LOCUS       sequence-359519-       16493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac Tet-ON plasmid for differentiating hiPSCs into lower motor 
            neurons via NGN2, ISL1, and LHX3 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16493)
  AUTHORS   Pantazis CB, Yang A, Lara E, McDonough JA, Blauwendraat C, Peng L, 
            Oguro H, Kanaujiya J, Zou J, Sebesta D, Pratt G, Cross E, Blockwick 
            J, Buxton P, Kinner-Bibeau L, Medura C, Tompkins C, Hughes S, 
            Santiana M, Faghri F, Nalls MA, Vitale D, Ballard S, Qi YA, Ramos 
            DM, Anderson KM, Stadler J, Narayan P, Papademetriou J, Reilly L, 
            Nelson MP, Aggarwal S, Rosen LU, Kirwan P, Pisupati V, Coon SL, 
            Scholz SW, Priebe T, Ottl M, Dong J, Meijer M, Janssen LJM, Lourenco
            VS, van der Kant R, Crusius D, Paquet D, Raulin AC, Bu G, Held A, 
            Wainger BJ, Gabriele RMC, Casey JM, Wray S, Abu-Bonsrah D, Parish 
            CL, Beccari MS, Cleveland DW, Li E, Rose IVL, Kampmann M, Calatayud 
            Aristoy C, Verstreken P, Heinrich L, Chen MY, Schule B, Dou D, 
            Holzbaur ELF, Zanellati MC, Basundra R, Deshmukh M, Cohen S, Khanna 
            R, Raman M, Nevin ZS, Matia M, Van Lent J, Timmerman V, Conklin BR, 
            Johnson Chase K, Zhang K, Funes S, Bosco DA, Erlebach L, Welzer M, 
            Kronenberg-Versteeg D, Lyu G, Arenas E, Coccia E, Sarrafha L, 
            Ahfeldt T, Marioni JC, Skarnes WC, Cookson MR, Ward ME, Merkle FT
  TITLE     A reference human induced pluripotent stem cell line for large-scale
            collaborative studies.
  JOURNAL   Cell Stem Cell. 2022 Dec 1;29(12):1685-1702.e22. doi: 
            10.1016/j.stem.2022.11.004.
  PUBMED    36459969
REFERENCE   2  (bases 1 to 16493)
  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"; doi: 
            "10.1016/j.stem.2022.11"; journalName: "Cell Stem Cell"; date: 
            "2022-12-1- 1"; volume: "29"; issue: "12"; pages: "1685-1702.e22"
COMMENT     Sequence Label: PB-TO-hNIL
FEATURES             Location/Qualifiers
     source          1..16493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(522..1313,1314..1382))
                     /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     1145..1164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1383..1487)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1513..1968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1650..1671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1862..1881
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2095..2117
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2109..2126
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2110..2126
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2256..2318
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3423..3800
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    3430..3448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3466..3484
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3502..3520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3538..3556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3574..3592
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3610..3628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3646..3664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3709..3733
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4644..4700
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      5491..5500
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5772..5825
                     /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"
     polyA_signal    7071..7192
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7108..7127)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7162..7181
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7241..7257)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7265..7281
                     /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(7289..7295,7296..7313,7314..7319))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7334..7355
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     7781..7800
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    7799..7854
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     7900..7919
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(8010..8756)
                     /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"
     primer_bind     complement(8788..8807)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          8815..9831
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(8869..8891)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        10111..10490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    10491..10539
                     /note="synthetic polyadenylation signal"
     misc_feature    10553..10644
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     10593..10612
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    complement(11856..11889)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        11890..12101
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          12102..13040
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     12997..13017
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             13063..13659
                     /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(13063..13082)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     13559..13579
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             13669..13722
                     /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             13729..13755
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             13771..13797
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      13819..13828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             13825..14535
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     polyA_signal    14555..14779
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(14780..14813)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   15329..15363
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(15854..15870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    15878..15894
                     /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(15902..15908,15909..15926,15927..15932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15947..15968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(join(16256..16493,1..351))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(16336..16355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
       61 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      121 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      181 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
      241 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      301 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      361 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      421 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      481 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      541 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      601 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      661 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      721 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      781 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      841 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      901 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      961 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1021 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1081 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1141 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1201 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1261 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1321 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1381 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1441 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1501 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     1561 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     1621 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1681 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     1741 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     1801 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     1861 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     1921 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     1981 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     2041 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     2101 cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt cattcacgtt tttgaacccg
     2161 tggaggacgg gcagactcgc ggtgcaaatg tgttttacag cgtgatggag cagatgaaga
     2221 tgctcgacac gctgcagaac acgcagctag attaacccta gaaagataat catattgtga
     2281 cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg
     2341 aggtttattt attaatttga atagatatta agttttatta tatttacact tacatactaa
     2401 taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa acaaaaactc
     2461 aaaatttctt ctataaagta acaaaacttt tatgagggac agcccccccc caaagccccc
     2521 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     2581 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     2641 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     2701 gacacctggg gggatacggg gaaaaggcct ccacggcctt aattaaggga ggtggtccct
     2761 gcagttacgc caatgataac ccccgccaga aaaatcttag tagccttccc tttttgtttt
     2821 ccgtgcccca actcggcgga ttgactcggc cccttccgga aacacccgaa tcaacttcta
     2881 gtcaaattat tgttcacgcc gcaatgaccc acccctggcc cgcgtctgtg gaactgaccc
     2941 ctggtgtaca ggagagttcg ctgctgaaag tggtcccaaa ggggtactag tttttaagct
     3001 cccaactccc cctcccccag cgtctggagg attccacacc ctcgcaccgc aggggcgagg
     3061 aagtgggcgg agtccggttt tggcgccagc cgctgaggct gccaagcaga aaagccaccg
     3121 ctgaggagac tccggtcact gtcctcgccc cgcctccccc ttccctcccc ttggggacca
     3181 ccgggcgcca cgccgcgaac ggtaagtgcc gcggtcgtcg gcgcctccgc cctcccccta
     3241 gggccccaat tcccagcggg cgcggcgcgc ggcccctccc cccgccgggc gcgcgcccgc
     3301 tgccccgccc ttcgtggccg cccggcgtgg gcggtgccac ccctcccccc ggcggccccg
     3361 cgcgcagctc ccggctccct cccccttcgg atgtggcttg agctgtaggc gcggagggta
     3421 ccagtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     3481 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     3541 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     3601 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     3661 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     3721 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     3781 ccacttccta ccctcgtaaa ggatccgccg ccaccatgtt cgtcaaatcc gagaccttgg
     3841 agttgaagga ggaagaggac gtgttagtgc tgctcggatc ggcctccccc gccttggcgg
     3901 ccctgacccc gctgtcatcc agcgccgacg aagaagagga ggaggagccg ggcgcgtcag
     3961 gcggggcgcg tcggcagcgc ggggctgagg ccgggcaggg ggcgcggggc ggcgtggctg
     4021 cgggtgcgga gggctgccgg cccgcacggc tgctgggtct ggtacacgat tgcaaacggc
     4081 gcccttcccg ggcgcgggcc gtctcccgag gcgccaagac ggccgagacg gtgcagcgca
     4141 tcaagaagac ccgtagactg aaggccaaca accgcgagcg aaaccgcatg cacaacctca
     4201 acgcggcact ggacgcgctg cgcgaggtgc tccccacgtt ccccgaggac gccaagctca
     4261 ccaagatcga gaccctgcgc ttcgcccaca actacatctg ggcactcacc gagaccctgc
     4321 gcctggcgga tcactgcggg ggcggcggcg ggggcctgcc gggggcgctc ttctccgagg
     4381 cagtgttgct gagcccggga ggcgccagcg ccgccctgag cagcagcgga gacagcccct
     4441 cgcccgcctc cacgtggagt tgcaccaaca gccccgcgcc gtcctcctcc gtgtcctcca
     4501 attccacctc cccctacagc tgcactttat cgcccgccag cccggccggg tcagacatgg
     4561 actattggca gcccccacct cccgacaagc accgctatgc acctcacctc cccatagcca
     4621 gggattgtat cttgggatcc ggagctacta acttcagcct gctgaagcag gctggagacg
     4681 tggaggagaa ccctggacct atgggagaca tgggagatcc accaaaaaaa aaacgtctga
     4741 tttccctatg tgttggttgc ggcaatcaga ttcacgatca gtatattctg agggtttctc
     4801 cggatttgga atggcatgcg gcatgtttga aatgtgcgga gtgtaatcag tatttggacg
     4861 agagctgtac atgctttgtt agggatggga aaacctactg taaaagagat tatatcaggt
     4921 tgtacgggat caaatgcgcc aagtgcagca tcggcttcag caagaacgac ttcgtgatgc
     4981 gtgcccgctc caaggtgtat cacatcgagt gtttccgctg tgtggcctgc agccgccagc
     5041 tcatccctgg ggacgaattt gcgcttcggg aggacggtct cttctgccga gcagaccacg
     5101 atgtggtgga gagggccagt ctaggcgctg gcgacccgct cagtcccctg catccagcgc
     5161 ggccactgca aatggcagcg gagcccatct ccgccaggca gccggccctg cggccccacg
     5221 tccacaagca gccggagaag accacccgcg tgcggactgt gctgaacgag aagcagctgc
     5281 acaccttgcg gacctgctac gccgcaaacc cgcggccaga tgcgctcatg aaggagcaac
     5341 tggtagagat gacgggcctc agtccccgtg tgatccgggt ctggtttcaa aacaagcggt
     5401 gcaaggacaa gaagcgaagc atcatgatga agcaactcca gcagcagcag cccaatgaca
     5461 aaactaatat ccaggggatg acaggaactc ccatggtggc tgccagtcca gagagacacg
     5521 acggtggctt acaggctaac ccagtggaag tacaaagtta ccagccacct tggaaagtac
     5581 tgagcgactt cgccttgcag agtgacatag atcagcctgc ttttcagcaa ctggtcaatt
     5641 tttcagaagg aggaccgggc tctaattcca ctggcagtga agtagcatca atgtcctctc
     5701 aacttccaga tacacctaac agcatggtag ccagtcctat tgaggcattg atgcacagca
     5761 gaggctctgg cgagggcaga ggcagcctgc tgacatgtgg cgacgtggaa gagaaccctg
     5821 gccccggtac catggaagcc agaggcgaac tgggccctgc cagagaatct gccggcggag
     5881 atctgctgct ggccctgctg gctagaaggg ccgacctgag aagagaaatc cccctgtgcg
     5941 ccggctgcga ccagcacatc ctggaccggt ttatcctgaa ggccctggac agacactggc
     6001 acagcaagtg cctgaagtgc agcgactgcc acacccccct ggccgagaga tgctttagca
     6061 gaggcgagag cgtgtactgc aaggacgact tcttcaagag attcggcacc aagtgcgccg
     6121 cctgccagct gggaattcct cctacacagg tcgtgcggag agcccaggac ttcgtgtacc
     6181 atctgcactg cttcgcctgc gtcgtgtgca agagacagct ggccaccggc gacgagttct
     6241 acctgatgga agatagccgg ctcgtgtgta aagccgacta cgagacagcc aagcagagag
     6301 aggccgaggc caccgccaaa agacccagaa ccaccatcac cgccaagcag ctggaaaccc
     6361 tgaagtccgc ctacaacacc agccccaagc ccgccagaca tgtgcgggaa cagctgagca
     6421 gcgagacagg cctggacatg agagtggtgc aagtgtggtt ccagaacaga cgggccaaag
     6481 agaagcggct gaagaaggac gccggcaggc agagatgggg ccagtacttc cggaacatga
     6541 agcggtccag aggcggcagc aagagcgaca aggacagcgt gcaggaaggc caggacagcg
     6601 acgccgaggt gtccttccca gatgagccta gcctggccga aatgggccca gccaatggcc
     6661 tgtatggcag cctgggagag ccaacccagg ctctgggcag accttctggc gccctgggca
     6721 acttctctct ggaacacggc ggactggccg gacccgagca gtatagagaa ctgaggcccg
     6781 gcagccctta cggcgtgcca ccttctccag ctgcccctca gtctctgcca ggacctcagc
     6841 ctctgctgtc cagcctggtg taccccgata caagcctggg cctggtgcca tctggcgctc
     6901 caggcggacc tcctccaatg agagtgctgg ccggcaatgg ccctagcagc gatctgagca
     6961 caggcagctc tggcggctac cccgattttc ctgccagccc tgcctcctgg ctggacgaag
     7021 tggatcacgc ccagttctga gtttaaactc gcgaatcgat cgcccacccc aacttgttta
     7081 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     7141 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     7201 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     7261 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     7321 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     7381 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7441 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     7501 ttcggctgcg gcgagcggta tcagctcact caaaggacgg ccgcacatgt aaggaaaatt
     7561 ttagggatgt taaagaaaaa aataacacaa aacaaaatat aaaaaaaatc taacctcaag
     7621 tcaaggcttt tctatggaat aaggaatgga cagcaggggg ctgtttcata tactgatgac
     7681 ctctttatag ccaacctttg ttcatggcag ccagcatatg ggcatatgtt gccaaactct
     7741 aaaccaaata ctcattctga tgttttaaat gatttgccct cccatatgtc cttccgagtg
     7801 agagacacaa aaaattccaa cacactattg caatgaaaat aaatttcctt tattagccag
     7861 aagtcagatg ctcaaggggc ttcatgatgt ccccataatt tttggcagag ggaaaaagat
     7921 ctcagtggta tttgtgagcc agggcattgg ccacaccagt caccaccttc tgataggcag
     7981 cctgcacctg aggagtgaat tcacgcgttt tagcctggca gcatatcgag atcgaagtca
     8041 tcgagagcgt cagcagggag catatccaga tcaaaatcat ccagggcatc ggcgggcagc
     8101 atgtccaggt cgaagtcgtc cagggcgtcg gtgggtccgc cgctctcgca cttcagctgc
     8161 ttctcgaggc cgcagatgat cagttccagg ccgaacagga aggcgggctc ggcgccctgt
     8221 ctgtcgaaca gctcgatggc ctgcttcagc agggggggca tgctgtcggt ggtgggtgtc
     8281 tctctttcct ctttggcgac ctggtgttcc tgttcttcca gcacgcagcc cagggtgaag
     8341 tggcccacgg cgctcagggc gtacagggcg ttttccaggc tgaagccctg ctggcacagg
     8401 aaggccagct ggttttccag tgtctcgtac tgcttctcgg tgggtctggt gcccaggtgc
     8461 actttggcgc cgtcccggtg gctcagcagg gcgcatctgt agctcttggc gttgttccgc
     8521 aggaagtcct gccagctctc gccttccaga gggcagctgt gggtgtggtg ccggtccagc
     8581 atctcgatgg gcagggcgtc cagcagggcc cgcttgttct tcacgtgcca gtacagggtg
     8641 ggctgttcca cgcccagttt ctgggccagc ttccgggtgg tcaggccctc gatgcccacg
     8701 ccgttcagca gttccagggc gctgttgatc actttgctct tgtccagccg gctcatgtcg
     8761 acattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca ggagctgtag
     8821 gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg tcacacgcca
     8881 gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc gcggacagcc
     8941 ccgaggctgg agagggagaa ggggacggcg gcgcggcgac gcacgaaggc cctccccgcc
     9001 catttccttc ctgccggcgc cgcaccgctt cgccccgcgc ccgctagagg gggtgcggcg
     9061 gcgcctccca gatttcggct ccgcacagat ttgggacaaa ggaagtccct gcgccctctc
     9121 gcacgattac cataaaaggc aatggctgcg gctcgccgcg cctcgacagc cgccggcgct
     9181 ccgggggccg ccgcgcccct cccccgagcc cyccccggcc cgaggcggcc ccgccccgcc
     9241 cggcaccccc acctgccgcc accccccgcc cggcacggcg agccccgcgc cacgccccgt
     9301 acggagcccc gcacccgaag ccgggccgtg ctcagcaact cggggagggg ggtgcagggg
     9361 gggttgcagc ccgaccgacg cgcccacacc ccctgctcac ccccccacgc acacaccccg
     9421 cacgcagcct ttgttcccct cgcagccccc cccgcaccgc ggggcaccgc ccccggccgc
     9481 gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc agcgctcaca
     9541 gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca cgcacgcacc
     9601 ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc tttcacgcag
     9661 ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg gaggagaagg
     9721 gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg gcgggcggcg
     9781 cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc ccgcccgccg
     9841 cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag gaaactttcg
     9901 gagcgcgccg ctctgattgg ctgccgccgc mccycyccsc cycsccccsc cccscccccc
     9961 sccccscccc sccccsccyg gcgcgcgccc cccccccccc cccgccccca tcgctgcaca
    10021 aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg agatggggag
    10081 agtgaagcag aacgtggggc tcacctcgac catggtaata gcgatgacta atacgtagat
    10141 gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca
    10201 tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc
    10261 caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg
    10321 cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca
    10381 gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac
    10441 taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc aataaaatat
    10501 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg
    10561 ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa
    10621 gtgcaggtgc cagaacattt ctctgagctc acggggacag ccccccccca aagcccccag
    10681 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
    10741 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
    10801 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
    10861 cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga atgacagaat
    10921 catagaacgg cctgggttgc aaaggagcac agtgctcatc cagatccaac cccctgctat
    10981 gtgcagggtc atcaaccagc agcccaggct gcccagagcc acatccagcc tggccttgaa
    11041 tgcctgcagg gatggggcat ccacagcctc cttgggcaac ctgttcagtg cgtcaccacc
    11101 ctctggggga aaaactgcct cctcatatcc aacccaaacc tcccctgtct cagtgtaaag
    11161 ccattccccc ttgtcctatc aagggggagt ttgctgtgac attgttggtc tggggtgaca
    11221 catgtttgcc aattcagtgc atcacggaga ggcagatctt ggggataagg aagtgcagga
    11281 cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca agtcacagcg
    11341 ttcagaacag ccttaaggat aagaagatag gatagaagga caaagagcaa gttaaaaccc
    11401 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca
    11461 tgtttgatgg tgtctggatg caagcagaag gggtggaaga gcttgcctgg agagatacag
    11521 ctgggtcagt aggactggga caggcagctg gagaattgcc atgtagatgt tcatacaatc
    11581 gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc caacccaccc
    11641 accgtgccca ctggccatgt ccctcagtgc cacatcccca cagttcttca tcacctccag
    11701 ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga
    11761 aggtaaatct tgctaaatcc agcccgaccc tcccctggca caacgtaagg ccattatctc
    11821 tcatccaact ccaggacgga gtcagtgaga atattataac ttcgtatagc atacattata
    11881 cgaagttatg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
    11941 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
    12001 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
    12061 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
    12121 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
    12181 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
    12241 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
    12301 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
    12361 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
    12421 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
    12481 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
    12541 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
    12601 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
    12661 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
    12721 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
    12781 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcgc
    12841 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
    12901 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
    12961 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
    13021 gtttttttct tccatttcag gtgtcgtgat ttaaatccca ccatgaccga gtacaagccc
    13081 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
    13141 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
    13201 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
    13261 gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
    13321 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
    13381 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
    13441 accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
    13501 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
    13561 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
    13621 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga
    13681 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtccc tgccgctaaa
    13741 cgggtcaaac tcgacggcgg ttctggaggt cctgctgcaa agcgcgttaa gttggatgga
    13801 gggtcagggg gagccaccgc caccatggtg tctaagggcg aagagctgat taaggagaac
    13861 atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc
    13921 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc
    13981 ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc
    14041 ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc
    14101 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
    14161 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc
    14221 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac
    14281 cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc
    14341 catctgatcg caaacgccaa gaccacatat agatccaaga aacccgctaa gaacctcaag
    14401 atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag
    14461 acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg
    14521 gggcacaagc ttaattaggc ggccgctaag aattctgtgc cttctagttg ccagccatct
    14581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
    14641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
    14701 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
    14761 gatgcggtgg gctctatgga taacttcgta tagcatacat tatacgaagt tatttggtat
    14821 ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca
    14881 gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct
    14941 cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc
    15001 tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccctgattc
    15061 tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata
    15121 taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga
    15181 ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca
    15241 cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt
    15301 agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta
    15361 gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc
    15421 ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt
    15481 gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa
    15541 cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg
    15601 ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg
    15661 gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc
    15721 ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat
    15781 cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct
    15841 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
    15901 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
    15961 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
    16021 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
    16081 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
    16141 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
    16201 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
    16261 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
    16321 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
    16381 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
    16441 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgc
//
