LOCUS       sequence-359517-       15248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac Tet-ON plasmid for differentiating hiPSCs into GABAergic 
            neuron via ASCL1 and DLX2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15248)
  AUTHORS   van Voorst TW, van Boven MA, Marinus KI, Colon-Mercado JM, 
            Schretzmeir J, Haag C, Toonen RF, Koopmans F, Ward ME, Smit AB, van 
            Kesteren RE, Verhage M, Cornelisse LN
  TITLE     One-step induction of human GABAergic neurons promotes presynaptic 
  JOURNAL   bioRxiv [Preprint]. 2025 Jul 7:2025.06.30.662293. doi: 
            10.1101/2025.06.30.662293.
  PUBMED    40672309
REFERENCE   2  (bases 1 to 15248)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Jul 7:2025.06.30.662293. doi: 
            10.1101/2025.06.30.662293."
COMMENT     Sequence Label: PB-TO-ASCL1-DLX2
FEATURES             Location/Qualifiers
     source          1..15248
                     /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..2679)
                     /label=chicken beta-actin promoter
     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             5633..6229
                     /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(5633..5652)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6129..6149
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6239..6292
                     /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             6299..6325
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             6341..6367
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      6389..6398
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6395..7105
                     /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    7125..7349
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(7350..7383)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   7899..7933
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8424..8440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8448..8464
                     /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(8472..8478,8479..8496,8497..8502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8517..8538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(8826..9414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8906..8925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9585..10376,10377..10445))
                     /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     10208..10227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10446..10550)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10576..11031)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10713..10734)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10925..10944
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11158..11180
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11172..11189
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11173..11189
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   11319..11381
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    12495..12616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      13341..13350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        13386..13836
                     /label=TRE3G BI promoter
                     /note="3rd-generation Tet-responsive bidirectional promoter
                     that can be activated by binding of Tet-On(R) 3G"
     protein_bind    13522..13540
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13558..13576
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13594..13612
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13630..13648
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13666..13684
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13702..13720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    13738..13756
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             14790..14816
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     polyA_signal    14889..15010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(15059..15075)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    15083..15099
                     /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(15107..15113,15114..15131,15132..15137))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15152..15173
                     /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 mcctctccgc cycsccccsc
     2521 cccsccccyc sccccscccc ccccccccyg 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 ccatgaccga
     5641 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     5701 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     5761 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     5821 ggtgtgggtc gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga
     5881 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     5941 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     6001 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     6061 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     6121 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     6181 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga
     6241 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtccc
     6301 tgccgctaaa cgggtcaaac tcgacggcgg ttctggaggt cctgctgcaa agcgcgttaa
     6361 gttggatgga gggtcagggg gagccaccgc caccatggtg tctaagggcg aagagctgat
     6421 taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa
     6481 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
     6541 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg
     6601 cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc
     6661 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     6721 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     6781 cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga
     6841 gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt
     6901 gggcgggagc catctgatcg caaacgccaa gaccacatat agatccaaga aacccgctaa
     6961 gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc
     7021 caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc
     7081 tagcaaactg gggcacaagc ttaattaggc ggccgctaag aattctgtgc cttctagttg
     7141 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     7201 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     7261 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     7321 gcatgctggg gatgcggtgg gctctatgga taacttcgta tagcatacat tatacgaagt
     7381 tatttggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     7441 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     7501 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     7561 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     7621 tccctgattc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
     7681 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
     7741 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
     7801 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
     7861 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
     7921 tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt ttttccggct
     7981 cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc gtgccttttc
     8041 ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct gcattgacgt
     8101 tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc acgcctttaa
     8161 cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt tccggacaca
     8221 gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg acagccggaa
     8281 ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga
     8341 ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg
     8401 cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     8461 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     8521 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     8581 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     8641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     8701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     8761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     8821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     8881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     8941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     9001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     9061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     9121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     9181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     9241 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     9301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     9361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     9421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     9481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     9541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     9601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     9661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     9721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     9781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     9841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     9901 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     9961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
    10021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    10081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    10141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    10201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
    10261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
    10321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
    10381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    10441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
    10501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
    10561 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
    10621 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
    10681 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
    10741 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
    10801 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
    10861 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
    10921 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
    10981 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
    11041 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
    11101 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
    11161 agtcacgacg ttgtaaaacg acggccagtg agcgcgcctc gttcattcac gtttttgaac
    11221 ccgtggagga cgggcagact cgcggtgcaa atgtgtttta cagcgtgatg gagcagatga
    11281 agatgctcga cacgctgcag aacacgcagc tagattaacc ctagaaagat aatcatattg
    11341 tgacgtacgt taaagataat catgtgtaaa attgacgcat gtgttttatc ggtctgtata
    11401 tcgaggttta tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac
    11461 taataataaa ttcaacaaac aatttattta tgtttattta tttattaaaa aaaacaaaaa
    11521 ctcaaaattt cttctataaa gtaacaaaac ttttatgagg gacagccccc ccccaaagcc
    11581 cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc
    11641 cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg
    11701 cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct
    11761 gcagacacct ggggggatac ggggaaaagg cctccacggc cttaattaag ggaggtggtc
    11821 cctgcagtta cgccaatgat aacccccgcc agaaaaatct tagtagcctt ccctttttgt
    11881 tttccgtgcc ccaactcggc ggattgactc ggccccttcc ggaaacaccc gaatcaactt
    11941 ctagtcaaat tattgttcac gccgcaatga cccacccctg gcccgcgtct gtggaactga
    12001 cccctggtgt acaggagagt tcgctgctga aagtggtccc aaaggggtac tagtttttaa
    12061 gctcccaact ccccctcccc cagcgtctgg aggattccac accctcgcac cgcaggggcg
    12121 aggaagtggg cggagtccgg ttttggcgcc agccgctgag gctgccaagc agaaaagcca
    12181 ccgctgagga gactccggtc actgtcctcg ccccgcctcc cccttccctc cccttgggga
    12241 ccaccgggcg ccacgccgcg aacggtaagt gccgcggtcg tcggcgcctc cgccctcccc
    12301 ctagggcccc aattcccagc gggcgcggcg cgcggcccct ccccccgccg ggcgcgcgcc
    12361 cgctgccccg cccttcgtgg ccgcccggcg tgggcggtgc cacccctccc cccggcggcc
    12421 ccgcgcgcag ctcccggctc cctccccctt cggatgtggc ttgagctgta ggcgcggagg
    12481 gtacccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
    12541 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
    12601 ctgcaataaa caagttgggg tgggcgatcg attcgcgagt ttttatctag agaaccagtt
    12661 ggtaaagtcc agcagctctt gttcctctgg gctaagaggg tcgtaggagc cctcgtcgga
    12721 ggagtaggac gagaccggag aacccgccat agagttcaag tcgttggagt agttggggga
    12781 gatggtgggc gacaggacgc ccgcctgaaa ggcagcgctc accgcgtcgt gctcgtccag
    12841 cagctgctgc agcgcgcgga tgtactcgac cgccgagcgc agcgtctcca ccttgctcat
    12901 cttcttgttg gccgcgccgt tggggacatg ctcccggagg gtggcaaaac ccaggttgac
    12961 caacttgacc cggttgcgct cgcgctcgtt gcggcgcgcc acggcggccg gctgctgctg
    13021 tggcaggctg tagccgaagc cgctgaagtt gagccggcgt ttgcagcgca tcagttccgg
    13081 agaggacgag cgctggcgct tgacctgctt ggccgctgac ttgtgaccgc cccctgaggg
    13141 ctggctgtcg gccaccgggc tcagctgcgg cgcctgctgc ggcggcgcct gcggctgttg
    13201 ctgctgcgcg ctctgagctg ccgcggccgc cgccgctgcc gccgccgccg cggtcgcaaa
    13261 gaagcaggct gcgggaggca ggaagggctg cgggggctgc ggctgctggc cggctccact
    13321 ctccatcttg ccagagctct ccatggtggc gggacccccg cggagatctg ggccccggtg
    13381 tcgactttac gagggtagga agtggtacgg aaagttggta taagacaaaa gtgttgtgga
    13441 attgctccag gcgatctgac ggttcactaa acgagctctg cttttatagg cgcccaccgt
    13501 acacgcctaa agcttatacg ttctctatca ctgataggga gtaaactgga tatacgttct
    13561 ctatcactga tagggagtaa actgtagata cgttctctat cactgatagg gagtaaactg
    13621 gtcatacgtt ctctatcact gatagggagt aaactcctta tacgttctct atcactgata
    13681 gggagtaaag tctgcatacg ttctctatca ctgataggga gtaaactctt catacgttct
    13741 ctatcactga tagggagtaa actcgagtat gtcgaggtgg cgtgtacggt gggaggccta
    13801 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agaattcgga tccgccacca
    13861 tgactggagt ctttgacagt ctggtggctg atatgcactc gacccagatc accgcctcca
    13921 gcacgtacca ccagcaccag cagcccccga gcggtgcggg cgccggccct ggcggcaaca
    13981 gcaacagcag cagcagcaac agcagcctgc acaagcccca ggagtcgcca accctcccgg
    14041 tgtccacggc tacggacagc agctactaca ccaaccagca gcacccggcg ggcggcggcg
    14101 gcgggggggc ctcgccctac gcgcacatgg gctcctacca gtaccacgcc agcggcctca
    14161 acaatgtctc ctactccgcc aaaagcagct acgacctggg ctacaccgcc gcgtacacct
    14221 cctacgcgcc ctacggcacc agttcgtctc cggtcaacaa cgagccggac aaggaagacc
    14281 ttgagcctga aatccgaata gtgaacggga agccaaagaa agtccggaaa ccacgcacca
    14341 tctactccag tttccagctg gcggcccttc aacgacgctt ccagaagacc cagtatctgg
    14401 ccctgccaga gcgagccgag ctggcggcgt ccctgggcct cacccaaact caggtcaaaa
    14461 tctggttcca gaaccgccga tccaagttca agaagatgtg gaaaagcggc gagataccca
    14521 ccgagcagca ccctggagcc agcgcttctc ctccttgtgc ctccccgccg gtctcggcgc
    14581 cagcatcctg ggacttcggc gcgccgcagc ggatggctgg cggcggcccg ggcagcggag
    14641 gcggcggtgc gggcagctct ggctccagcc cgagcagcgc cgcctcggcc tttctgggaa
    14701 actacccgtg gtatcaccag gcttcgggct ccgcttcaca cctgcaggcc acagcgccac
    14761 ttctgcatcc ttcgcagact ccgcaggcgc accatcacca ccatcaccac caccacgcag
    14821 gcgggggcgc cccggtgagc gcggggacga ttttctaagt ttaaactcgc gaatcgatcg
    14881 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
    14941 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
    15001 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
    15061 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
    15121 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
    15181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
    15241 gccaacgc
//
