LOCUS       sequence-256140-       11355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_human_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA2 targeting human TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11355)
  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 11355)
  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-2
FEATURES             Location/Qualifiers
     source          1..11355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        73..321
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     73..93
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     244..263
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        343..418
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        515..800
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1029..1256
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1277..1303
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1307..1327
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1337..1360,1361..1363,1364..3919,3920..3922,
                     3923..5437)
                     /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(1337..1360,1361..1363,1364..3850,3851..3853,
                     3854..5437)
                     /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             7679..7732
                     /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             7733..8440
                     /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(7875..7893)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8468..8485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8474..8681
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8690..8830
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8690..8819
                     /label=AAV2 ITR
     rep_origin      8905..9360
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8992..9011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9202..9223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9377..9396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9496..9518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9556..9574)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9642..9746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9747..9815,9816..10607)
                     /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(9965..9984)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(10778..11355,1..11)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11267..11286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
       61 ttgctcacat gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag
      121 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
      181 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      241 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      301 ttgtggaaag gacgaaacac cgttatacta tttgaagacc ccgttttaga gctagaaata
      361 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      421 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      481 gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc
      541 cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga
      601 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      661 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      721 ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      781 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      841 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      901 tgggggcggg gggggggagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
      961 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     1021 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     1081 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     1141 cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg
     1201 gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg
     1261 gaccggtgcc accatgtacc catacgatgt tccagattac gcttcgccga agaaaaagcg
     1321 caaggtcgaa gcgtccgaca agaagtacag catcggcctg gccatcggca ccaactctgt
     1381 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     1441 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     1501 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     1561 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     1621 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     1681 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     1741 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     1801 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     1861 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     1921 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     1981 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     2041 cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     2101 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     2161 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     2221 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     2281 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     2341 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     2401 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     2461 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     2521 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     2581 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     2641 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     2701 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     2761 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     2821 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     2881 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     2941 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     3001 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     3061 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     3121 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     3181 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     3241 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     3301 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     3361 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     3421 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     3481 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     3541 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     3601 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     3661 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     3721 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     3781 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     3841 cgatgtggac gccatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     3901 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     3961 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     4021 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     4081 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     4141 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     4201 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     4261 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     4321 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     4381 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     4441 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     4501 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     4561 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     4621 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     4681 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     4741 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     4801 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     4861 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     4921 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     4981 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     5041 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     5101 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     5161 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     5221 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     5281 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     5341 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     5401 cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa
     5461 ggccggccag ggaggaggat ccctgcccac ctgcagctgt cttgatcgag ttatacaaaa
     5521 agacaaaggc ccatattata cacaccttgg ggcaggacca agtgttgctg ctgtcaggga
     5581 aatcatggag aataggtatg gtcaaaaagg aaacgcaata aggatagaaa tagtagtgta
     5641 caccggtaaa gaagggaaaa gctctcatgg gtgtccaatt gctaagtggg ttttaagaag
     5701 aagcagtgat gaagaaaaag ttctttgttt ggtccggcag cgtacaggcc accactgtcc
     5761 aactgctgtg atggtggtgc tcatcatggt gtgggatggc atccctcttc caatggccga
     5821 ccggctatac acagagctca cagagaatct aaagtcatac aatgggcacc ctaccgacag
     5881 aagatgcacc ctcaatgaaa atcgtacctg tacatgtcaa ggaattgatc cagagacttg
     5941 tggagcttca ttctcttttg gctgttcatg gagtatgtac tttaatggct gtaagtttgg
     6001 tagaagccca agccccagaa gatttagaat tgatccaagc tctcccttac atgaaaaaaa
     6061 ccttgaagat aacttacaga gtttggctac acgattagct ccaatttata agcagtatgc
     6121 tccagtagct taccaaaatc aggtggaata tgaaaatgtt gcccgagaat gtcggcttgg
     6181 cagcaaggaa ggtcgaccct tctctggggt cactgcttgc ctggacttct gtgctcatcc
     6241 ccacagggac attcacaaca tgaataatgg aagcactgtg gtttgtacct taactcgaga
     6301 agataaccgc tctttgggtg ttattcctca agatgagcag ctccatgtgc tacctcttta
     6361 taagctttca gacacagatg agtttggctc caaggaagga atggaagcca agatcaaatc
     6421 tggggccatc gaggtcctgg caccccgccg caaaaaaaga acgtgtttca ctcagcctgt
     6481 tccccgttct ggaaagaaga gggctgcgat gatgacagag gttcttgcac ataagataag
     6541 ggcagtggaa aagaaaccta ttccccgaat caagcggaag aataactcaa caacaacaaa
     6601 caacagtaag ccttcgtcac tgccaacctt agggagtaac actgagaccg tgcaacctga
     6661 agtaaaaagt gaaaccgaac cccattttat cttaaaaagt tcagacaaca ctaaaactta
     6721 ttcgctgatg ccatccgctc ctcacccagt gaaagaggca tctccaggct tctcctggtc
     6781 cccgaagact gcttcagcca caccagctcc actgaagaat gacgcaacag cctcatgcgg
     6841 gttttcagaa agaagcagca ctccccactg tacgatgcct tcgggaagac tcagtggtgc
     6901 caatgctgca gctgctgatg gccctggcat ttcacagctt ggcgaagtgg ctcctctccc
     6961 caccctgtct gctcctgtga tggagcccct cattaattct gagccttcca ctggtgtgac
     7021 tgagccgcta acgcctcatc agccaaacca ccagccctcc ttcctcacct ctcctcaaga
     7081 ccttgcctct tctccaatgg aagaagatga gcagcattct gaagcagatg agcctccatc
     7141 agacgaaccc ctatctgatg accccctgtc acctgctgag gagaaattgc cccacattga
     7201 tgagtattgg tcagacagtg agcacatctt tttggatgca aatattggtg gggtggccat
     7261 cgcacctgct cacggctcgg ttttgattga gtgtgcccgg cgagagctgc acgctaccac
     7321 tcctgttgag caccccaacc gtaatcatcc aacccgcctc tcccttgtct tttaccagca
     7381 caaaaaccta aataagcccc aacatggttt tgaactaaac aagattaagt ttgaggctaa
     7441 agaagctaag aataagaaaa tgaaggcctc agagcaaaaa gaccaggcag ctaatgaagg
     7501 tccagaacag tcctctgaag taaatgaatt gaaccaaatt ccttctcata aagcattaac
     7561 attaacccat gacaatgttg tcaccgtgtc cccttatgct ctcacacacg ttgcggggcc
     7621 ctataaccat tgggtcatgg ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga
     7681 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa
     7741 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     7801 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     7861 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     7921 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     7981 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     8041 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     8101 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     8161 aaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     8221 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     8281 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     8341 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     8401 ccgccactcc accggcggca tggacgagct gtacaagtaa gaattctaac tagagctcgc
     8461 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     8521 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     8581 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     8641 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     8701 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     8761 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     8821 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     8881 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     8941 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     9001 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     9061 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     9121 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     9181 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     9241 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     9301 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     9361 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     9421 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     9481 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     9541 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     9601 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     9661 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     9721 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     9781 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     9841 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     9901 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     9961 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
    10021 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
    10081 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
    10141 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
    10201 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    10261 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    10321 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    10381 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    10441 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
    10501 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    10561 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    10621 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    10681 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    10741 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    10801 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    10861 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    10921 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    10981 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    11041 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    11101 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    11161 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    11221 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    11281 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    11341 cgtcaggggg gcgga
//
