LOCUS       sequence-374863-        9611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9-POLB (iFE).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9611)
  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 9611)
  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: SpCas9 iFE
FEATURES             Location/Qualifiers
     source          1..9611
                     /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             6134..6154
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6177..6194)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6183..6390
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6399..6539
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6399..6528
                     /label=AAV2 ITR
     rep_origin      6614..7069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6701..6720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6911..6932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7086..7105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7205..7227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7265..7283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7351..7455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7456..7524,7525..8316)
                     /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(7674..7693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8487..9075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8976..8995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9150..9455)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     9305..9327
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     9319..9336
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9320..9336
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9346..9365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9346..9364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9412..9432)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9412..9430)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9451..9467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9451..9467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9464..9486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9475..9491
                     /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(9499..9505,9506..9523,9524..9529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9544..9565
                     /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 ggggggsgcg
      421 cgccgggggg gggggggggg gggggggggg gggggggggs 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 aaaaagggcg gcggatctgg
     5101 tggtgggtct ggcggaggct cgtctaagcg aaaggctccg caggagaccc ttaacggtgg
     5161 catcacggat atgctgacgg agctggctaa ttttgaaaaa aatgtttcac aagctatcca
     5221 caagtacaac gcctacagaa aagctgcttc tgtaatagcc aaatatcctc ataaaatcaa
     5281 atcaggggcc gaggccaaaa aactgccggg ggtggggacg aaaatcgcag aaaaaataga
     5341 tgagtttttg gcgacgggta aattgcgaaa gctggaaaag atcaggcagg acgacacctc
     5401 ttctagtatt aatttcctca cgagagtaag cggtatcgga cctagtgccg ccaggaagtt
     5461 cgtagatgag gggattaaga cattggaaga tctccggaag aacgaggaca agttgaatca
     5521 tcaccagcga ataggactta aatatttcgg agacttcgaa aagcgcattc cacgagagga
     5581 gatgctgcaa atgcaagaca tagtgcttaa tgaagtcaaa aaagtagaca gcgagtacat
     5641 agctactgtc tgcggatctt ttaggcgggg tgctgagtct tctggtgata tggatgtact
     5701 gctgactcat ccatctttta caagcgagag cactaagcaa ccaaaactgc ttcaccaagt
     5761 cgtcgagcaa ctgcagaagg tacactttat cactgacact ctgtctaaag gtgaaacgaa
     5821 gttcatgggc gtctgccagt tgccgagtaa aaatgatgaa aaagaatatc cgcatcggag
     5881 aatagacatc cgactcatcc cgaaggacca atactactgt ggggtcttgt acttcacggg
     5941 ctcagacatt ttcaacaaga atatgagggc acacgccctg gaaaagggtt ttacaataaa
     6001 tgagtacacg atcagacctc tgggagtcac cggggttgcg ggagagccgc ttccggttga
     6061 ctcagaaaag gacattttcg actatatcca gtggaaatac agagaaccaa aggacagaag
     6121 tgagggtggt tctccaaaga agaagcggaa ggtctagcct agagctcgct gatcagcctc
     6181 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     6241 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     6301 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     6361 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     6421 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     6481 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     6541 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     6601 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6661 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     6721 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6781 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     6841 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     6901 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     6961 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     7021 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     7081 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     7141 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     7201 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     7261 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     7321 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7381 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     7441 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7501 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7561 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7621 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7681 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     7741 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7801 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7861 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     7921 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     7981 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     8041 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     8101 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     8161 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     8221 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     8281 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8341 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8401 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8461 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     8521 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     8581 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     8641 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     8701 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     8761 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     8821 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     8881 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     8941 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     9001 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     9061 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     9121 ccttttgctc acatgtcaga caaatggctc tagagaccgg cgccgctaca gggcgcgtcc
     9181 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     9241 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     9301 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     9361 tagggcgaat tgggtaccgg gccccccctc gaggtcctcc agcttttgtt ccctttagtg
     9421 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     9481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     9541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccaccggtg
     9601 gtctcttcta g
//
