LOCUS       sequence-418440-       12354 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac vector expressing SpCas9-ABE8e with tracrRNA for stable 
            cell line establishment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12354)
  AUTHORS   Zhang H, Kelly K, Lee J, Echeverria D, Cooper D, Panwala R, Amrani 
            N, Chen Z, Gaston N, Wagh A, Newby GA, Xie J, Liu DR, Gao G, Wolfe 
            SA, Khvorova A, Watts JK, Sontheimer EJ
  TITLE     Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery
            and genome editing in vivo.
  JOURNAL   Nucleic Acids Res. 2023 Nov 30:gkad1125. doi: 10.1093/nar/gkad1125.
  PUBMED    38033325
REFERENCE   2  (bases 1 to 12354)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2023 Nov 30:gkad1125. doi: 10.1093/nar/gkad1125."
COMMENT     Sequence Label: pEJS2194-RP_PiggyBac_SpCas9ABE8e-tracrRNA-Puro
FEATURES             Location/Qualifiers
     source          1..12354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    162..395
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     enhancer        448..733
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1038..1265
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1319..1339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1934..1957,1958..1960,1961..6034)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1934..6034
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6077..6097
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6162..6182)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    6358..6582
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(6661..6909)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6719..6738)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6889..6909)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    6957..7074
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7123..7633
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7552..7570
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7655..8254
                     /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(7655..7674)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8151..8171
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     repeat_region   8872..8906
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(9055..9073)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9094..9110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9094..9110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9107..9129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9118..9134
                     /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(9142..9148,9149..9166,9167..9172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9187..9208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9325..9342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9496..10084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9576..9595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10255..11046,11047..11115))
                     /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     10878..10897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11116..11220)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11246..11701)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11383..11404)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     11595..11614
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11828..11850
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11842..11859
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11843..11859
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11866..11885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11866..11884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   11951..12013
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ccggtgcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
       61 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
      121 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
      181 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
      241 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
      301 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
      361 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
      421 ctcatttgca ccactgctgt gggtacccgt tacataactt acggtaaatg gcccgcctgg
      481 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
      541 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      601 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
      661 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      721 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      781 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      841 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
      901 gggggggggg gggggggggg ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      961 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1021 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1081 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1141 gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat
     1201 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt
     1261 ttcaggttgg accggtgcca ccatgaaacg gacagccgat ggaagcgagt tcgagtcacc
     1321 aaagaagaag cggaaagtct ctgaggtgga gttttcccac gagtactgga tgagacatgc
     1381 cctgaccctg gccaagaggg cacgggatga gagggaggtg cctgtgggag ccgtgctggt
     1441 gctgaacaat agagtgatcg gcgagggctg gaacagagcc atcggcctgc acgacccaac
     1501 agcccatgcc gaaattatgg ccctgagaca gggcggcctg gtcatgcaga actacagact
     1561 gattgacgcc accctgtacg tgacattcga gccttgcgtg atgtgcgccg gcgccatgat
     1621 ccactctagg atcggccgcg tggtgtttgg cgtgaggaac tcaaaaagag gcgccgcagg
     1681 ctccctgatg aacgtgctga actaccccgg catgaatcac cgcgtcgaaa ttaccgaggg
     1741 aatcctggca gatgaatgtg ccgccctgct gtgcgatttc tatcggatgc ctagacaggt
     1801 gttcaatgct cagaagaagg cccagagctc catcaactcc ggaggatcta gcggaggctc
     1861 ctctggctct gagacacctg gcacaagcga gagcgcaaca cctgaaagca gcgggggcag
     1921 cagcgggggg tcagacaaga agtacagcat cggcctggcc atcggcacca actctgtggg
     1981 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     2041 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     2101 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     2161 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     2221 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     2281 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     2341 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     2401 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     2461 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     2521 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     2581 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     2641 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     2701 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     2761 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     2821 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     2881 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     2941 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     3001 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     3061 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     3121 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     3181 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     3241 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     3301 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     3361 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     3421 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     3481 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     3541 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     3601 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     3661 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     3721 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     3781 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     3841 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     3901 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     3961 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     4021 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     4081 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     4141 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     4201 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     4261 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     4321 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     4381 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     4441 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     4501 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     4561 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     4621 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     4681 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     4741 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     4801 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     4861 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     4921 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     4981 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     5041 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     5101 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     5161 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     5221 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     5281 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     5341 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     5401 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     5461 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     5521 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     5581 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     5641 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     5701 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     5761 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     5821 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     5881 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     5941 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     6001 gacacggatc gacctgtctc agctgggagg tgactctggc ggctcaaaaa gaaccgccga
     6061 cggcagcgaa ttcgagccca agaagaagag gaaagtctaa gatctgataa tcaacctctg
     6121 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     6181 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     6241 ttctcctcct tgtataaatc ctggttagtt cttgccacgg cggaactcat cgccgcctgc
     6301 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgcgactg
     6361 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     6421 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     6481 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     6541 aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgagaa aaaaagcacc
     6601 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatgctc
     6661 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     6721 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     6781 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     6841 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     6901 taggccctcg cggccgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt
     6961 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     7021 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     7081 tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct
     7141 tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa
     7201 acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg
     7261 gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg
     7321 ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac
     7381 tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc
     7441 tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt
     7501 gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc
     7561 attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga
     7621 cctctctccc cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg
     7681 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     7741 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     7801 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     7861 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     7921 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     7981 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     8041 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     8101 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     8161 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     8221 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     8281 gaccgaaagg agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc
     8341 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     8401 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     8461 agcctgggag ctctctggct aactagggaa cccactgctt aagcttctag ccaacaagct
     8521 cgtcatcgct ttgcagaaga gcagagagga tatgctcatc gtctaaagaa ctacccattt
     8581 tattatatat tagtcacgat atctataaca agaaaatata tatataataa gttatcacgt
     8641 aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa
     8701 aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt
     8761 accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc
     8821 acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca
     8881 attttacgca gactatcttt ctagggttaa aaaagatttg cgctttactc gacctaaact
     8941 ttaaacacgt catagaatct tcgtttgaca aaaaccacat tgtggccaag ctgtgtgacg
     9001 cgacgcgcgc taaagaatgg caaaccaagt cgcgcgaggt accagctttt gttcccttta
     9061 gtgagggtta atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     9121 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     9181 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     9241 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     9301 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     9361 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     9421 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     9481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     9541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     9601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     9661 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     9721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     9781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     9841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     9901 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     9961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
    10021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
    10081 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
    10141 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
    10201 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
    10261 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
    10321 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
    10381 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
    10441 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
    10501 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
    10561 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
    10621 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
    10681 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
    10741 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
    10801 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
    10861 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
    10921 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
    10981 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
    11041 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    11101 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    11161 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    11221 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
    11281 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
    11341 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
    11401 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
    11461 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
    11521 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
    11581 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
    11641 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
    11701 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
    11761 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
    11821 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
    11881 tagggcgaat tggagctcgg tattcacgac agcaggctga ataataaaaa aattagaaac
    11941 tattatttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta
    12001 aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga
    12061 tattaagttt tattatattt acacttacat actaataata aattcaacaa acaatttatt
    12121 tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa
    12181 aacttttaaa cattctctct tttacaaaaa taaacttatt ttgtacttta aaaacagtca
    12241 tgttgtatta taaaataagt aattagctta acctatacat aatagaaaca aattatactt
    12301 attagtcagt cagaaacaac tttggcacat atcaatatta tgctctcgtt aata
//
