LOCUS       sequence-497004-       10211 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Expresses EPEC DarT2(G49D)-nSpCas9(D10A) in mammalian cells and 
            gfp-dropout cassette for cloning sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10211)
  AUTHORS   Gupta D, Patinios C, Bassett HV, Kibe A, Collins SP, Kamm C, Wang Y,
            Zhao C, Vollen K, Toussaint C, Calvin I, Cullot G, Aird EJ, Polkoff 
            KM, Nguyen-Vo TP, Migur A, Schut F, Al'Abri IS, Achmedov T, Del Re 
            A, Corn JE, Saliba AE, Crook N, Stepanova AN, Alonso JM, Beisel CL
  TITLE     Targeted DNA ADP-ribosylation triggers templated repair in bacteria 
            and base mutagenesis in eukaryotes.
  JOURNAL   Nat Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w.
  PUBMED    40908325
REFERENCE   2  (bases 1 to 10211)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w."
COMMENT     Sequence Label: pCBS-4559
FEATURES             Location/Qualifiers
     source          1..10211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      171..759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     660..679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        821..1069
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     821..841
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     992..1011
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     RBS             1228..1239
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1246..1959
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1971..2042
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2058..2085
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2102..2177
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2274..2559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2784..3011
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      3023..3032
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3032..3097
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3104..3124
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3896..3919,3920..3922,3923..7996)
                     /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             3896..7996
                     /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             7997..8044
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8072..8089)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8078..8285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8294..8434
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8294..8423
                     /label=AAV2 ITR
     rep_origin      8509..8964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8596..8615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8806..8827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8981..9000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9100..9122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9160..9178)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9246..9350
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9351..9419,9420..10211)
                     /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(9569..9588)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
       61 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      121 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      181 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      241 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      301 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      361 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      421 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      481 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      541 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      601 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
      661 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
      721 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
      781 ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat
      841 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
      901 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
      961 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     1021 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggtcttcgtg
     1081 aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt
     1141 gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac
     1201 ggcaggttcg gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa
     1261 gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat
     1321 aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag
     1381 ttcatctgta ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact
     1441 tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag
     1501 tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg
     1561 tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg
     1621 aaaggcattg actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt
     1681 aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt
     1741 aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac
     1801 actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc
     1861 gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc
     1921 gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac
     1981 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2041 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat agtgaagacc
     2101 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     2161 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
     2221 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca
     2281 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     2341 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     2401 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     2461 gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact
     2521 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     2581 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     2641 taattatttt gtgcagcgat gggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     2701 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     2761 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     2821 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     2881 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     2941 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     3001 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     3061 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     3121 ggtcggtatc cacggagtcc cagcagccgc ctatgattac tcagccagct tgaatccaca
     3181 gaaagctctt atctggcgca ttgtccatcg tgataacatt ccttggatct tagacaatgg
     3241 tctgcactgc ggtaatagct tggttcaagc cgagaactgg attaacatcg acaatcccga
     3301 gttgattggt aaacgcgctg ggcacccggt tcccgtaggc accgggggta cgttgcacga
     3361 ttatgtcccg ttctacttta cgccttttag tcctatgctt atgaatatcc attcaggccg
     3421 cggcggcatt aaacgccgcc ccaatgaaga gattgtaatc ttagttagca acttacgcaa
     3481 cgtcgcagcc catgacgtgc cctttgtttt taccgactca cacgcctatt ataattggac
     3541 aaactactat acgtcgttaa actcattgga ccagatcgat tggccaattt tgcaagcccg
     3601 cgatttccgc cgtgacccag atgacccggc taagtttgaa cgctaccaag cagaggcctt
     3661 aatctggcaa cactgtccga tctcattact tgacggtatc atctgttact cggaagaggt
     3721 ccgcttgcag ttggagcagt ggttgtttca acgtaatttg acaatgtcag tacacacccg
     3781 ttctggctgg tatttttcga gtggcggatc gtctggtggc agttcaggct cggaaacacc
     3841 aggcacaagc gagtccgcga cacccgaatc gagtggaggg agctccgggg gttccgacaa
     3901 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga
     3961 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     4021 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     4081 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     4141 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     4201 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     4261 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     4321 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     4381 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     4441 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     4501 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     4561 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     4621 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     4681 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     4741 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     4801 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     4861 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     4921 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     4981 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     5041 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     5101 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     5161 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     5221 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     5281 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     5341 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     5401 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     5461 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     5521 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     5581 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     5641 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     5701 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     5761 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     5821 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     5881 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     5941 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     6001 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     6061 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     6121 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     6181 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     6241 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     6301 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     6361 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     6421 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     6481 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     6541 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     6601 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     6661 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     6721 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     6781 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     6841 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     6901 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     6961 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     7021 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     7081 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     7141 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     7201 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     7261 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     7321 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     7381 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     7441 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     7501 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     7561 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     7621 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     7681 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     7741 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     7801 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     7861 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     7921 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     7981 tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     8041 aaagtaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     8101 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     8161 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     8221 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     8281 ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     8341 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     8401 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     8461 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     8521 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     8581 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     8641 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     8701 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     8761 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     8821 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     8881 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     8941 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     9001 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     9061 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     9121 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     9181 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     9241 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     9301 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     9361 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     9421 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     9481 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     9541 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     9601 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     9661 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     9721 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     9781 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     9841 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     9901 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     9961 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
    10021 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
    10081 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
    10141 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
    10201 agcattggta a
//
