LOCUS       sequence-256272-       11375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_mouse_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA10 targeting mouse TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11375)
  AUTHORS   Kressler C, Gasparoni G, Nordstrom K, Hamo D, Salhab A, 
            Dimitropoulos C, Tierling S, Reinke P, Volk HD, Walter J, Hamann A, 
            Polansky JK
  TITLE     Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce 
            Physiological FOXP3 Expression but Not a Functional Treg Phenotype.
  JOURNAL   Front Immunol. 2021 Jan 7;11:609891. doi: 10.3389/fimmu.2020.609891.
            eCollection 2020.
  PUBMED    33488615
REFERENCE   2  (bases 1 to 11375)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front 
            Immunol."; date: "2021-01-7"; volume: "11:609891. doi"; pages: " 
            10.3389/fimmu.2020.609891. eCollection 2020"
COMMENT     Sequence Label: pSpdCas9-huTET1CD-T2A-mCherry(PX458)-SgmouseTS...
FEATURES             Location/Qualifiers
     source          1..11375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          133..360
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             381..407
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             411..431
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(441..464,465..467,468..3023,3024..3026,3027..4541)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(441..464,465..467,468..2954,2955..2957,2958..4541)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6783..6836
                     /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             6837..7544
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(6979..6997)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7572..7589)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7578..7785
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7794..7934
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7794..7923
                     /label=AAV2 ITR
     rep_origin      8009..8464
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8096..8115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8306..8327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8481..8500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8600..8622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8660..8678)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8746..8850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8851..8919,8920..9711)
                     /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(9069..9088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9882..10470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10371..10390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10532..10780
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10532..10552
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10703..10722
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10802..10877
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        10974..11259
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
       61 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      121 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      181 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      241 ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg
      301 gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag
      361 gttggaccgg tgccaccatg tacccatacg atgttccaga ttacgcttcg ccgaagaaaa
      421 agcgcaaggt cgaagcgtcc gacaagaagt acagcatcgg cctggccatc ggcaccaact
      481 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
      541 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
      601 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
      661 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
      721 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
      781 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
      841 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
      901 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
      961 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     1021 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     1081 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     1141 atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     1201 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     1261 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     1321 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     1381 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     1441 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     1501 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     1561 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     1621 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     1681 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     1741 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     1801 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     1861 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     1921 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     1981 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     2041 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     2101 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     2161 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     2221 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     2281 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     2341 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     2401 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     2461 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     2521 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     2581 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     2641 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     2701 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     2761 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     2821 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     2881 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     2941 actacgatgt ggacgccatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca
     3001 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     3061 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     3121 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     3181 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     3241 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     3301 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     3361 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     3421 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     3481 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     3541 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     3601 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     3661 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     3721 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     3781 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     3841 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     3901 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     3961 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     4021 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     4081 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     4141 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     4201 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     4261 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     4321 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     4381 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     4441 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     4501 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga
     4561 aaaaggccgg ccagggagga ggatccctgc ccacctgcag ctgtcttgat cgagttatac
     4621 aaaaagacaa aggcccatat tatacacacc ttggggcagg accaagtgtt gctgctgtca
     4681 gggaaatcat ggagaatagg tatggtcaaa aaggaaacgc aataaggata gaaatagtag
     4741 tgtacaccgg taaagaaggg aaaagctctc atgggtgtcc aattgctaag tgggttttaa
     4801 gaagaagcag tgatgaagaa aaagttcttt gtttggtccg gcagcgtaca ggccaccact
     4861 gtccaactgc tgtgatggtg gtgctcatca tggtgtggga tggcatccct cttccaatgg
     4921 ccgaccggct atacacagag ctcacagaga atctaaagtc atacaatggg caccctaccg
     4981 acagaagatg caccctcaat gaaaatcgta cctgtacatg tcaaggaatt gatccagaga
     5041 cttgtggagc ttcattctct tttggctgtt catggagtat gtactttaat ggctgtaagt
     5101 ttggtagaag cccaagcccc agaagattta gaattgatcc aagctctccc ttacatgaaa
     5161 aaaaccttga agataactta cagagtttgg ctacacgatt agctccaatt tataagcagt
     5221 atgctccagt agcttaccaa aatcaggtgg aatatgaaaa tgttgcccga gaatgtcggc
     5281 ttggcagcaa ggaaggtcga cccttctctg gggtcactgc ttgcctggac ttctgtgctc
     5341 atccccacag ggacattcac aacatgaata atggaagcac tgtggtttgt accttaactc
     5401 gagaagataa ccgctctttg ggtgttattc ctcaagatga gcagctccat gtgctacctc
     5461 tttataagct ttcagacaca gatgagtttg gctccaagga aggaatggaa gccaagatca
     5521 aatctggggc catcgaggtc ctggcacccc gccgcaaaaa aagaacgtgt ttcactcagc
     5581 ctgttccccg ttctggaaag aagagggctg cgatgatgac agaggttctt gcacataaga
     5641 taagggcagt ggaaaagaaa cctattcccc gaatcaagcg gaagaataac tcaacaacaa
     5701 caaacaacag taagccttcg tcactgccaa ccttagggag taacactgag accgtgcaac
     5761 ctgaagtaaa aagtgaaacc gaaccccatt ttatcttaaa aagttcagac aacactaaaa
     5821 cttattcgct gatgccatcc gctcctcacc cagtgaaaga ggcatctcca ggcttctcct
     5881 ggtccccgaa gactgcttca gccacaccag ctccactgaa gaatgacgca acagcctcat
     5941 gcgggttttc agaaagaagc agcactcccc actgtacgat gccttcggga agactcagtg
     6001 gtgccaatgc tgcagctgct gatggccctg gcatttcaca gcttggcgaa gtggctcctc
     6061 tccccaccct gtctgctcct gtgatggagc ccctcattaa ttctgagcct tccactggtg
     6121 tgactgagcc gctaacgcct catcagccaa accaccagcc ctccttcctc acctctcctc
     6181 aagaccttgc ctcttctcca atggaagaag atgagcagca ttctgaagca gatgagcctc
     6241 catcagacga acccctatct gatgaccccc tgtcacctgc tgaggagaaa ttgccccaca
     6301 ttgatgagta ttggtcagac agtgagcaca tctttttgga tgcaaatatt ggtggggtgg
     6361 ccatcgcacc tgctcacggc tcggttttga ttgagtgtgc ccggcgagag ctgcacgcta
     6421 ccactcctgt tgagcacccc aaccgtaatc atccaacccg cctctccctt gtcttttacc
     6481 agcacaaaaa cctaaataag ccccaacatg gttttgaact aaacaagatt aagtttgagg
     6541 ctaaagaagc taagaataag aaaatgaagg cctcagagca aaaagaccag gcagctaatg
     6601 aaggtccaga acagtcctct gaagtaaatg aattgaacca aattccttct cataaagcat
     6661 taacattaac ccatgacaat gttgtcaccg tgtcccctta tgctctcaca cacgttgcgg
     6721 ggccctataa ccattgggtc atggccggcc aggcaaaaaa gaaaaaggaa ttcggcagtg
     6781 gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga
     6841 gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     6901 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     6961 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     7021 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     7081 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     7141 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     7201 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     7261 agaaaaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga
     7321 agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca
     7381 agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca
     7441 agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg
     7501 agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaagaattc taactagagc
     7561 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     7621 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     7681 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     7741 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     7801 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     7861 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     7921 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     7981 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     8041 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     8101 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     8161 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8221 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8281 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8341 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     8401 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     8461 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     8521 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     8581 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     8641 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     8701 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     8761 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     8821 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     8881 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     8941 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     9001 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     9061 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     9121 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     9181 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     9241 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     9301 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     9361 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     9421 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     9481 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     9541 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     9601 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     9661 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     9721 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     9781 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     9841 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     9901 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     9961 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    10021 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    10081 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    10141 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    10201 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    10261 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    10321 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    10381 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    10441 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    10501 ctggcctttt gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata
    10561 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
    10621 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
    10681 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
    10741 tttcttggct ttatatatct tgtggaaagg acgaaacacc gttctgccat tgacgtcatg
    10801 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
    10861 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
    10921 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca
    10981 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
    11041 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
    11101 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
    11161 gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact
    11221 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
    11281 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
    11341 taattatttt gtgcagcgat gggggcgggg ggggg
//
