LOCUS       sequence-167644-        9478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9 fused to DHFR domain on both N- and C-termini in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9478)
  AUTHORS   Maji B, Moore CL, Zetsche B, Volz SE, Zhang F, Shoulders MD, 
            Choudhary A
  TITLE     Multidimensional chemical control of CRISPR-Cas9.
  JOURNAL   Nat Chem Biol. 2017 Jan;13(1):9-11. doi: 10.1038/nchembio.2224. Epub
            2016 Oct 31.
  PUBMED    27820801
REFERENCE   2  (bases 1 to 9478)
  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"; doi: 
            "10.1038/nchembio.2224"; journalName: "Nat Chem Biol"; date: 
            "2017-01"; volume: "13"; issue: "1"; pages: "9-11"
COMMENT     Sequence Label: DHFR-SpCas9-DHFR
FEATURES             Location/Qualifiers
     source          1..9478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1254..1319
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1326..1346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1377..1850
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1869..5969
                     /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             1869..5969
                     /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             5970..6017
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6018..6494
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     primer_bind     complement(6519..6536)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6525..6732
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6741..6881
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6741..6870
                     /label=AAV2 ITR
     rep_origin      6956..7411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7043..7062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7253..7274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7428..7447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7547..7569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7607..7625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7693..7797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7798..7866,7867..8658)
                     /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(8016..8035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8829..9417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9318..9337
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
     1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc ggatccatca
     1381 gtctgattgc ggcgttagcg gtagattacg ttatcggcat ggaaaacgcc atgccgtgga
     1441 acctgcctgc cgatctcgcc tggtttaaac gcaacacctt aaataaaccc gtgattatgg
     1501 gccgccatac ctgggaatca atcggtcgtc cgttgccagg acgcaaaaat attatcctca
     1561 gcagtcaacc gagtacggac gatcgcgtaa cgtgggtgaa gtcggtggat gaagccatcg
     1621 cggcgtgtgg tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt attgaacagt
     1681 tcttgccaaa agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg gaaggcgaca
     1741 cccatttccc ggattacgag ccggatgact gggaatcggt attcagcgaa ttccacgatg
     1801 ctgatgcgca gaactctcac agctattgct ttgagattct ggagcggcga ggaagcggaa
     1861 gcggatccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     1921 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     1981 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     2041 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     2101 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     2161 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     2221 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     2281 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     2341 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     2401 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     2461 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     2521 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     2581 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     2641 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     2701 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     2761 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     2821 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     2881 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     2941 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     3001 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     3061 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     3121 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     3181 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     3241 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     3301 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     3361 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     3421 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     3481 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     3541 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     3601 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     3661 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     3721 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     3781 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     3841 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     3901 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     3961 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     4021 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     4081 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     4141 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     4201 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     4261 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     4321 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     4381 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     4441 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     4501 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     4561 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     4621 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     4681 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     4741 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     4801 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     4861 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     4921 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     4981 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     5041 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     5101 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     5161 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     5221 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     5281 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     5341 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     5401 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     5461 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     5521 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     5581 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     5641 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     5701 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     5761 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     5821 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     5881 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     5941 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     6001 aggcaaaaaa gaaaaagatc agtctgattg cggcgttagc ggtagatcac gttatcggca
     6061 tggaaaccgt catgccgtgg aacctgcctg ccgatctcgc ctggtttaaa cgcaacacct
     6121 taaataaacc cgtgattatg ggccgccata cctgggaatc aatcggtcgt ccgttgccag
     6181 gacgcaaaaa tattatcctc agcagtcaac cgagtacgga cgatcgcgta acgtgggtga
     6241 agtcggtgga tgaagccatc gcggcgtgtg gtgacgtacc agaaatcatg gttattggcg
     6301 gcggtcgcgt ttatgaacag ttcttgccaa aagcgcaaaa actgtatctg acgcatatcg
     6361 acgcagaagt ggaaggcgac acccatttcc cggattacga gccggatgac tgggaatcgg
     6421 tattcagcga attccacgat gctgatgcgc agaactctca cagctattgc tttgagattc
     6481 tggagcggcg ataagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     6541 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     6601 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     6661 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     6721 gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     6781 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     6841 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     6901 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     6961 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     7021 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     7081 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     7141 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     7201 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     7261 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     7321 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     7381 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     7441 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     7501 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     7561 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     7621 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     7681 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     7741 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     7801 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     7861 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     7921 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     7981 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     8041 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     8101 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     8161 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     8221 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     8281 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     8341 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     8401 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     8461 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     8521 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     8581 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     8641 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     8701 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     8761 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     8821 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     8881 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     8941 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     9001 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     9061 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     9121 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     9181 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     9241 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     9301 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     9361 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     9421 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
