LOCUS       sequence-256194-       11516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_human_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA11 targeting human TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11516)
  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 11516)
  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)-SghuTSDR-11
FEATURES             Location/Qualifiers
     source          1..11516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        83..331
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     83..103
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     254..273
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        353..428
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        525..810
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1200..1427
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1448..1474
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1478..1498
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1508..1531,1532..1534,1535..4090,4091..4093,
                     4094..5608)
                     /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(1508..1531,1532..1534,1535..4021,4022..4024,
                     4025..5608)
                     /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             7850..7903
                     /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             7904..8611
                     /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(8046..8064)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8639..8656)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8645..8852
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8861..9001
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8861..8990
                     /label=AAV2 ITR
     rep_origin      9076..9531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9163..9182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9373..9394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9548..9567)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9667..9689
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9727..9745)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9813..9917
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9918..9986,9987..10778)
                     /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(10136..10155)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(10949..11516,1..21)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11438..11457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
       61 tgctggcctt ttgctcacat gtgagggcct atttcccatg attccttcat atttgcatat
      121 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      181 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      241 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      301 tttatatatc ttgtggaaag gacgaaacac cgagtcgggg gctgtgacaa cagttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      421 gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt
      481 tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg
      541 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg
      601 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      661 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      721 tggcccgcct ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac
      781 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
      841 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
      901 tgtgcagcga tgggggcggg gggggggggg ggsgcgcgcc rggggggggg gggggggggg
      961 gggggggggg gggggggggg gggggggggg gggggggggg sgranagggg gggggggggg
     1021 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     1081 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     1141 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     1201 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     1261 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     1321 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat
     1381 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc
     1441 caccatgtac ccatacgatg ttccagatta cgcttcgccg aagaaaaagc gcaaggtcga
     1501 agcgtccgac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
     1561 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     1621 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     1681 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     1741 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     1801 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     1861 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     1921 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     1981 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     2041 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     2101 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     2161 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     2221 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     2281 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     2341 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     2401 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     2461 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     2521 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     2581 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     2641 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     2701 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     2761 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     2821 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     2881 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     2941 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     3001 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     3061 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     3121 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     3181 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     3241 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     3301 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     3361 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     3421 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     3481 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     3541 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     3601 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     3661 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     3721 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     3781 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     3841 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     3901 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     3961 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     4021 cgccatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     4081 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     4141 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     4201 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     4261 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     4321 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     4381 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     4441 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     4501 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     4561 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     4621 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     4681 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     4741 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     4801 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     4861 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     4921 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     4981 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     5041 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     5101 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     5161 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     5221 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     5281 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     5341 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     5401 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     5461 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     5521 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     5581 gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
     5641 gggaggagga tccctgccca cctgcagctg tcttgatcga gttatacaaa aagacaaagg
     5701 cccatattat acacaccttg gggcaggacc aagtgttgct gctgtcaggg aaatcatgga
     5761 gaataggtat ggtcaaaaag gaaacgcaat aaggatagaa atagtagtgt acaccggtaa
     5821 agaagggaaa agctctcatg ggtgtccaat tgctaagtgg gttttaagaa gaagcagtga
     5881 tgaagaaaaa gttctttgtt tggtccggca gcgtacaggc caccactgtc caactgctgt
     5941 gatggtggtg ctcatcatgg tgtgggatgg catccctctt ccaatggccg accggctata
     6001 cacagagctc acagagaatc taaagtcata caatgggcac cctaccgaca gaagatgcac
     6061 cctcaatgaa aatcgtacct gtacatgtca aggaattgat ccagagactt gtggagcttc
     6121 attctctttt ggctgttcat ggagtatgta ctttaatggc tgtaagtttg gtagaagccc
     6181 aagccccaga agatttagaa ttgatccaag ctctccctta catgaaaaaa accttgaaga
     6241 taacttacag agtttggcta cacgattagc tccaatttat aagcagtatg ctccagtagc
     6301 ttaccaaaat caggtggaat atgaaaatgt tgcccgagaa tgtcggcttg gcagcaagga
     6361 aggtcgaccc ttctctgggg tcactgcttg cctggacttc tgtgctcatc cccacaggga
     6421 cattcacaac atgaataatg gaagcactgt ggtttgtacc ttaactcgag aagataaccg
     6481 ctctttgggt gttattcctc aagatgagca gctccatgtg ctacctcttt ataagctttc
     6541 agacacagat gagtttggct ccaaggaagg aatggaagcc aagatcaaat ctggggccat
     6601 cgaggtcctg gcaccccgcc gcaaaaaaag aacgtgtttc actcagcctg ttccccgttc
     6661 tggaaagaag agggctgcga tgatgacaga ggttcttgca cataagataa gggcagtgga
     6721 aaagaaacct attccccgaa tcaagcggaa gaataactca acaacaacaa acaacagtaa
     6781 gccttcgtca ctgccaacct tagggagtaa cactgagacc gtgcaacctg aagtaaaaag
     6841 tgaaaccgaa ccccatttta tcttaaaaag ttcagacaac actaaaactt attcgctgat
     6901 gccatccgct cctcacccag tgaaagaggc atctccaggc ttctcctggt ccccgaagac
     6961 tgcttcagcc acaccagctc cactgaagaa tgacgcaaca gcctcatgcg ggttttcaga
     7021 aagaagcagc actccccact gtacgatgcc ttcgggaaga ctcagtggtg ccaatgctgc
     7081 agctgctgat ggccctggca tttcacagct tggcgaagtg gctcctctcc ccaccctgtc
     7141 tgctcctgtg atggagcccc tcattaattc tgagccttcc actggtgtga ctgagccgct
     7201 aacgcctcat cagccaaacc accagccctc cttcctcacc tctcctcaag accttgcctc
     7261 ttctccaatg gaagaagatg agcagcattc tgaagcagat gagcctccat cagacgaacc
     7321 cctatctgat gaccccctgt cacctgctga ggagaaattg ccccacattg atgagtattg
     7381 gtcagacagt gagcacatct ttttggatgc aaatattggt ggggtggcca tcgcacctgc
     7441 tcacggctcg gttttgattg agtgtgcccg gcgagagctg cacgctacca ctcctgttga
     7501 gcaccccaac cgtaatcatc caacccgcct ctcccttgtc ttttaccagc acaaaaacct
     7561 aaataagccc caacatggtt ttgaactaaa caagattaag tttgaggcta aagaagctaa
     7621 gaataagaaa atgaaggcct cagagcaaaa agaccaggca gctaatgaag gtccagaaca
     7681 gtcctctgaa gtaaatgaat tgaaccaaat tccttctcat aaagcattaa cattaaccca
     7741 tgacaatgtt gtcaccgtgt ccccttatgc tctcacacac gttgcggggc cctataacca
     7801 ttgggtcatg gccggccagg caaaaaagaa aaaggaattc ggcagtggag agggcagagg
     7861 aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca agggcgagga
     7921 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     7981 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     8041 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     8101 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     8161 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     8221 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     8281 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga aaaccatggg
     8341 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     8401 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     8461 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     8521 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     8581 caccggcggc atggacgagc tgtacaagta agaattctaa ctagagctcg ctgatcagcc
     8641 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     8701 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     8761 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     8821 gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag
     8881 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     8941 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     9001 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     9061 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     9121 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     9181 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     9241 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     9301 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     9361 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     9421 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     9481 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     9541 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     9601 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     9661 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     9721 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     9781 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     9841 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     9901 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     9961 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    10021 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    10081 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    10141 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    10201 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    10261 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    10321 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    10381 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    10441 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    10501 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    10561 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    10621 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
    10681 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    10741 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    10801 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    10861 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    10921 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    10981 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    11041 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    11101 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    11161 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    11221 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    11281 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    11341 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    11401 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    11461 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtca
//
