcctgtaca gtgggaggcc tatataagca gagctccttt agtgaaccct cagatggcca
    15121 gagataagag agaaaagaag agtaagaaga aatataagag ccaccacaag tttgtacaaa
    15181 aaagcaggct tagccgccat ggatgtattc atgaaaggac tttcaaaggc caaggaggga
    15241 gttgtggctg ctgctgagaa aaccaaacag ggtgtggcag aagcagcagg aaagacaaaa
    15301 gagggtgttc tctatgtagg ctccaaaacc aaggagggag tggtgcatgg tgtgacaaca
    15361 gtggctgaga agaccaaaga gcaagtgaca aatgttggag gagcagtggt gacgggtgtg
    15421 acagcagtag cccagaagac agtggaggga gcagggagca ttgcagcagc cactggcttt
    15481 gtcaaaaagg accagttggg caagaatgaa gaaggagccc cacaggaagg aattctggaa
    15541 gatatgcctg tggatcctga caatgaggct tatgaaatgc cttctgagga agggtatcaa
    15601 gactacgaac ctgaagccca gagcaccgtg cccagagcca gggacccacc tgtggccacc
    15661 agcaaaggcg aggaactctt tacgggggta gtaccgattc ttgtggagtt ggacggtgat
    15721 gtcaatggtc acaagttttc tgtccgaggc gaaggggagg gcgatgcgac caatggtaag
    15781 cttacattga agttcatttg tacgacaggc aagctgccag taccttggcc aacgttggtg
    15841 acgacactta catacggagt acagtgcttc agccgctatc ctgatcacat gaaacgacat
    15901 gattttttta aatccgcaat gcccgaaggg tacgtgcagg aacggacaat ttcctttaaa
    15961 gacgacggga catataagac aagggcggag gtgaagtttg aaggcgacac tcttgtgaat
    16021 aggatagaac tcaaaggcat tgattttaaa gaagatggga acattttggg tcataagctt
    16081 gagtataact tcaattccca caacgtctac ataacagccg acaaacaaaa aaacggaata
    16141 aaagcaaact tcaaaattag gcataacgtt gaggatggtt ccgtccagct cgccgatcat
    16201 tatcagcaga atacgcctat tggagatggg ccggttctcc tccccgataa tcactatctg
    16261 agtacccaga gtgtgctttc taaagatccc aacgaaaagc gcgatcatat ggttctcctc
    16321 gaatttgtca ctgcagcagg gattacccat ggtatggacg aattgtataa atga
//
