LOCUS       sequence-315469-       13136 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All-in-one prime editor piggyBac transposon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13136)
  AUTHORS   Eggenschwiler R, Gschwendtberger T, Felski C, Jahn C, Langer F, 
            Sterneckert J, Hermann A, Luhmann J, Steinemann D, Haase A, Martin 
            U, Petri S, Cantz T
  TITLE     A selectable all-in-one CRISPR prime editing piggyBac transposon 
            allows for highly efficient gene editing in human cell lines.
  JOURNAL   Sci Rep. 2021 Nov 12;11(1):22154. doi: 10.1038/s41598-021-01689-2.
  PUBMED    34773059
REFERENCE   2  (bases 1 to 13136)
  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: "Sci Rep."; 
            date: "2021-11-12"; volume: "11(1):22154. doi"; pages: " 
            10.1038/s41598-021-01689-2"
COMMENT     Sequence Label: PB-PE
FEATURES             Location/Qualifiers
     source          1..13136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..265
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     17..37
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     188..207
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        294..369
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        415..794
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        796..1071
                     /label=chicken beta-actin promoter
     intron          1072..2084
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2008..2030
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2092..2111
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2218..2238
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2239..4752,4753..4755,4756..6339)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2239..6339
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8512..8532
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8542..8598
                     /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"
     CDS             8599..9195
                     /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(8599..8618)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9095..9115
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             9202..10197
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(10219..10236)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10225..10449
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   10633..10695
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(10761..11349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10841..10860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11520..12311,12312..12380))
                     /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     12143..12162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12381..12485)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     12553..12571
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(12609..12631)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     12731..12750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   12774..12808
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 caaggtcggg caggaagagg gcctatttcc catgattcct tcatatttgc atatacgata
       61 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
      121 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
      181 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
      241 tatcttgtgg aaaggacgaa acaccgggtc ttcgtggagc tcatcgaaga cctgttttag
      301 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      361 agtcggtgca gagaccgaat cgaccagagg tctcgttttt ttctagacgc tagcgacatt
      421 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      481 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      541 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      601 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      661 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      721 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      781 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
      841 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
      901 cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg
      961 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
     1021 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
     1081 gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc ccggctctga
     1141 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1201 tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag ccttaaaggg
     1261 ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     1321 cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg cgggcgcggc
     1381 gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     1441 ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     1501 agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc cctccccgag
     1561 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     1621 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     1681 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     1741 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     1801 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     1861 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     1921 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     1981 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2041 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2101 tggttattgt gctgtctcat cattttggca aagaattgat agcggccgct aatacgactc
     2161 actataggga gagccgccac catgaaacgg acagccgacg gaagcgagtt cgagtcacca
     2221 aagaagaagc ggaaagtcga caagaagtac agcatcggcc tggacatcgg caccaactct
     2281 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     2341 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     2401 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     2461 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     2521 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     2581 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     2641 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     2701 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     2761 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     2821 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     2881 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     2941 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     3001 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     3061 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     3121 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     3181 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     3241 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     3301 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     3361 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     3421 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     3481 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     3541 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     3601 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     3661 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     3721 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     3781 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     3841 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     3901 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     3961 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     4021 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     4081 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     4141 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     4201 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     4261 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     4321 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     4381 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     4441 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     4501 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     4561 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     4621 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     4681 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     4741 tacgatgtgg acgctatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     4801 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     4861 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     4921 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     4981 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     5041 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     5101 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     5161 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     5221 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     5281 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     5341 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     5401 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     5461 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     5521 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     5581 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     5641 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     5701 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     5761 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     5821 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     5881 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     5941 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     6001 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     6061 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     6121 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     6181 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     6241 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     6301 tacgagacac ggatcgacct gtctcagctg ggaggtgact ctggaggatc tagcggagga
     6361 tcctctggca gcgagacacc aggaacaagc gagtcagcaa caccagagag cagtggcggc
     6421 agcagcggcg gcagcagcac cctaaatata gaagatgagt atcggctaca tgaaacctca
     6481 aaagagccag atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg
     6541 gaaaccgggg gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca
     6601 acctctaccc ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc
     6661 aagccccaca tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg
     6721 aacacgcccc tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat
     6781 ctgagagaag tcaacaagcg ggtggaggac atccacccca ccgtgcccaa cccttacaac
     6841 ctcttgagcg ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc
     6901 tttttctgcc tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat
     6961 ccagagatgg gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac
     7021 agtcccaccc tgtttaatga ggcactgcac agggacctag cagacttcag aatccagcac
     7081 ccagacttga tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta
     7141 gactgccaac aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc
     7201 tcggccaaga aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa
     7261 gagggtcaga gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccc
     7321 aagacccctc gacaactaag ggagttccta gggaaggcag gcttctgtcg cctcttcatc
     7381 cctgggtttg cagaaatggc agcccccctg taccctctca ccaaaccggg gactctgttt
     7441 aattggggcc cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc
     7501 ccagccctgg ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag
     7561 ggctacgcca aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac
     7621 ctgtccaaaa agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca
     7681 gccattgccg tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt
     7741 ctggcccccc atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac
     7801 gcccggatga ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggcccg
     7861 gtggtagccc tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac
     7921 tgccttgata tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc
     7981 ccagacgccg accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt
     8041 aaggcgggag ctgcggtgac caccgagaca gaggtaatct gggctaaagc cctgccagcc
     8101 gggacatccg ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa
     8161 ggtaagaagc taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat
     8221 ggagaaatat acagaaggcg tgggtggctc acatcagaag gcaaagagat caaaaataaa
     8281 gacgagatct tggccctact aaaagccctc tttctgccca aaagacttag cataatccat
     8341 tgtccaggac atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa
     8401 gcggcccgaa aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat
     8461 tcatcaccct ctggcggctc aaaaagaacc gccgacggca gcgaattcga gcccaagaag
     8521 aagaggaaag tcggctccgg cgctaccaat ttctccctgc tcaagcaagc cggagacgtc
     8581 gaggaaaacc caggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     8641 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     8701 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     8761 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     8821 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     8881 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     8941 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     9001 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     9061 ggggtgcccg ccttcctgga gacatccgcg ccccgcaacc tccccttcta cgagcggctc
     9121 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     9181 cgcaagcccg gtgccggctc catgcccacg ctactgcggg tttatataga cggtcctcac
     9241 gggatgggga aaaccaccac cacgcaactg ctggtggccc tgggttcgcg cgacgatatc
     9301 gtctacgtac ccgagccgat gacttactgg caggtgctgg gggcttccga gacaatcgcg
     9361 aacatctaca ccacacaaca ccgcctcgac cagggtgaga tatcggccgg ggacgcggcg
     9421 gtggtaatga caagcgccca gataacaatg ggcatgcctt atgccgtgac cgacgccgtt
     9481 ctggctcctc atatcggggg ggaggctggg agctcacatg ccccgccccc ggccctcacc
     9541 ctcatcttcg accgccatcc catcgccgcc ctcctgtgct acccggccgc gcgatacctt
     9601 atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc gccgaccttg
     9661 cccggcacaa acatcgtgtt gggggccctt ccggaggaca gacacatcga ccgcctggcc
     9721 aaacgccagc gccccggcga gcggcttgac ctggctatgc tggccgcgat tcgccgcgtt
     9781 tacgggctgc ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg gcgggaggat
     9841 tggggacagc tttcggggac ggccgtgccg ccccagggtg ccgagcccca gagcaacgcg
     9901 ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc cgagttgctg
     9961 gcccccaacg gcgacctgta caacgtgttt gcctgggcct tggacgtctt ggccaaacgc
    10021 ctccgtccca tgcacgtctt tatcctggat tacgaccaat cgcccgccgg ctgccgggac
    10081 gccctgctgc aacttacctc cgggatggtc cagacccacg tcaccacccc cggctccata
    10141 ccgacgatct gcgacctggc gcgcacgttt gcccgggaga tgggggaggc taactgagct
    10201 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
    10261 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
    10321 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
    10381 ggcaggacag caagggggag gattgggagg acaatagcag gcatgctggg gatgcggtgg
    10441 gctctatggg gcgcgcctaa aagttttgtt actttataga agaaattttg agtttttgtt
    10501 tttttttaat aaataaataa acataaataa attgtttgtt gaatttatta ttagtatgta
    10561 agtgtaaata taataaaact taatatctat tcaaattaat aaataaacct cgatatacag
    10621 accgataaaa cacatgcgtc aattttacgc atgattatct ttaacgtacg tcacaatatg
    10681 attatctttc tagggttaaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
    10741 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
    10801 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
    10861 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
    10921 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
    10981 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
    11041 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
    11101 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
    11161 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
    11221 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
    11281 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
    11341 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
    11401 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
    11461 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
    11521 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
    11581 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
    11641 agtgctgcaa tgataccgcg actcccacgc tcaccggctc cagatttatc agcaataaac
    11701 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
    11761 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
    11821 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
    11881 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
    11941 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
    12001 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
    12061 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
    12121 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
    12181 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
    12241 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
    12301 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
    12361 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
    12421 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
    12481 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
    12541 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
    12601 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
    12661 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
    12721 cttaactatg cggcatcaga gcagattgta ctgagagtgc accaatttgt taaccctaga
    12781 aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct ctttctaaat
    12841 agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag ctcccgtgag
    12901 gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg cgtgagtcaa
    12961 aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga taattatatt
    13021 gttatttcat gttctactta cgtgataact tattatatat atattttctt gttatagata
    13081 tcatgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggacc cggtac
//
