LOCUS       sequence-376721-        9650 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9 and POLB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9650)
  AUTHORS   Nakade S, Nakamae K, Tang T-C, Yu D, Sakuma T, Yamamoto T, Lu TK
  TITLE     Frame Editors for Precise, Template-Free Frameshifting
  JOURNAL   bioRxiv 2022.12.05.518807
REFERENCE   2  (bases 1 to 9650)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2022.12.05.518807"
COMMENT     Sequence Label: Cas9_P2A_POLB
FEATURES             Location/Qualifiers
     source          1..9650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..572
                     /label=chicken beta-actin promoter
     intron          573..800
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      812..821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             821..886
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             893..913
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             938..5038
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             938..5038
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5039..5086
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5105..5161
                     /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             6173..6193
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6216..6233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6222..6429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6438..6578
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6438..6567
                     /label=AAV2 ITR
     rep_origin      6653..7108
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6740..6759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6950..6971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7125..7144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7244..7266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7304..7322)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7390..7494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7495..7563,7564..8355)
                     /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     complement(7713..7732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8526..9114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9015..9034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9189..9494)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     9344..9366
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9358..9375
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9359..9375
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9385..9404
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9385..9403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9451..9471)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9451..9469)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9490..9506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9490..9506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9503..9525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9514..9530
                     /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(9538..9544,9545..9562,9563..9568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9583..9604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      421 cgccrggcgg ggcggggsgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
      481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      601 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      661 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
      721 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
      781 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg
      841 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
      901 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct
      961 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     1021 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     1081 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     1141 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     1201 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     1261 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     1321 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     1381 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     1441 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     1501 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     1561 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     1621 gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac
     1681 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     1741 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     1801 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     1861 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     1921 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     1981 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     2041 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     2101 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     2161 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     2221 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     2281 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     2341 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     2401 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     2461 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     2521 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     2581 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     2641 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     2701 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     2761 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     2821 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     2881 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     2941 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     3001 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     3061 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     3121 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     3181 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     3241 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     3301 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     3361 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     3421 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga
     3481 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
     3541 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     3601 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     3661 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     3721 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     3781 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     3841 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     3901 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     3961 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     4021 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     4081 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     4141 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     4201 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     4261 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     4321 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     4381 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     4441 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     4501 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     4561 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     4621 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     4681 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     4741 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     4801 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     4861 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     4921 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     4981 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa
     5041 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagataa gcgctggaag
     5101 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     5161 ttctaagcga aaggctccgc aggagaccct taacggtggc atcacggata tgctgacgga
     5221 gctggctaat tttgaaaaaa atgtttcaca agctatccac aagtacaacg cctacagaaa
     5281 agctgcttct gtaatagcca aatatcctca taaaatcaaa tcaggggccg aggccaaaaa
     5341 actgccgggg gtggggacga aaatcgcaga aaaaatagat gagtttttgg cgacgggtaa
     5401 attgcgaaag ctggaaaaga tcaggcagga cgacacctct tctagtatta atttcctcac
     5461 gagagtaagc ggtatcggac ctagtgccgc caggaagttc gtagatgagg ggattaagac
     5521 attggaagat ctccggaaga acgaggacaa gttgaatcat caccagcgaa taggacttaa
     5581 atatttcgga gacttcgaaa agcgcattcc acgagaggag atgctgcaaa tgcaagacat
     5641 agtgcttaat gaagtcaaaa aagtagacag cgagtacata gctactgtct gcggatcttt
     5701 taggcggggt gctgagtctt ctggtgatat ggatgtactg ctgactcatc catcttttac
     5761 aagcgagagc actaagcaac caaaactgct tcaccaagtc gtcgagcaac tgcagaaggt
     5821 acactttatc actgacactc tgtctaaagg tgaaacgaag ttcatgggcg tctgccagtt
     5881 gccgagtaaa aatgatgaaa aagaatatcc gcatcggaga atagacatcc gactcatccc
     5941 gaaggaccaa tactactgtg gggtcttgta cttcacgggc tcagacattt tcaacaagaa
     6001 tatgagggca cacgccctgg aaaagggttt tacaataaat gagtacacga tcagacctct
     6061 gggagtcacc ggggttgcgg gagagccgct tccggttgac tcagaaaagg acattttcga
     6121 ctatatccag tggaaataca gagaaccaaa ggacagaagt gagggtggtt ctccaaagaa
     6181 gaagcggaag gtctagccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     6241 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     6301 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     6361 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     6421 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     6481 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     6541 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     6601 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     6661 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6721 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6781 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6841 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     6901 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6961 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     7021 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7081 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     7141 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     7201 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     7261 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     7321 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     7381 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     7441 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     7501 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     7561 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     7621 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     7681 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     7741 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     7801 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     7861 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     7921 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     7981 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     8041 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     8101 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     8161 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     8221 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     8281 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     8341 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     8401 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     8461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     8521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     8581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     8641 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     8701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     8761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     8821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     8881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     8941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     9001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     9061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     9121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcagac
     9181 aaatggctct agagaccggc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     9241 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     9301 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     9361 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtaccggg
     9421 ccccccctcg aggtcctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     9481 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     9541 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     9601 attaattgcg ttgcgctcac tgcccgcttt ccaccggtgg tctcttctag
//
