LOCUS       sequence-497001-        9983 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Expresses EPEC DarT2(G49D)-nSpCas9(D10A) and sg#2 targeting PTEN 
            gene in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9983)
  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 9983)
  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-6756
FEATURES             Location/Qualifiers
     source          1..9983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(71..90)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     281..302
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(456..475)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     575..597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(635..653)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        721..825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(826..894,895..1686)
                     /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(1044..1063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1857..2445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2346..2365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2507..2755
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2507..2527
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2678..2697
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2777..2852
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2949..3234
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          3459..3686
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      3698..3707
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3707..3772
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3779..3799
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4571..4594,4595..4597,4598..8671)
                     /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             4571..8671
                     /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             8672..8719
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8735..8788
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(8789..8791,8792..9502)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(8789..8791,8792..9502)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9092..9111)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9439..9460
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(9530..9547)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9536..9743
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   9752..9892
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   9752..9881
                     /label=AAV2 ITR
     rep_origin      join(9967..9983,1..439)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
       61 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      121 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      181 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      241 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      301 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
      361 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      421 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
      481 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
      541 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
      601 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
      661 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
      721 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      781 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
      841 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      901 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      961 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1021 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1081 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1141 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     1201 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     1261 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1321 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     1381 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1441 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1501 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     1561 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1621 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1681 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     1741 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     1801 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1861 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1921 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1981 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2041 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2101 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     2161 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     2221 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     2281 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     2341 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2401 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2461 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc
     2521 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     2581 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2641 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2701 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgaata
     2761 tatcttcacc tttagcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2821 atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta gaaatagcaa
     2881 gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca aatggctcta
     2941 gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3001 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3061 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3121 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
     3181 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     3241 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     3301 tatttattta ttttttaatt attttgtgca gcgatggggg cggggcgagg cggagaggtg
     3361 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     3421 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     3481 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     3541 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     3601 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     3661 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
     3721 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     3781 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgcctatg attactcagc
     3841 cagcttgaat ccacagaaag ctcttatctg gcgcattgtc catcgtgata acattccttg
     3901 gatcttagac aatggtctgc actgcggtaa tagcttggtt caagccgaga actggattaa
     3961 catcgacaat cccgagttga ttggtaaacg cgctgggcac ccggttcccg taggcaccgg
     4021 gggtacgttg cacgattatg tcccgttcta ctttacgcct tttagtccta tgcttatgaa
     4081 tatccattca ggccgcggcg gcattaaacg ccgccccaat gaagagattg taatcttagt
     4141 tagcaactta cgcaacgtcg cagcccatga cgtgcccttt gtttttaccg actcacacgc
     4201 ctattataat tggacaaact actatacgtc gttaaactca ttggaccaga tcgattggcc
     4261 aattttgcaa gcccgcgatt tccgccgtga cccagatgac ccggctaagt ttgaacgcta
     4321 ccaagcagag gccttaatct ggcaacactg tccgatctca ttacttgacg gtatcatctg
     4381 ttactcggaa gaggtccgct tgcagttgga gcagtggttg tttcaacgta atttgacaat
     4441 gtcagtacac acccgttctg gctggtattt ttcgagtggc ggatcgtctg gtggcagttc
     4501 aggctcggaa acaccaggca caagcgagtc cgcgacaccc gaatcgagtg gagggagctc
     4561 cgggggttcc gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
     4621 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     4681 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     4741 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     4801 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     4861 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     4921 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     4981 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     5041 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     5101 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     5161 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     5221 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     5281 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     5341 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     5401 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     5461 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     5521 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     5581 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     5641 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     5701 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     5761 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     5821 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     5881 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     5941 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     6001 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     6061 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     6121 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     6181 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     6241 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     6301 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     6361 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     6421 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     6481 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     6541 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     6601 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     6661 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     6721 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     6781 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     6841 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     6901 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     6961 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     7021 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     7081 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     7141 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     7201 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     7261 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     7321 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     7381 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     7441 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     7501 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     7561 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     7621 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     7681 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     7741 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     7801 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     7861 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     7921 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     7981 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     8041 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     8101 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     8161 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     8221 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     8281 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     8341 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     8401 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     8461 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     8521 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     8581 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     8641 acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg
     8701 ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg
     8761 cggtgacgtc gaggagaatc ctggcccagt gagcaagggc gaggagctgt tcaccggggt
     8821 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     8881 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     8941 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     9001 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     9061 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     9121 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     9181 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     9241 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     9301 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     9361 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     9421 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     9481 cggcatggac gagctgtaca aggaattcta actagagctc gctgatcagc ctcgactgtg
     9541 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     9601 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     9661 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     9721 gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact
     9781 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     9841 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     9901 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     9961 catagtacgc gccctgtagc ggc
//
