LOCUS       sequence-249107-       12299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eSpCas9(1.1) and sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12299)
  AUTHORS   Adachi K, Kopp W, Wu G, Heising S, Greber B, Stehling M, 
            Arauzo-Bravo MJ, Boerno ST, Timmermann B, Vingron M, Scholer HR
  TITLE     Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive 
            Pluripotency.
  JOURNAL   Cell Stem Cell. 2018 Aug 2;23(2):266-275.e6. doi: 
            10.1016/j.stem.2018.05.020. Epub 2018 Jun 14.
  PUBMED    29910149
REFERENCE   2  (bases 1 to 12299)
  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: 
            "10.1016/j.stem.2018.05.020"; journalName: "Cell Stem Cell"; date: 
            "2018-08-2- 2"; volume: "23"; issue: "2"; pages: "266-275.e6"
COMMENT     Sequence Label: KA2963_eSpCas9-PGKHygdtkCh
FEATURES             Location/Qualifiers
     source          1..12299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        74..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     74..94
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     245..264
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        341..416
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        513..798
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        800..1073
                     /label=chicken beta-actin promoter
     intron          1074..1301
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1313..1322
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1322..1387
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1394..1414
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1439..3976,3977..3979,3980..4441,4442..4444,4445..4612,
                     4613..4615,4616..5539)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5540..5587
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5615..5632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5621..5828
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5944..6443
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5979..6002)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6376..6395
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6462..7484
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             6462..7484
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             7500..8492
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     CDS             8499..8555
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             8556..9263
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(8701..8719)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     8969..8988
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    9578..9699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9615..9634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9669..9688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9850..10305
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9937..9956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10147..10168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10322..10341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10441..10463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10501..10519)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10587..10691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10692..10760,10761..11552)
                     /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(10910..10929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(11723..12299,1..12)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12212..12231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
       61 tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      121 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      181 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      241 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      301 cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc
      361 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      421 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
      481 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
      541 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
      601 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      661 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      721 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      781 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      841 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      901 ggggcggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
      961 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1021 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1081 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1141 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1201 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     1261 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat
     1321 ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga
     1381 cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga
     1441 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     1501 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     1561 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     1621 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     1681 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     1741 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     1801 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     1861 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     1921 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     1981 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     2041 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     2101 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     2161 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     2221 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     2281 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     2341 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     2401 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     2461 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     2521 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     2581 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     2641 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     2701 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     2761 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     2821 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     2881 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     2941 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     3001 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     3061 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     3121 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     3181 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     3241 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     3301 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     3361 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     3421 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     3481 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     3541 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     3601 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     3661 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     3721 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     3781 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     3841 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     3901 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     3961 gcctcagagc tttctggccg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     4021 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     4081 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     4141 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     4201 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     4261 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     4321 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     4381 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     4441 tgcgctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     4501 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     4561 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga aggcgcctct
     4621 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     4681 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     4741 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     4801 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     4861 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     4921 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     4981 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     5041 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     5101 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     5161 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     5221 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     5281 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     5341 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     5401 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     5461 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     5521 gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     5581 gaaaaagtaa gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     5641 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5701 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5761 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     5821 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5881 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctg caggaattct
     5941 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     6001 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta
     6061 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag
     6121 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag
     6181 cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc
     6241 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa
     6301 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt
     6361 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     6421 cttcctcatc tccgggcctt tcgaccgatc cagccgccac catgaaaaag cctgaactca
     6481 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc
     6541 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg
     6601 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact
     6661 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc
     6721 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg
     6781 aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc
     6841 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacgt
     6901 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg
     6961 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg
     7021 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg
     7081 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat
     7141 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc
     7201 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc
     7261 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag
     7321 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta
     7381 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg
     7441 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaaggatct ggaagatcca
     7501 tgcccacgct actgcgggtt tatatagacg gtccccacgg gatggggaaa accaccacca
     7561 cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt ctacgtaccc gagccgatga
     7621 cttactggcg ggtgctgggg gcttccgaga caatcgcgaa catctacacc acacaacacc
     7681 gcctcgacca gggtgagata tcggccgggg acgcggcggt ggtaatgaca agcgcccaga
     7741 taacaatggg catgccttat gccgtgaccg acgccgttct ggctcctcat atcggggggg
     7801 aggctgggag ctcacatgcc ccgcccccgg ccctcaccct catcttcgac cgccatccca
     7861 tcgccgccct cctgtgctac ccggccgcgc ggtaccttat gggcagcatg accccccagg
     7921 ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc cggcaccaac atcgtgcttg
     7981 gggcccttcc ggaggacaga cacatcgacc gcctggccaa acgccagcgc cccggcgagc
     8041 ggctggacct ggctatgctg gctgcgattc gccgcgttta cgggctactt gccaatacgg
     8101 tgcggtatct gcagtgcggc gggtcgtggc gggaggactg gggacagctt tcggggacgg
     8161 ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg cccacgaccc catatcgggg
     8221 acacgttatt taccctgttt cgggcccccg agttgctggc ccccaacggc gacctgtata
     8281 acgtgtttgc ctgggccttg gacgtcttgg ccaaacgcct ccgttccatg cacgtcttta
     8341 tcctggatta cgaccaatcg cccgccggct gccgggacgc cctgctgcaa cttacctccg
     8401 ggatggtcca gacccacgtc accacccccg gctccatacc gacgatatgc gacctggcgc
     8461 gcacgtttgc ccgggagatg ggggaggcta acgctagtgc caccaacttc tccctgctga
     8521 agcaggccgg cgacgtggag gagaaccccg gccccatggt gagcaagggc gaggaggata
     8581 acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg
     8641 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     8701 ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc
     8761 agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     8821 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     8881 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     8941 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     9001 aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga
     9061 ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca
     9121 agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc
     9181 acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg
     9241 gcggcatgga cgagctgtac aaggctagct gagcggccgc tgtacaagta aagcggccag
     9301 cttatcgact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     9361 agtcgagaaa ttgatgatct attaaacaat aaagatgtcc acatggaagt ttttcctgtc
     9421 atactttgtt aagaagggtg agaacagagt acctacattt tgaatggaag gattggagct
     9481 acgggggtgg gggtggggtg ggattagata aatgcctgct ctttactgaa ggctctttac
     9541 tattgcttta tgataatgtt tcatagttgg ataactaaac ttgtttattg cagcttataa
     9601 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     9661 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga attgactcaa
     9721 atgatgtcaa ttagtctatc agaagctcat ctggtctccc ttccgggcct gcaggggcgc
     9781 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     9841 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     9901 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     9961 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
    10021 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
    10081 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
    10141 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
    10201 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
    10261 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
    10321 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
    10381 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
    10441 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
    10501 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
    10561 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
    10621 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
    10681 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
    10741 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
    10801 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
    10861 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
    10921 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
    10981 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
    11041 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
    11101 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
    11161 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
    11221 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
    11281 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
    11341 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
    11401 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
    11461 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
    11521 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
    11581 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
    11641 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
    11701 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
    11761 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
    11821 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
    11881 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
    11941 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
    12001 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
    12061 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
    12121 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
    12181 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    12241 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgg
//
