LOCUS       sequence-382841-       11339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9 and T4pol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11339)
  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 11339)
  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_T4pol
FEATURES             Location/Qualifiers
     source          1..11339
                     /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"
     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             7862..7882
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7905..7922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7911..8118
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8127..8267
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8127..8256
                     /label=AAV2 ITR
     rep_origin      8342..8797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8429..8448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8639..8660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8814..8833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8933..8955
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8993..9011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9079..9183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9184..9252,9253..10044)
                     /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(9402..9421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10215..10803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10704..10723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10878..11183)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     11033..11055
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11047..11064
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11048..11064
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11074..11093
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11074..11092
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11140..11160)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11140..11158)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11179..11195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11179..11195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11192..11214)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11203..11219
                     /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(11227..11233,11234..11251,11252..11257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11272..11293
                     /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 cgccrggsgg ggsggggsgg ggsgrggggs ggggsggggs grggsggrgr ggkgcggcgg
      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 taaggaattc tatattagta tcgagacagt gggaaacaac attgtagagc ggtatatcga
     5221 tgaaaacggc aaagaaagga ctcgagaggt cgaatacctt ccgaccatgt ttcgccactg
     5281 taaagaagaa tctaagtaca aagatattta cgggaagaat tgcgctcccc aaaaatttcc
     5341 ctccatgaag gatgctcgag actggatgaa gcgcatggag gacataggtc tcgaagcatt
     5401 ggggatgaac gattttaagt tggcttacat ctccgacact tacgggtcag aaatagtgta
     5461 cgataggaag ttcgttcgcg tggcaaattg cgatatagag gtcactggag ataagttccc
     5521 ggacccgatg aaggcggagt acgaaattga cgctataaca cactatgact caatcgacga
     5581 ccggttctat gtatttgacc tgctcaattc catgtacggg tctgtaagca agtgggacgc
     5641 taaactcgcg gctaaacttg actgtgaagg aggggatgaa gtacctcagg aaatcttgga
     5701 cagggtaatc tacatgccct ttgacaatga acgagatatg cttatggagt acattaattt
     5761 gtgggagcaa aagcgccccg caatatttac aggctggaac atagaagggt tcgatgtacc
     5821 gtatattatg aatcgggtaa agatgatcct cggagagaga agcatgaaaa gattttcacc
     5881 tattggcaga gtgaaatcta agttgataca aaacatgtat ggctcaaaag agatctattc
     5941 aatagatgga gttagcatac tcgactacct ggatctgtat aaaaagtttg cttttaccaa
     6001 cttgcctagc ttctcccttg aaagtgtcgc ccaacacgag accaagaaag gtaagctgcc
     6061 gtacgatggc ccgattaata aactgcgcga gaccaaccat caaagatata ttagctacaa
     6121 cataattgat gtcgaatctg tgcaagccat tgataagata aggggcttta tcgaccttgt
     6181 cctgtcaatg tcctattacg ccaaaatgcc gttctcaggt gtaatgtcac ccataaagac
     6241 gtgggatgcg atcatcttca attctctcaa gggagagcac aaggtgatcc cccaacaggg
     6301 gtcccacgta aagcagtcct tcccgggagc ttttgtcttt gagccaaagc cgatcgcccg
     6361 aaggtatatc atgtctttcg accttacgtc actttaccct tcaattattc gacaagtgaa
     6421 tatatcaccc gagactatcc ggggccagtt taaggtacac ccaatccatg agtacatagc
     6481 cggtacagcc ccaaaaccca gtgacgaata ctcttgcagc cctaatgggt ggatgtacga
     6541 caaacaccag gagggcataa tcccaaagga aatcgcgaaa gtatttttcc aacggaaaga
     6601 ctggaagaaa aagatgttcg cggaggaaat gaacgccgag gctattaaaa aaatcattat
     6661 gaagggagcg ggtagctgtt ctaccaagcc agaggtagag cgctacgtca aattcagtga
     6721 tgacttcctt aatgagctga gtaactacac agagtctgta ctgaactcac tgattgagga
     6781 atgtgaaaaa gccgcaacac ttgctaatac caatcaactg aatcggaaga tcctgattaa
     6841 ttcactgtat ggcgccttgg gcaacattca tttcagatac tacgacctca ggaatgccac
     6901 ggccattaca attttcggtc aggtcgggat ccagtggatc gcccgaaaaa tcaacgagta
     6961 cctcaataaa gtgtgtggta ccaatgacga ggattttatc gcagcaggcg ataccgatag
     7021 cgtgtatgtt tgcgtcgaca aggtcattga aaaggtaggg ctggatcggt ttaaggagca
     7081 gaatgatctt gtcgagttta tgaaccagtt tggtaaaaaa aagatggaac cgatgataga
     7141 tgtagcgtac cgagaacttt gtgactacat gaataatcgc gagcacttga tgcacatgga
     7201 cagggaagcg atttcatgcc ccccactcgg ttcaaagggc gtagggggtt tctggaaagc
     7261 taagaaacgg tacgccctca acgtctatga catggaagac aagaggttcg cggaacctca
     7321 tttgaagata atggggatgg agacgcaaca gtcctcaact ccaaaggctg tgcaagaggc
     7381 tctggaagaa agcatacgac gcatactcca ggagggggaa gagagtgttc aggagtatta
     7441 taaaaacttt gaaaaggagt accgccagct tgactacaag gtaatcgcgg aggttaagac
     7501 tgcgaatgat atcgccaaat atgatgataa aggatggccc ggtttcaaat gccctttcca
     7561 tatacgaggg gtcctcacct accgccgcgc cgtgtctggt ctgggggtcg caccaattct
     7621 cgacggaaat aaggttatgg tactcccact ccgcgagggg aatccgtttg gtgacaaatg
     7681 catcgcctgg ccgtctggta cggagctccc caaggaaata cgcagcgacg tcctcagttg
     7741 gatcgaccac tccacactgt ttcagaagtc attcgttaaa cctctggccg ggatgtgtga
     7801 atccgcgggt atggactacg aggagaaagc ttcattggac tttcttttcg gtggtggttc
     7861 tccaaagaag aagcggaagg tctagcctag agctcgctga tcagcctcga ctgtgccttc
     7921 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     7981 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     8041 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     8101 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc
     8161 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     8221 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     8281 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     8341 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     8401 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     8461 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     8521 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     8581 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     8641 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     8701 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     8761 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     8821 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     8881 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     8941 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     9001 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     9061 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     9121 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     9181 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     9241 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     9301 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     9361 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     9421 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     9481 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     9541 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     9601 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     9661 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     9721 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     9781 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     9841 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     9901 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     9961 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
    10021 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
    10081 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
    10141 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
    10201 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
    10261 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
    10321 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
    10381 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
    10441 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
    10501 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
    10561 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
    10621 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
    10681 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
    10741 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
    10801 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
    10861 atgtcagaca aatggctcta gagaccggcg ccgctacagg gcgcgtccca ttcgccattc
    10921 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
    10981 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
    11041 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
    11101 ggtaccgggc cccccctcga ggtcctccag cttttgttcc ctttagtgag ggttaattgc
    11161 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
    11221 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
    11281 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc caccggtggt ctcttctag
//
