LOCUS       sequence-388956-       16523 bp    DNA     circular SYN 14-APR-2025
DEFINITION  PiggyBac plasmid with tet-inducible expression of transcription 
            factors for motor neuron differentiation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16523)
  AUTHORS   Held A, Adler M, Marques C, Reyes CJ, Kavuturu AS, Quadros ARAA, 
            Ndayambaje IS, Lara E, Ward M, Lagier-Tourenne C, Wainger BJ
  TITLE     iPSC motor neurons, but not other derived cell types, capture gene 
            expression changes in postmortem sporadic ALS motor neurons.
  JOURNAL   Cell Rep. 2023 Aug 30;42(9):113046. doi: 
            10.1016/j.celrep.2023.113046.
  PUBMED    37651231
REFERENCE   2  (bases 1 to 16523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2023-08-30"; volume: "42(9):113046. doi"; pages: " 
            10.1016/j.celrep.2023.113046"
COMMENT     Sequence Label: PB-tetO-hNIL
FEATURES             Location/Qualifiers
     source          1..16523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(441..1232,1233..1301))
                     /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     1064..1083
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1302..1406)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1432..1887)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1569..1590)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1781..1800
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2014..2036
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2028..2045
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2029..2045
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2175..2237
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3342..3719
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    3349..3367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3385..3403
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3421..3439
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3457..3475
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3493..3511
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3529..3547
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3565..3583
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3628..3652
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4563..4619
                     /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      5410..5419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5691..5744
                     /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    7020..7141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7057..7076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7111..7130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7190..7206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7214..7230
                     /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(7238..7244,7245..7262,7263..7268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7283..7304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     7730..7749
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    7748..7803
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     7849..7868
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(7959..8705)
                     /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(8737..8756)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          8764..9780
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(8818..8840)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        10060..10439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    10440..10488
                     /note="synthetic polyadenylation signal"
     misc_feature    10502..10593
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     10542..10561
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     protein_bind    complement(11805..11838)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        11839..12050
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          12051..12989
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     12946..12966
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             13012..13608
                     /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(13012..13031)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     13508..13528
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             13618..13671
                     /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             13678..13704
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             13720..13746
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     regulatory      13768..13777
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             13774..14484
                     /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    14504..14728
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(14729..14762)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   15278..15312
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(15803..15819)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    15827..15843
                     /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(15851..15857,15858..15875,15876..15881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15896..15917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(join(16205..16523,1..270))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(16285..16304)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
       61 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      121 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
      181 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
      241 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
      301 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
      361 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      421 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
      481 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
      541 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
      601 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
      661 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
      721 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
      781 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
      841 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
      901 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
      961 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1021 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1081 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1141 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1201 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1261 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1321 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1381 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     1441 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     1501 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     1561 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     1621 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     1681 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     1741 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     1801 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     1861 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     1921 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     1981 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     2041 ccagtgagcg cgcctcgttc attcacgttt ttgaacccgt ggaggacggg cagactcgcg
     2101 gtgcaaatgt gttttacagc gtgatggagc agatgaagat gctcgacacg ctgcagaaca
     2161 cgcagctaga ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg
     2221 tgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa
     2281 tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt
     2341 tatttatgtt tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     2401 caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc
     2461 ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg
     2521 catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg
     2581 ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg
     2641 aaaaggcctc cacggcctta attaagggag gtggtccctg cagttacgcc aatgataacc
     2701 cccgccagaa aaatcttagt agccttccct ttttgttttc cgtgccccaa ctcggcggat
     2761 tgactcggcc ccttccggaa acacccgaat caacttctag tcaaattatt gttcacgccg
     2821 caatgaccca cccctggccc gcgtctgtgg aactgacccc tggtgtacag gagagttcgc
     2881 tgctgaaagt ggtcccaaag gggtactagt ttttaagctc ccaactcccc ctcccccagc
     2941 gtctggagga ttccacaccc tcgcaccgca ggggcgagga agtgggcgga gtccggtttt
     3001 ggcgccagcc gctgaggctg ccaagcagaa aagccaccgc tgaggagact ccggtcactg
     3061 tcctcgcccc gcctccccct tccctcccct tggggaccac cgggcgccac gccgcgaacg
     3121 gtaagtgccg cggtcgtcgg cgcctccgcc ctccccctag ggccccaatt cccagcgggc
     3181 gcggcgcgcg gcccctcccc ccgccgggcg cgcgcccgct gccccgccct tcgtggccgc
     3241 ccggcgtggg cggtgccacc cctccccccg gcggccccgc gcgcagctcc cggctccctc
     3301 ccccttcgga tgtggcttga gctgtaggcg cggagggtac cagtttactc cctatcagtg
     3361 atagagaacg tatgaagagt ttactcccta tcagtgatag agaacgtatg cagactttac
     3421 tccctatcag tgatagagaa cgtataagga gtttactccc tatcagtgat agagaacgta
     3481 tgaccagttt actccctatc agtgatagag aacgtatcta cagtttactc cctatcagtg
     3541 atagagaacg tatatccagt ttactcccta tcagtgatag agaacgtata agctttaggc
     3601 gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     3661 caattccaca acacttttgt cttataccaa ctttccgtac cacttcctac cctcgtaaag
     3721 gatccgccgc caccatgttc gttaagtccg agacactcga gttgaaggag gaagaagatg
     3781 tactggtttt gctcggtagc gcgagtccgg ctttggcagc cctcaccccc ctgtcaagct
     3841 ctgcagacga ggaagaagag gaagaaccgg gcgcttcagg gggtgcaaga agacaacgcg
     3901 gggctgaagc tggtcaaggt gccagagggg gagtcgccgc gggagccgag ggatgccgcc
     3961 ctgctaggct tttgggcctt gtacacgatt gtaagagaag accaagcaga gcgcgcgccg
     4021 tatcccgggg cgcaaaaaca gcggagacgg tccaacgaat caagaagacc cgccgcttga
     4081 aagcgaataa ccgcgagaga aacaggatgc ataacttgaa cgcggccctt gacgctcttc
     4141 gagaagttct ccctaccttc ccggaagacg cgaagctgac caagatagag acactcaggt
     4201 ttgcccataa ctatatctgg gcgctcacgg agacgctccg ccttgcggac cattgtgggg
     4261 gaggcggcgg tggattgccc ggggcactgt tttctgaagc cgtcttgctc tcccctggcg
     4321 gggcctcagc ggcgctgagc tcctccgggg attctccttc acccgcctcc acgtggagtt
     4381 gcaccaacag ccccgcgccg tcctcctccg tgtcctccaa ttccacctcc ccctacagct
     4441 gcactttatc gcccgccagc ccggccgggt cagacatgga ctattggcag cccccacctc
     4501 ccgacaagca ccgctatgca cctcacctcc ccatagccag ggattgtatc ttgggatccg
     4561 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     4621 tgggagacat gggagatcca ccaaaaaaaa aacgtctgat ttccctatgt gttggttgcg
     4681 gcaatcagat tcacgatcag tatattctga gggtttctcc ggatttggaa tggcatgcgg
     4741 catgtttgaa atgtgcggag tgtaatcagt atttggacga gagctgtaca tgctttgtta
     4801 gggatgggaa aacctactgt aaaagagatt atatcaggtt gtacgggatc aaatgcgcca
     4861 agtgcagcat cggcttcagc aagaacgact tcgtgatgcg tgcccgctcc aaggtgtatc
     4921 acatcgagtg tttccgctgt gtggcctgca gccgccagct catccctggg gacgaatttg
     4981 cgcttcggga ggacggtctc ttctgccgag cagaccacga tgtggtggag agggccagtc
     5041 taggcgctgg cgacccgctc agtcccctgc atccagcgcg gccactgcaa atggcagcgg
     5101 agcccatctc cgccaggcag ccggccctgc ggccccacgt ccacaagcag ccggagaaga
     5161 ccacccgcgt gcggactgtg ctgaacgaga agcagctgca caccttgcgg acctgctacg
     5221 ccgcaaaccc gcggccagat gcgctcatga aggagcaact ggtagagatg acgggcctca
     5281 gtccccgtgt gatccgggtc tggtttcaaa acaagcggtg caaggacaag aagcgaagca
     5341 tcatgatgaa gcaactccag cagcagcagc ccaatgacaa aactaatatc caggggatga
     5401 caggaactcc catggtggct gccagtccag agagacacga cggtggctta caggctaacc
     5461 cagtggaagt acaaagttac cagccacctt ggaaagtact gagcgacttc gccttgcaga
     5521 gtgacataga tcagcctgct tttcagcaac tggtcaattt ttcagaagga ggaccgggct
     5581 ctaattccac tggcagtgaa gtagcatcaa tgtcctctca acttccagat acacctaaca
     5641 gcatggtagc cagtcctatt gaggcattga tgcacagcag aggctctggc gagggcagag
     5701 gcagcctgct gacatgtggc gacgtggaag agaaccctgg ccccggtacc atggaagcca
     5761 gaggcgaact gggccctgcc agagaatctg ccggcggaga tctgctgctg gccctgctgg
     5821 ctagaagggc cgacctgaga agagaaatcc ccctgtgcgc cggctgcgac cagcacatcc
     5881 tggaccggtt tatcctgaag gccctggaca gacactggca cagcaagtgc ctgaagtgca
     5941 gcgactgcca cacccccctg gccgagagat gctttagcag aggcgagagc gtgtactgca
     6001 aggacgactt cttcaagaga ttcggcacca agtgcgccgc ctgccagctg ggaattcctc
     6061 ctacacaggt cgtgcggaga gcccaggact tcgtgtacca tctgcactgc ttcgcctgcg
     6121 tcgtgtgcaa gagacagctg gccaccggcg acgagttcta cctgatggaa gatagccggc
     6181 tcgtgtgtaa agccgactac gagacagcca agcagagaga ggccgaggcc accgccaaaa
     6241 gacccagaac caccatcacc gccaagcagc tggaaaccct gaagtccgcc tacaacacca
     6301 gccccaagcc cgccagacat gtgcgggaac agctgagcag cgagacaggc ctggacatga
     6361 gagtggtgca agtgtggttc cagaacagac gggccaaaga gaagcggctg aagaaggacg
     6421 ccggcaggca gagatggggc cagtacttcc ggaacatgaa gcggtccaga ggcggcagca
     6481 agagcgacaa ggacagcgtg caggaaggcc aggacagcga cgccgaggtg tccttcccag
     6541 atgagcctag cctggccgaa atgggcccag ccaatggcct gtatggcagc ctgggagagc
     6601 caacccaggc tctgggcaga ccttctggcg ccctgggcaa cttctctctg gaacacggcg
     6661 gactggccgg acccgagcag tatagagaac tgaggcccgg cagcccttac ggcgtgccac
     6721 cttctccagc tgcccctcag tctctgccag gacctcagcc tctgctgtcc agcctggtgt
     6781 accccgatac aagcctgggc ctggtgccat ctggcgctcc aggcggacct cctccaatga
     6841 gagtgctggc cggcaatggc cctagcagcg atctgagcac aggcagctct ggcggctacc
     6901 ccgattttcc tgccagccct gcctcctggc tggacgaagt ggatcacgcc cagttctgaa
     6961 tcgatcgccc accccaactt gtttattgcg tttaaactcg cgaatcgatc gcccacccca
     7021 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     7081 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     7141 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     7201 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     7261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     7321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     7381 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     7441 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggacggc cgcacatgta
     7501 aggaaaattt tagggatgtt aaagaaaaaa ataacacaaa acaaaatata aaaaaaatct
     7561 aacctcaagt caaggctttt ctatggaata aggaatggac agcagggggc tgtttcatat
     7621 actgatgacc tctttatagc caacctttgt tcatggcagc cagcatatgg gcatatgttg
     7681 ccaaactcta aaccaaatac tcattctgat gttttaaatg atttgccctc ccatatgtcc
     7741 ttccgagtga gagacacaaa aaattccaac acactattgc aatgaaaata aatttccttt
     7801 attagccaga agtcagatgc tcaaggggct tcatgatgtc cccataattt ttggcagagg
     7861 gaaaaagatc tcagtggtat ttgtgagcca gggcattggc cacaccagtc accaccttct
     7921 gataggcagc ctgcacctga ggagtgaatt cacgcgtttt agcctggcag catatcgaga
     7981 tcgaagtcat cgagagcgtc agcagggagc atatccagat caaaatcatc cagggcatcg
     8041 gcgggcagca tgtccaggtc gaagtcgtcc agggcgtcgg tgggtccgcc gctctcgcac
     8101 ttcagctgct tctcgaggcc gcagatgatc agttccaggc cgaacaggaa ggcgggctcg
     8161 gcgccctgtc tgtcgaacag ctcgatggcc tgcttcagca gggggggcat gctgtcggtg
     8221 gtgggtgtct ctctttcctc tttggcgacc tggtgttcct gttcttccag cacgcagccc
     8281 agggtgaagt ggcccacggc gctcagggcg tacagggcgt tttccaggct gaagccctgc
     8341 tggcacagga aggccagctg gttttccagt gtctcgtact gcttctcggt gggtctggtg
     8401 cccaggtgca ctttggcgcc gtcccggtgg ctcagcaggg cgcatctgta gctcttggcg
     8461 ttgttccgca ggaagtcctg ccagctctcg ccttccagag ggcagctgtg ggtgtggtgc
     8521 cggtccagca tctcgatggg cagggcgtcc agcagggccc gcttgttctt cacgtgccag
     8581 tacagggtgg gctgttccac gcccagtttc tgggccagct tccgggtggt caggccctcg
     8641 atgcccacgc cgttcagcag ttccagggcg ctgttgatca ctttgctctt gtccagccgg
     8701 ctcatgtcga cattctttgc caaaatgatg agacagcaca ataaccagca cgttgcccag
     8761 gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag agccgccggt
     8821 cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc cgtccccccg
     8881 cggacagccc cgaggctgga gagggagaag gggacggcgg cgcggcgacg cacgaaggcc
     8941 ctccccgccc atttccttcc tgccggcgcc gcaccgcttc gccccgcgcc cgctagaggg
     9001 ggtgcggcgg cgcctcccag atttcggctc cgcacagatt tgggacaaag gaagtccctg
     9061 cgccctctcg cacgattacc ataaaaggca atggctgcgg ctcgccgcgc ctcgacagcc
     9121 gccggcgctc cgggggccgc cgcgcccctc ccccgagccc tccccggccc gaggcggccc
     9181 cgccccgccc ggcaccccca cctgccgcca ccccccgccc ggcacggcga gccccgcgcc
     9241 acgccccgta cggagccccg cacccgaagc cgggccgtgc tcagcaactc ggggaggggg
     9301 gtgcaggggg ggttgcagcc cgaccgacgc gcccacaccc cctgctcacc cccccacgca
     9361 cacaccccgc acgcagcctt tgttcccctc gcagcccccc ccgcaccgcg gggcaccgcc
     9421 cccggccgcg ctcccctcgc gcacactgcg gagcgcacaa agccccgcgc cgcgcccgca
     9481 gcgctcacag ccgccgggca gcgcggagcc gcacgcggcg ctccccacgc acacacacac
     9541 gcacgcaccc cccgagccgc tccccccgca caaagggccc tcccggagcc cctcaaggct
     9601 ttcacgcagc cacagaaaag aaacaagccg tcattaaacc aagcgctaat tacagcccgg
     9661 aggagaaggg ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc agagccgggg
     9721 cgggcggcgc gaggcggcgg cggagcgggg cacggggcga aggcagcgcg cagcgactcc
     9781 cgcccgccgc gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg ccataaaagg
     9841 aaactttcgg agcgcgccgc tctgattggc tgccgccgcm cctcyccgcc ycsccccscc
     9901 ccsccccycs ccccsccccs ccccsccygg cgcgcgcccc cccccccccc ccgcccccat
     9961 cgctgcacaa aataattaaa aaataaataa atacaaaatt gggggtgggg agggggggga
    10021 gatggggaga gtgaagcaga acgtggggct cacctcgacc atggtaatag cgatgactaa
    10081 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
    10141 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
    10201 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
    10261 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
    10321 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
    10381 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
    10441 ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac
    10501 taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc
    10561 ccagtgcaag tgcaggtgcc agaacatttc tctgagctca cggggacagc ccccccccaa
    10621 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
    10681 gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc
    10741 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
    10801 gcctgcagac acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa
    10861 tgacagaatc atagaacggc ctgggttgca aaggagcaca gtgctcatcc agatccaacc
    10921 ccctgctatg tgcagggtca tcaaccagca gcccaggctg cccagagcca catccagcct
    10981 ggccttgaat gcctgcaggg atggggcatc cacagcctcc ttgggcaacc tgttcagtgc
    11041 gtcaccaccc tctgggggaa aaactgcctc ctcatatcca acccaaacct cccctgtctc
    11101 agtgtaaagc cattccccct tgtcctatca agggggagtt tgctgtgaca ttgttggtct
    11161 ggggtgacac atgtttgcca attcagtgca tcacggagag gcagatcttg gggataagga
    11221 agtgcaggac agcatggacg tgggacatgc aggtgttgag ggctctggga cactctccaa
    11281 gtcacagcgt tcagaacagc cttaaggata agaagatagg atagaaggac aaagagcaag
    11341 ttaaaaccca gcatggagag gagcacaaaa aggccacaga cactgctggt ccctgtgtct
    11401 gagcctgcat gtttgatggt gtctggatgc aagcagaagg ggtggaagag cttgcctgga
    11461 gagatacagc tgggtcagta ggactgggac aggcagctgg agaattgcca tgtagatgtt
    11521 catacaatcg tcaaatcatg aaggctggaa aagccctcca agatccccaa gaccaacccc
    11581 aacccaccca ccgtgcccac tggccatgtc cctcagtgcc acatccccac agttcttcat
    11641 cacctccagg gacggtgacc cccccacctc cgtgggcagc tgtgccactg cagcaccgct
    11701 ctttggagaa ggtaaatctt gctaaatcca gcccgaccct cccctggcac aacgtaaggc
    11761 cattatctct catccaactc caggacggag tcagtgagaa tattataact tcgtatagca
    11821 tacattatac gaagttatgg gcagagcgca catcgcccac agtccccgag aagttggggg
    11881 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
    11941 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
    12001 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
    12061 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
    12121 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
    12181 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
    12241 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
    12301 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
    12361 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
    12421 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
    12481 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
    12541 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
    12601 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
    12661 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
    12721 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
    12781 agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
    12841 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
    12901 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
    12961 tggttcaaag tttttttctt ccatttcagg tgtcgtgatt taaatcccac catgaccgag
    13021 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
    13081 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
    13141 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
    13201 gtgtgggtcg cggacgacgg cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa
    13261 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
    13321 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
    13381 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
    13441 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
    13501 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
    13561 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atcgggagag
    13621 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc accggtccct
    13681 gccgctaaac gggtcaaact cgacggcggt tctggaggtc ctgctgcaaa gcgcgttaag
    13741 ttggatggag ggtcaggggg agccaccgcc accatggtgt ctaagggcga agagctgatt
    13801 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
    13861 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
    13921 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
    13981 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
    14041 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
    14101 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
    14161 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag
    14221 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
    14281 ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag
    14341 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
    14401 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
    14461 agcaaactgg ggcacaagct taattaggcg gccgctaaga attctgtgcc ttctagttgc
    14521 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
    14581 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
    14641 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
    14701 catgctgggg atgcggtggg ctctatggat aacttcgtat agcatacatt atacgaagtt
    14761 atttggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
    14821 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
    14881 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
    14941 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
    15001 ccctgattct ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa
    15061 ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg
    15121 tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag
    15181 cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc
    15241 gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact
    15301 atctttctag ggttaatcta gctgcatcag gatcatatcg tcgggtcttt tttccggctc
    15361 agtcatcgcc caagctggcg ctatctgggc atcggggagg aagaagcccg tgccttttcc
    15421 cgcgaggttg aagcggcatg gaaagagttt gccgaggatg actgctgctg cattgacgtt
    15481 gagcgaaaac gcacgtttac catgatgatt cgggaaggtg tggccatgca cgcctttaac
    15541 ggtgaactgt tcgttcaggc cacctgggat accagttcgt cgcggctttt ccggacacag
    15601 ttccggatgg tcagcccgaa gcgcatcagc aacccgaaca ataccggcga cagccggaac
    15661 tgccgtgccg gtgtgcagat taatgacagc ggtgcggcgc tgggatatta cgtcagcgag
    15721 gacgggtatc ctggctggat gccgcagaaa tggacatgga taccccgtga gttacccggc
    15781 gggcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
    15841 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
    15901 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
    15961 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
    16021 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
    16081 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
    16141 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
    16201 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
    16261 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
    16321 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
    16381 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
    16441 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
    16501 gtaactatcg tcttgagtcc aac
//
