LOCUS       sequence-359511-       16346 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, bsd selection, mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16346)
  TITLE     Characterization of iPSC lines for the iPSC Neurodegenerative 
            Disease Initiative (iNDI)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 16346)
  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: 
            "Unpublished"
COMMENT     Sequence Label: PB-TO-hNIL BSD-mApple
FEATURES             Location/Qualifiers
     source          1..16346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     351..370
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    369..424
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     470..489
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(580..1326)
                     /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(1358..1377)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          1385..2401
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(1439..1461)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        2681..3060
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    3061..3109
                     /note="synthetic polyadenylation signal"
     misc_feature    3123..3214
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     3163..3182
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    complement(4426..4459)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4460..4671
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          4672..5610
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5567..5587
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6068..6121
                     /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             6128..6154
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6170..6196
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      6245..6254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6978..7202
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(7203..7236)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   7752..7786
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8277..8293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8301..8317
                     /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(8325..8331,8332..8349,8350..8355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8370..8391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(8679..9267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8759..8778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9438..10229,10230..10298))
                     /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     10061..10080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10299..10403)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10429..10884)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10566..10587)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10778..10797
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11011..11033
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11025..11042
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11026..11042
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   11172..11234
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        12339..12716
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    12346..12364
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12382..12400
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12418..12436
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12454..12472
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12490..12508
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12526..12544
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12562..12580
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     12625..12649
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             13560..13616
                     /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      14407..14416
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             14688..14741
                     /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    15987..16108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(16024..16043)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     16078..16097
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(16157..16173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    16181..16197
                     /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(16205..16211,16212..16229,16230..16235))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    16250..16271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
       61 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggacgg ccgcacatgt
      121 aaggaaaatt ttagggatgt taaagaaaaa aataacacaa aacaaaatat aaaaaaaatc
      181 taacctcaag tcaaggcttt tctatggaat aaggaatgga cagcaggggg ctgtttcata
      241 tactgatgac ctctttatag ccaacctttg ttcatggcag ccagcatatg ggcatatgtt
      301 gccaaactct aaaccaaata ctcattctga tgttttaaat gatttgccct cccatatgtc
      361 cttccgagtg agagacacaa aaaattccaa cacactattg caatgaaaat aaatttcctt
      421 tattagccag aagtcagatg ctcaaggggc ttcatgatgt ccccataatt tttggcagag
      481 ggaaaaagat ctcagtggta tttgtgagcc agggcattgg ccacaccagt caccaccttc
      541 tgataggcag cctgcacctg aggagtgaat tcacgcgttt tagcctggca gcatatcgag
      601 atcgaagtca tcgagagcgt cagcagggag catatccaga tcaaaatcat ccagggcatc
      661 ggcgggcagc atgtccaggt cgaagtcgtc cagggcgtcg gtgggtccgc cgctctcgca
      721 cttcagctgc ttctcgaggc cgcagatgat cagttccagg ccgaacagga aggcgggctc
      781 ggcgccctgt ctgtcgaaca gctcgatggc ctgcttcagc agggggggca tgctgtcggt
      841 ggtgggtgtc tctctttcct ctttggcgac ctggtgttcc tgttcttcca gcacgcagcc
      901 cagggtgaag tggcccacgg cgctcagggc gtacagggcg ttttccaggc tgaagccctg
      961 ctggcacagg aaggccagct ggttttccag tgtctcgtac tgcttctcgg tgggtctggt
     1021 gcccaggtgc actttggcgc cgtcccggtg gctcagcagg gcgcatctgt agctcttggc
     1081 gttgttccgc aggaagtcct gccagctctc gccttccaga gggcagctgt gggtgtggtg
     1141 ccggtccagc atctcgatgg gcagggcgtc cagcagggcc cgcttgttct tcacgtgcca
     1201 gtacagggtg ggctgttcca cgcccagttt ctgggccagc ttccgggtgg tcaggccctc
     1261 gatgcccacg ccgttcagca gttccagggc gctgttgatc actttgctct tgtccagccg
     1321 gctcatgtcg acattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca
     1381 ggagctgtag gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg
     1441 tcacacgcca gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc
     1501 gcggacagcc ccgaggctgg agagggagaa ggggacggcg gcgcggcgac gcacgaaggc
     1561 cctccccgcc catttccttc ctgccggcgc cgcaccgctt cgccccgcgc ccgctagagg
     1621 gggtgcggcg gcgcctccca gatttcggct ccgcacagat ttgggacaaa ggaagtccct
     1681 gcgccctctc gcacgattac cataaaaggc aatggctgcg gctcgccgcg cctcgacagc
     1741 cgccggcgct ccgggggccg ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc
     1801 ccgccccgcc cggcaccccc acctgccgcc accccccgcc cggcacggcg agccccgcgc
     1861 cacgccccgt acggagcccc gcacccgaag ccgggccgtg ctcagcaact cggggagggg
     1921 ggtgcagggg gggttgcagc ccgaccgacg cgcccacacc ccctgctcac ccccccacgc
     1981 acacaccccg cacgcagcct ttgttcccct cgcagccccc cccgcaccgc ggggcaccgc
     2041 ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc
     2101 agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca
     2161 cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc
     2221 tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg
     2281 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     2341 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     2401 ccgcccgccg cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     2461 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc mccycyccgc cycsccccsc
     2521 cccsccccyc sccccscccc cccccccccg gcgcgcgccc cccccccccc cccgccccca
     2581 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     2641 agatggggag agtgaagcag aacgtggggc tcacctcgac catggtaata gcgatgacta
     2701 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     2761 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     2821 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     2881 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     2941 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     3001 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     3061 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
     3121 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
     3181 cccagtgcaa gtgcaggtgc cagaacattt ctctgagctc acggggacag ccccccccca
     3241 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     3301 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     3361 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     3421 agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga gagatttaga
     3481 atgacagaat catagaacgg cctgggttgc aaaggagcac agtgctcatc cagatccaac
     3541 cccctgctat gtgcagggtc atcaaccagc agcccaggct gcccagagcc acatccagcc
     3601 tggccttgaa tgcctgcagg gatggggcat ccacagcctc cttgggcaac ctgttcagtg
     3661 cgtcaccacc ctctggggga aaaactgcct cctcatatcc aacccaaacc tcccctgtct
     3721 cagtgtaaag ccattccccc ttgtcctatc aagggggagt ttgctgtgac attgttggtc
     3781 tggggtgaca catgtttgcc aattcagtgc atcacggaga ggcagatctt ggggataagg
     3841 aagtgcagga cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca
     3901 agtcacagcg ttcagaacag ccttaaggat aagaagatag gatagaagga caaagagcaa
     3961 gttaaaaccc agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc
     4021 tgagcctgca tgtttgatgg tgtctggatg caagcagaag gggtggaaga gcttgcctgg
     4081 agagatacag ctgggtcagt aggactggga caggcagctg gagaattgcc atgtagatgt
     4141 tcatacaatc gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc
     4201 caacccaccc accgtgccca ctggccatgt ccctcagtgc cacatcccca cagttcttca
     4261 tcacctccag ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc
     4321 tctttggaga aggtaaatct tgctaaatcc agcccgaccc tcccctggca caacgtaagg
     4381 ccattatctc tcatccaact ccaggacgga gtcagtgaga atattataac ttcgtatagc
     4441 atacattata cgaagttatg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     4501 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     4561 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     4621 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     4681 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     4741 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     4801 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     4861 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     4921 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     4981 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     5041 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     5101 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     5161 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     5221 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     5281 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     5341 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     5401 tagttctcgc gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     5461 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     5521 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     5581 gtggttcaaa gtttttttct tccatttcag gtgtcgtgat ttaaatccca ccatgaagac
     5641 cttcaacatc tcccagcagg atctagaatt agtagaagta gcgacagaga agattacaat
     5701 gctttatgag gataataaac atcatgtggg agcggcaatt cgtacgaaaa caggagaaat
     5761 catttcggca gtacatattg aagcgtatat aggacgagta actgtttgtg cagaagccat
     5821 tgcgattggt agtgcagttt cgaatggaca aaaggatttt gacacgattg tagctgttag
     5881 acacccttat tctgacgaag tagatagaag tattcgagtg gtaagtcctt gtggtatgtg
     5941 tagggagttg atttcagact atgcaccaga ttgttttgtg ttaatagaaa tgaatggcaa
     6001 gttagtcaaa actacgattg aagaactcat tccactcaaa tatacccgaa atggatcggg
     6061 atcgggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     6121 accggtccct gccgctaaac gggtcaaact cgacggcggt tctggaggtc ctgctgcaaa
     6181 gcgcgttaag ttggatggag ggtcaggggg agccaccgcc accccggtcg ccacgctacc
     6241 ggtcgccacc atggtgtcca agggcgaaga gaacaacatg gccatcatca aagaattcat
     6301 gcggttcaag gtgcacatgg aaggcagcgt gaacggccac gagttcgaga ttgaaggcga
     6361 aggcgagggc agaccctacg aggcttttca gaccgccaag ctgaaagtga ccaaaggcgg
     6421 ccctctgcct tttgcctggg acattctgag ccctcagttt atgtacggca gcaaggtgta
     6481 catcaagcac cccgccgaca ttcccgacta cttcaagctg agcttccccg agggcttcag
     6541 atgggagaga gtgatgaact tcgaggacgg cggcatcatc cacgtgaacc aggatagctc
     6601 tctgcaggac ggcgtgttca tctacaaagt gaagctgcgg ggcaccaact ttccctctga
     6661 tggccccgtg atgcagaaaa agaccatggg ctgggaagcc agcgaggaaa gaatgtaccc
     6721 tgaggatggc gccctgaagt ccgagatcaa gaagcggctg aagctgaagg atggcggcca
     6781 ctatgccgcc gaagtgaaaa ccacctacaa ggccaagaaa cccgtgcagc tgcctggcgc
     6841 ctacatcgtg gatatcaagc tggacatcgt gtcccacaac gaggactaca ccatcgtgga
     6901 acagtacgag agagccgaag gcagacacag cacaggcgga atggacgagc tgtacaagta
     6961 agcggccgct aagaattctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     7021 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     7081 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     7141 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     7201 ggataacttc gtatagcata cattatacga agttatttgg tatggctttt tccccgtatc
     7261 cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt
     7321 gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag
     7381 cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt
     7441 aattacatcc ctgggggctt tggggggggg ctgtccctga ttctataaca agaaaatata
     7501 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     7561 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     7621 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     7681 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     7741 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa tctagctgca
     7801 tcaggatcat atcgtcgggt cttttttccg gctcagtcat cgcccaagct ggcgctatct
     7861 gggcatcggg gaggaagaag cccgtgcctt ttcccgcgag gttgaagcgg catggaaaga
     7921 gtttgccgag gatgactgct gctgcattga cgttgagcga aaacgcacgt ttaccatgat
     7981 gattcgggaa ggtgtggcca tgcacgcctt taacggtgaa ctgttcgttc aggccacctg
     8041 ggataccagt tcgtcgcggc ttttccggac acagttccgg atggtcagcc cgaagcgcat
     8101 cagcaacccg aacaataccg gcgacagccg gaactgccgt gccggtgtgc agattaatga
     8161 cagcggtgcg gcgctgggat attacgtcag cgaggacggg tatcctggct ggatgccgca
     8221 gaaatggaca tggatacccc gtgagttacc cggcgggcgc gcttggcgta atcatggtca
     8281 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     8341 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     8401 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     8461 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     8521 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     8581 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     8641 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     8701 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     8761 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     8821 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     8881 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     8941 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     9001 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     9061 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     9121 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     9181 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     9241 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     9301 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     9361 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     9421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     9481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     9541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     9601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     9661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     9721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     9781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     9841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     9901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     9961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
    10021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
    10081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
    10141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
    10201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
    10261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
    10321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
    10381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
    10441 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
    10501 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
    10561 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
    10621 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
    10681 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
    10741 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
    10801 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
    10861 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
    10921 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
    10981 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
    11041 gtgagcgcgc ctcgttcatt cacgtttttg aacccgtgga ggacgggcag actcgcggtg
    11101 caaatgtgtt ttacagcgtg atggagcaga tgaagatgct cgacacgctg cagaacacgc
    11161 agctagatta accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgtgt
    11221 aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag
    11281 atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat
    11341 ttatgtttat ttatttatta aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa
    11401 aacttttatg agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc
    11461 cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat
    11521 ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt
    11581 tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa
    11641 aggcctccac ggccttaatt aagggaggtg gtccctgcag ttacgccaat gataaccccc
    11701 gccagaaaaa tcttagtagc cttccctttt tgttttccgt gccccaactc ggcggattga
    11761 ctcggcccct tccggaaaca cccgaatcaa cttctagtca aattattgtt cacgccgcaa
    11821 tgacccaccc ctggcccgcg tctgtggaac tgacccctgg tgtacaggag agttcgctgc
    11881 tgaaagtggt cccaaagggg tactagtttt taagctccca actccccctc ccccagcgtc
    11941 tggaggattc cacaccctcg caccgcaggg gcgaggaagt gggcggagtc cggttttggc
    12001 gccagccgct gaggctgcca agcagaaaag ccaccgctga ggagactccg gtcactgtcc
    12061 tcgccccgcc tcccccttcc ctccccttgg ggaccaccgg gcgccacgcc gcgaacggta
    12121 agtgccgcgg tcgtcggcgc ctccgccctc cccctagggc cccaattccc agcgggcgcg
    12181 gcgcgcggcc cctccccccg ccgggcgcgc gcccgctgcc ccgcccttcg tggccgcccg
    12241 gcgtgggcgg tgccacccct ccccccggcg gccccgcgcg cagctcccgg ctccctcccc
    12301 cttcggatgt ggcttgagct gtaggcgcgg agggtaccag tttactccct atcagtgata
    12361 gagaacgtat gaagagttta ctccctatca gtgatagaga acgtatgcag actttactcc
    12421 ctatcagtga tagagaacgt ataaggagtt tactccctat cagtgataga gaacgtatga
    12481 ccagtttact ccctatcagt gatagagaac gtatctacag tttactccct atcagtgata
    12541 gagaacgtat atccagttta ctccctatca gtgatagaga acgtataagc tttaggcgtg
    12601 tacggtgggc gcctataaaa gcagagctcg tttagtgaac cgtcagatcg cctggagcaa
    12661 ttccacaaca cttttgtctt ataccaactt tccgtaccac ttcctaccct cgtaaaggat
    12721 ccgccgccac catgttcgtc aaatccgaga ccttggagtt gaaggaggaa gaggacgtgt
    12781 tagtgctgct cggatcggcc tcccccgcct tggcggccct gaccccgctg tcatccagcg
    12841 ccgacgaaga agaggaggag gagccgggcg cgtcaggcgg ggcgcgtcgg cagcgcgggg
    12901 ctgaggccgg gcagggggcg cggggcggcg tggctgcggg tgcggagggc tgccggcccg
    12961 cacggctgct gggtctggta cacgattgca aacggcgccc ttcccgggcg cgggccgtct
    13021 cccgaggcgc caagacggcc gagacggtgc agcgcatcaa gaagacccgt agactgaagg
    13081 ccaacaaccg cgagcgaaac cgcatgcaca acctcaacgc ggcactggac gcgctgcgcg
    13141 aggtgctccc cacgttcccc gaggacgcca agctcaccaa gatcgagacc ctgcgcttcg
    13201 cccacaacta catctgggca ctcaccgaga ccctgcgcct ggcggatcac tgcgggggcg
    13261 gcggcggggg cctgccgggg gcgctcttct ccgaggcagt gttgctgagc ccgggaggcg
    13321 ccagcgccgc cctgagcagc agcggagaca gcccctcgcc cgcctccacg tggagttgca
    13381 ccaacagccc cgcgccgtcc tcctccgtgt cctccaattc cacctccccc tacagctgca
    13441 ctttatcgcc cgccagcccg gccgggtcag acatggacta ttggcagccc ccacctcccg
    13501 acaagcaccg ctatgcacct cacctcccca tagccaggga ttgtatcttg ggatccggag
    13561 ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatgg
    13621 gagacatggg agatccacca aaaaaaaaac gtctgatttc cctatgtgtt ggttgcggca
    13681 atcagattca cgatcagtat attctgaggg tttctccgga tttggaatgg catgcggcat
    13741 gtttgaaatg tgcggagtgt aatcagtatt tggacgagag ctgtacatgc tttgttaggg
    13801 atgggaaaac ctactgtaaa agagattata tcaggttgta cgggatcaaa tgcgccaagt
    13861 gcagcatcgg cttcagcaag aacgacttcg tgatgcgtgc ccgctccaag gtgtatcaca
    13921 tcgagtgttt ccgctgtgtg gcctgcagcc gccagctcat ccctggggac gaatttgcgc
    13981 ttcgggagga cggtctcttc tgccgagcag accacgatgt ggtggagagg gccagtctag
    14041 gcgctggcga cccgctcagt cccctgcatc cagcgcggcc actgcaaatg gcagcggagc
    14101 ccatctccgc caggcagccg gccctgcggc cccacgtcca caagcagccg gagaagacca
    14161 cccgcgtgcg gactgtgctg aacgagaagc agctgcacac cttgcggacc tgctacgccg
    14221 caaacccgcg gccagatgcg ctcatgaagg agcaactggt agagatgacg ggcctcagtc
    14281 cccgtgtgat ccgggtctgg tttcaaaaca agcggtgcaa ggacaagaag cgaagcatca
    14341 tgatgaagca actccagcag cagcagccca atgacaaaac taatatccag gggatgacag
    14401 gaactcccat ggtggctgcc agtccagaga gacacgacgg tggcttacag gctaacccag
    14461 tggaagtaca aagttaccag ccaccttgga aagtactgag cgacttcgcc ttgcagagtg
    14521 acatagatca gcctgctttt cagcaactgg tcaatttttc agaaggagga ccgggctcta
    14581 attccactgg cagtgaagta gcatcaatgt cctctcaact tccagataca cctaacagca
    14641 tggtagccag tcctattgag gcattgatgc acagcagagg ctctggcgag ggcagaggca
    14701 gcctgctgac atgtggcgac gtggaagaga accctggccc cggtaccatg gaagccagag
    14761 gcgaactggg ccctgccaga gaatctgccg gcggagatct gctgctggcc ctgctggcta
    14821 gaagggccga cctgagaaga gaaatccccc tgtgcgccgg ctgcgaccag cacatcctgg
    14881 accggtttat cctgaaggcc ctggacagac actggcacag caagtgcctg aagtgcagcg
    14941 actgccacac ccccctggcc gagagatgct ttagcagagg cgagagcgtg tactgcaagg
    15001 acgacttctt caagagattc ggcaccaagt gcgccgcctg ccagctggga attcctccta
    15061 cacaggtcgt gcggagagcc caggacttcg tgtaccatct gcactgcttc gcctgcgtcg
    15121 tgtgcaagag acagctggcc accggcgacg agttctacct gatggaagat agccggctcg
    15181 tgtgtaaagc cgactacgag acagccaagc agagagaggc cgaggccacc gccaaaagac
    15241 ccagaaccac catcaccgcc aagcagctgg aaaccctgaa gtccgcctac aacaccagcc
    15301 ccaagcccgc cagacatgtg cgggaacagc tgagcagcga gacaggcctg gacatgagag
    15361 tggtgcaagt gtggttccag aacagacggg ccaaagagaa gcggctgaag aaggacgccg
    15421 gcaggcagag atggggccag tacttccgga acatgaagcg gtccagaggc ggcagcaaga
    15481 gcgacaagga cagcgtgcag gaaggccagg acagcgacgc cgaggtgtcc ttcccagatg
    15541 agcctagcct ggccgaaatg ggcccagcca atggcctgta tggcagcctg ggagagccaa
    15601 cccaggctct gggcagacct tctggcgccc tgggcaactt ctctctggaa cacggcggac
    15661 tggccggacc cgagcagtat agagaactga ggcccggcag cccttacggc gtgccacctt
    15721 ctccagctgc ccctcagtct ctgccaggac ctcagcctct gctgtccagc ctggtgtacc
    15781 ccgatacaag cctgggcctg gtgccatctg gcgctccagg cggacctcct ccaatgagag
    15841 tgctggccgg caatggccct agcagcgatc tgagcacagg cagctctggc ggctaccccg
    15901 attttcctgc cagccctgcc tcctggctgg acgaagtgga tcacgcccag ttctgagttt
    15961 aaactcgcga atcgatcgcc caccccaact tgtttattgc agcttataat ggttacaaat
    16021 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
    16081 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
    16141 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
    16201 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
    16261 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
    16321 agctgcatta atgaatcggc caacgc
//
