LOCUS       sequence-488651-       16389 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Piggybac Tet-ON plasmid for differentiating hiPSCs into astrocytes 
            via NFIA and SOX9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16389)
  TITLE     Characterization of iPSC lines for the iPSC Neurodegenerative 
            Disease Initiative (iNDI)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 16389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 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            "Unpublished"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-NFIA.1-SOX9
FEATURES             Location/Qualifiers
     source          1..16389
                     /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"
     promoter        complement(2402..2680)
                     /label=chicken beta-actin promoter
     enhancer        2682..3061
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    3062..3110
                     /note="synthetic polyadenylation signal"
     misc_feature    3124..3215
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     3164..3183
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    complement(4427..4460)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4461..4672
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          4673..5611
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5568..5588
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5634..6230
                     /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(5634..5653)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6130..6150
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6240..6293
                     /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             6300..6326
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6342..6368
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      6390..6399
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6396..7106
                     /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    7126..7350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(7351..7384)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   7900..7934
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8425..8441)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8449..8465
                     /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(8473..8479,8480..8497,8498..8503))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8518..8539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(8827..9415)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8907..8926)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9586..10377,10378..10446))
                     /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     10209..10228
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10447..10551)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10577..11032)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10714..10735)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10926..10945
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11159..11181
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11173..11190
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11174..11190
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   11320..11382
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        12487..12864
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    12494..12512
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12530..12548
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12566..12584
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12602..12620
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12638..12656
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12674..12692
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    12710..12728
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     12773..12797
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             14416..14469
                     /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    16030..16151
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(16067..16086)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     16121..16140
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(16200..16216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    16224..16240
                     /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(16248..16254,16255..16272,16273..16278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    16293..16314
                     /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 acctctccgc ctcgccccgc
     2521 cccgcccctc gccccgcccc gccccgcctg gcgcgcgccc cccccccccc cccgccccca
     2581 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     2641 gagatgggga gagtgaagca gaacgtgggg ctcacctcga ccatggtaat agcgatgact
     2701 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     2761 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     2821 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     2881 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     2941 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     3001 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
     3061 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt
     3121 actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt
     3181 ccccagtgca agtgcaggtg ccagaacatt tctctgagct cacggggaca gccccccccc
     3241 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     3301 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     3361 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     3421 gagcctgcag acacctgggg ggatacgggg aaaaagcttt aggctgaaag agagatttag
     3481 aatgacagaa tcatagaacg gcctgggttg caaaggagca cagtgctcat ccagatccaa
     3541 ccccctgcta tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc
     3601 ctggccttga atgcctgcag ggatggggca tccacagcct ccttgggcaa cctgttcagt
     3661 gcgtcaccac cctctggggg aaaaactgcc tcctcatatc caacccaaac ctcccctgtc
     3721 tcagtgtaaa gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt
     3781 ctggggtgac acatgtttgc caattcagtg catcacggag aggcagatct tggggataag
     3841 gaagtgcagg acagcatgga cgtgggacat gcaggtgttg agggctctgg gacactctcc
     3901 aagtcacagc gttcagaaca gccttaagga taagaagata ggatagaagg acaaagagca
     3961 agttaaaacc cagcatggag aggagcacaa aaaggccaca gacactgctg gtccctgtgt
     4021 ctgagcctgc atgtttgatg gtgtctggat gcaagcagaa ggggtggaag agcttgcctg
     4081 gagagataca gctgggtcag taggactggg acaggcagct ggagaattgc catgtagatg
     4141 ttcatacaat cgtcaaatca tgaaggctgg aaaagccctc caagatcccc aagaccaacc
     4201 ccaacccacc caccgtgccc actggccatg tccctcagtg ccacatcccc acagttcttc
     4261 atcacctcca gggacggtga cccccccacc tccgtgggca gctgtgccac tgcagcaccg
     4321 ctctttggag aaggtaaatc ttgctaaatc cagcccgacc ctcccctggc acaacgtaag
     4381 gccattatct ctcatccaac tccaggacgg agtcagtgag aatattataa cttcgtatag
     4441 catacattat acgaagttat gggcagagcg cacatcgccc acagtccccg agaagttggg
     4501 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     4561 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     4621 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
     4681 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
     4741 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
     4801 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
     4861 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
     4921 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
     4981 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
     5041 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
     5101 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
     5161 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     5221 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     5281 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     5341 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     5401 ttagttctcg cgcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     5461 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     5521 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     5581 agtggttcaa agtttttttc ttccatttca ggtgtcgtga tttaaatccc accatgaccg
     5641 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     5701 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     5761 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     5821 aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg
     5881 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     5941 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     6001 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     6061 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     6121 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     6181 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggatcgggag
     6241 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaccggtcc
     6301 ctgccgctaa acgggtcaaa ctcgacggcg gttctggagg tcctgctgca aagcgcgtta
     6361 agttggatgg agggtcaggg ggagccaccg ccaccatggt gtctaagggc gaagagctga
     6421 ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac catcacttca
     6481 agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg agaatcaagg
     6541 tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc ttcctctacg
     6601 gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag cagtccttcc
     6661 ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg ctgaccgcta
     6721 cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc agaggggtga
     6781 acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag gccttcaccg
     6841 agacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg
     6901 tgggcgggag ccatctgatc gcaaacgcca agaccacata tagatccaag aaacccgcta
     6961 agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga atcaaggagg
     7021 ccaacaacga gacctacgtc gagcagcacg aggtggcagt ggccagatac tgcgacctcc
     7081 ctagcaaact ggggcacaag cttaattagg cggccgctaa gaattctgtg ccttctagtt
     7141 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     7201 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     7261 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     7321 ggcatgctgg ggatgcggtg ggctctatgg ataacttcgt atagcataca ttatacgaag
     7381 ttatttggta tggctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg
     7441 agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca
     7501 cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg
     7561 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct
     7621 gtccctgatt ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg
     7681 aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg
     7741 cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca
     7801 agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat
     7861 tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga
     7921 ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc
     7981 tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt
     8041 cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg
     8101 ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta
     8161 acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac
     8221 agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga
     8281 actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg
     8341 aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg
     8401 gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     8461 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     8521 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     8581 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     8641 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     8701 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     8761 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     8821 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     8881 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     8941 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     9001 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     9061 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     9121 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     9181 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     9241 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     9301 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     9361 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     9421 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     9481 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     9541 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     9601 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     9661 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     9721 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     9781 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     9841 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     9901 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     9961 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
    10021 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
    10081 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
    10141 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
    10201 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
    10261 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
    10321 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
    10381 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
    10441 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
    10501 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
    10561 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
    10621 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
    10681 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
    10741 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
    10801 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
    10861 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
    10921 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
    10981 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
    11041 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
    11101 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
    11161 cagtcacgac gttgtaaaac gacggccagt gagcgcgcct cgttcattca cgtttttgaa
    11221 cccgtggagg acgggcagac tcgcggtgca aatgtgtttt acagcgtgat ggagcagatg
    11281 aagatgctcg acacgctgca gaacacgcag ctagattaac cctagaaaga taatcatatt
    11341 gtgacgtacg ttaaagataa tcatgtgtaa aattgacgca tgtgttttat cggtctgtat
    11401 atcgaggttt atttattaat ttgaatagat attaagtttt attatattta cacttacata
    11461 ctaataataa attcaacaaa caatttattt atgtttattt atttattaaa aaaaacaaaa
    11521 actcaaaatt tcttctataa agtaacaaaa cttttatgag ggacagcccc cccccaaagc
    11581 ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct
    11641 ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg
    11701 gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc
    11761 tgcagacacc tggggggata cggggaaaag gcctccacgg ccttaattaa gggaggtggt
    11821 ccctgcagtt acgccaatga taacccccgc cagaaaaatc ttagtagcct tccctttttg
    11881 ttttccgtgc cccaactcgg cggattgact cggccccttc cggaaacacc cgaatcaact
    11941 tctagtcaaa ttattgttca cgccgcaatg acccacccct ggcccgcgtc tgtggaactg
    12001 acccctggtg tacaggagag ttcgctgctg aaagtggtcc caaaggggta ctagttttta
    12061 agctcccaac tccccctccc ccagcgtctg gaggattcca caccctcgca ccgcaggggc
    12121 gaggaagtgg gcggagtccg gttttggcgc cagccgctga ggctgccaag cagaaaagcc
    12181 accgctgagg agactccggt cactgtcctc gccccgcctc ccccttccct ccccttgggg
    12241 accaccgggc gccacgccgc gaacggtaag tgccgcggtc gtcggcgcct ccgccctccc
    12301 cctagggccc caattcccag cgggcgcggc gcgcggcccc tccccccgcc gggcgcgcgc
    12361 ccgctgcccc gcccttcgtg gccgcccggc gtgggcggtg ccacccctcc ccccggcggc
    12421 cccgcgcgca gctcccggct ccctccccct tcggatgtgg cttgagctgt aggcgcggag
    12481 ggtaccagtt tactccctat cagtgataga gaacgtatga agagtttact ccctatcagt
    12541 gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat aaggagttta
    12601 ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga tagagaacgt
    12661 atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact ccctatcagt
    12721 gatagagaac gtataagctt taggcgtgta cggtgggcgc ctataaaagc agagctcgtt
    12781 tagtgaaccg tcagatcgcc tggagcaatt ccacaacact tttgtcttat accaactttc
    12841 cgtaccactt cctaccctcg taaaggatcc gccgccacca tgtactcccc actgtgcttg
    12901 acacaagacg agttccaccc ctttattgag gccctcttgc ctcatgtgcg tgcgttcgct
    12961 tatacgtggt ttaatttgca agcgcgtaag aggaagtatt ttaagaagca cgagaaacga
    13021 atgtccaagg aagaggaacg cgctgtcaaa gacgagctcc tgagcgagaa gcctgaagtg
    13081 aaacaaaagt gggcttcccg tctgttggcc aaactgcgta aggacatacg gccggagtac
    13141 agagaagact tcgtgttgac cgtcacgggc aagaagccac cgtgctgcgt cctcagtaat
    13201 ccggatcaaa agggtaaaat gaggcggatc gattgtctgc gacaagccga caaggtgtgg
    13261 agactcgatc tggtcatggt catcctcttc aagggaatcc cacttgagtc cacagacgga
    13321 gaacgactgg tcaaaagtcc ccagtgttca aaccccggcc tgtgcgttca gcctcaccat
    13381 atcggcgtaa gcgtcaaaga gcttgacctg tacctggcct atttcgtcca cgcggctgac
    13441 tcttcccaga gcgagtctcc gtcacaaccc tcagatgcgg atatcaaaga tcaacctgag
    13501 aacggtcacc tggggtttca agattctttc gtgacctctg gcgtcttctc tgtgacggaa
    13561 ctggtcaggg tctcccaaac ccctattgca gccggcacag gacctaactt cagtcttagt
    13621 gacctggagt ctagctctta ttattctatg tctcccggcg ccatgcgacg tagccttcct
    13681 tctacgtcta gcaccagttc taccaaaaga ttgaaatcag ttgaagacga gatggattcc
    13741 ccaggggaag aacccttcta cacgggtcag ggcagatcac ctgggagcgg atcacaatct
    13801 tctgggtggc acgaggttga acccggcatg ccgagtccta caaccctcaa gaagagcgag
    13861 aaaagcggct tttcatcccc ttcacccagt caaacaagtt ctcttgggac tgcctttacc
    13921 caacaccata ggccagtgat caccggccct cgggcgtccc cacacgcgac tccttccaca
    13981 ctgcactttc ctaccagccc gatcatacaa caaccgggcc cctattttag ccatcccgct
    14041 atacgttacc atccacaaga aactttgaag gagttcgtgc agctggtgtg tccagacgcc
    14101 ggccaacaag ccggccaagt tggctttctg aaccctaacg gttcttcaca gggtaaagtc
    14161 cataatcctt tcttgcctac gcctatgctg ccacccccac ccccgccccc tatggcacgg
    14221 ccagtacccc ttcctgttcc cgataccaaa ccacccacta ctagcactga aggcggcgct
    14281 gctagcccta caagccccac ttatagcacc ccatccacaa gccctgcgaa tcggtttgtg
    14341 tctgtggggc ctagggaccc ttctttcgtg aacataccac agcaaaccca aagctggtat
    14401 ttgggcgggt caggcgaagg gcgcggatcc cttctcacct gtggggatgt tgaggaaaac
    14461 cctggaccaa tgaacttgtt ggatcctttt atgaaaatga cagatgaaca agagaaaggt
    14521 ttgagcggag ctccttcccc aacaatgtca gaagatagcg ctggtagccc atgtccaagc
    14581 gggtctggtt cagatacgga aaataccaga cctcaagaaa atacttttcc taaaggggaa
    14641 cctgacctta agaaagaaag tgaagaagat aaatttccag tttgtatacg ggaagctgtg
    14701 tcccaagtct tgaaggggta tgattggacc ttggtcccta tgcccgttcg ggtgaatggt
    14761 tctagtaaga ataaacccca tgtgaaaagg ccaatgaatg catttatggt ttgggctcaa
    14821 gcagcacggc ggaaactggc cgatcaatat ccacatctcc ataatgctga attgtcaaag
    14881 acccttggga aactttggcg gttgcttaat gaatccgaaa agcggccttt tgtcgaagaa
    14941 gccgaaaggt tgagagtaca acataagaag gatcatccag actataaata tcaaccccgt
    15001 aggagaaaat ctgttaagaa tggccaagca gaagccgaag aagcaacaga acaaactcat
    15061 ataagcccaa atgctatatt taaagcactc caagctgata gcccccattc tagttctggt
    15121 atgtccgaag tccatagccc tggggaacat agcggacaga gccaagggcc gcccactccc
    15181 cctacgactc caaagactga tgttcaaccc ggaaaagccg atctcaaacg ggaaggccgg
    15241 cctctgcccg aaggcgggcg ccaacccccg atagattttc gcgatgtcga cataggggaa
    15301 ttgagttcag atgtgatttc aaatatagaa acatttgacg tgaatgaatt cgatcaatat
    15361 cttccaccaa atggtcatcc cggtgttcct gcaacccatg gtcaagttac gtataccggg
    15421 agttatggaa tttcctctac tgccgcgact ccagcgtccg ctgggcatgt ctggatgtca
    15481 aaacaacaag ctcccccacc cccacctcaa caaccaccgc aagcgccccc agctccccaa
    15541 gcaccacccc aaccacaagc agccccaccc caacaacctg ccgcgccacc ccaacaaccc
    15601 caagcccata cccttactac attgtcctca gaacctggtc aaagccaacg gacacatatt
    15661 aaaaccgaac aactctctcc atcccattat agtgaacaac aacaacattc tccgcagcaa
    15721 attgcttatt caccttttaa tctgcctcat tactcaccat cttatccccc aataacgcgt
    15781 agtcaatacg attatactga tcatcagaat agttcatcat attattccca tgcagcggga
    15841 caagggacgg gattgtattc tacatttacg tatatgaatc cagcgcaaag acctatgtat
    15901 acacctattg ctgatacgag cggagtacca agcattcctc aaactcattc ccctcaacat
    15961 tgggagcagc cagtttatac tcaactgaca cgtccctagg tttaaactcg cgaatcgatc
    16021 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
    16081 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
    16141 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
    16201 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
    16261 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
    16321 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
    16381 ggccaacgc
//
