LOCUS       sequence-256220-       11349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inactivated control (targeted DNA demethylation_human_TSDR), 
            expression of dCas9-hudTET1CD-T2A-mCherry sgRNA6 targeting human 
            TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11349)
  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 11349)
  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-hudTET1CD-T2A-mCherry(PX458)-SghuTSDR-6
FEATURES             Location/Qualifiers
     source          1..11349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        59..134
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        231..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          740..967
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             988..1014
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1018..1038
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1048..1071,1072..1074,1075..3630,3631..3633,
                     3634..5148)
                     /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(1048..1071,1072..1074,1075..3561,3562..3564,
                     3565..5148)
                     /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             7390..7443
                     /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             7444..8151
                     /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(7586..7604)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8179..8196)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8185..8392
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8401..8541
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8401..8530
                     /label=AAV2 ITR
     rep_origin      8616..9071
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8703..8722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8913..8934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9088..9107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9207..9229
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9267..9285)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9353..9457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9458..9526,9527..10318)
                     /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(9676..9695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10489..11077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10978..10997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(11139..11349,1..38)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     11139..11159
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     11310..11329
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tcttggcttt atatatcttg tggaaaggac gaaacaccgc agcaggcttg gcccaggtgt
       61 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      121 caccgagtcg gtgctttttt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      181 cgtttttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa
      241 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      541 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      601 ttattttgtg cagcgatggg ggcggggggg ggggagaggt gcggcggcag ccaatcagag
      661 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
      721 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
      781 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
      841 gcgggacggc ccttctcctc cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg
      901 atggttggtt ggtggggtat taatgtttaa ttacctggag cacctgcctg aaatcacttt
      961 ttttcaggtt ggaccggtgc caccatgtac ccatacgatg ttccagatta cgcttcgccg
     1021 aagaaaaagc gcaaggtcga agcgtccgac aagaagtaca gcatcggcct ggccatcggc
     1081 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     1141 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     1201 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     1261 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     1321 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     1381 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     1441 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     1501 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     1561 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     1621 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     1681 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     1741 aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc
     1801 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     1861 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     1921 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     1981 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     2041 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     2101 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     2161 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     2221 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     2281 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     2341 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     2401 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     2461 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     2521 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     2581 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     2641 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     2701 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     2761 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     2821 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     2881 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     2941 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     3001 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     3061 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     3121 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     3181 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     3241 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     3301 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     3361 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     3421 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     3481 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     3541 ctgtccgact acgatgtgga cgccatcgtg cctcagagct ttctgaagga cgactccatc
     3601 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     3661 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     3721 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     3781 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     3841 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     3901 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     3961 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     4021 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     4081 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     4141 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     4201 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     4261 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     4321 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     4381 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     4441 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     4501 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     4561 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     4621 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     4681 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     4741 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     4801 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     4861 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     4921 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     4981 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     5041 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     5101 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
     5161 gccacgaaaa aggccggcca gggaggagga tccctgccca cctgcagctg tcttgatcga
     5221 gttatacaaa aagacaaagg cccatattat acacaccttg gggcaggacc aagtgttgct
     5281 gctgtcaggg aaatcatgga gaataggtat ggtcaaaaag gaaacgcaat aaggatagaa
     5341 atagtagtgt acaccggtaa agaagggaaa agctctcatg ggtgtccaat tgctaagtgg
     5401 gttttaagaa gaagcagtga tgaagaaaaa gttctttgtt tggtccggca gcgtacaggc
     5461 caccactgtc caactgctgt gatggtggtg ctcatcatgg tgtgggatgg catccctctt
     5521 ccaatggccg accggctata cacagagctc acagagaatc taaagtcata caatgggcac
     5581 cctaccgaca gaagatgcac cctcaatgaa aatcgtacct gtacatgtca aggaattgat
     5641 ccagagactt gtggagcttc attctctttt ggctgttcat ggagtatgta ctttaatggc
     5701 tgtaagtttg gtagaagccc aagccccaga agatttagaa ttgatccaag ctctccctta
     5761 catgaaaaaa accttgaaga taacttacag agtttggcta cacgattagc tccaatttat
     5821 aagcagtatg ctccagtagc ttaccaaaat caggtggaat atgaaaatgt tgcccgagaa
     5881 tgtcggcttg gcagcaagga aggtcgaccc ttctctgggg tcactgcttg cctggacttc
     5941 tgtgctcatc cctacagggc cattcacaac atgaataatg gaagcactgt ggtttgtacc
     6001 ttaactcgag aagataaccg ctctttgggt gttattcctc aagatgagca gctccatgtg
     6061 ctacctcttt ataagctttc agacacagat gagtttggct ccaaggaagg aatggaagcc
     6121 aagatcaaat ctggggccat cgaggtcctg gcaccccgcc gcaaaaaaag aacgtgtttc
     6181 actcagcctg ttccccgttc tggaaagaag agggctgcga tgatgacaga ggttcttgca
     6241 cataagataa gggcagtgga aaagaaacct attccccgaa tcaagcggaa gaataactca
     6301 acaacaacaa acaacagtaa gccttcgtca ctgccaacct tagggagtaa cactgagacc
     6361 gtgcaacctg aagtaaaaag tgaaaccgaa ccccatttta tcttaaaaag ttcagacaac
     6421 actaaaactt attcgctgat gccatccgct cctcacccag tgaaagaggc atctccaggc
     6481 ttctcctggt ccccgaagac tgcttcagcc acaccagctc cactgaagaa tgacgcaaca
     6541 gcctcatgcg ggttttcaga aagaagcagc actccccact gtacgatgcc ttcgggaaga
     6601 ctcagtggtg ccaatgctgc agctgctgat ggccctggca tttcacagct tggcgaagtg
     6661 gctcctctcc ccaccctgtc tgctcctgtg atggagcccc tcattaattc tgagccttcc
     6721 actggtgtga ctgagccgct aacgcctcat cagccaaacc accagccctc cttcctcacc
     6781 tctcctcaag accttgcctc ttctccaatg gaagaagatg agcagcattc tgaagcagat
     6841 gagcctccat cagacgaacc cctatctgat gaccccctgt cacctgctga ggagaaattg
     6901 ccccacattg atgagtattg gtcagacagt gagcacatct ttttggatgc aaatattggt
     6961 ggggtggcca tcgcacctgc tcacggctcg gttttgattg agtgtgcccg gcgagagctg
     7021 cacgctacca ctcctgttga gcaccccaac cgtaatcatc caacccgcct ctcccttgtc
     7081 ttttaccagc acaaaaacct aaataagccc caacatggtt ttgaactaaa caagattaag
     7141 tttgaggcta aagaagctaa gaataagaaa atgaaggcct cagagcaaaa agaccaggca
     7201 gctaatgaag gtccagaaca gtcctctgaa gtaaatgaat tgaaccaaat tccttctcat
     7261 aaagcattaa cattaaccca tgacaatgtt gtcaccgtgt ccccttatgc tctcacacac
     7321 gttgcggggc cctataacca ttgggtcatg gccggccagg caaaaaagaa aaaggaattc
     7381 ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
     7441 ccagtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     7501 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     7561 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     7621 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     7681 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     7741 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     7801 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     7861 atgcagaaga aaaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     7921 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     7981 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     8041 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     8101 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta agaattctaa
     8161 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     8221 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     8281 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     8341 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     8401 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     8461 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     8521 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     8581 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     8641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     8701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     8761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     8821 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     8881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     8941 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     9001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     9061 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     9121 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     9181 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     9241 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     9301 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     9361 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     9421 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     9481 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     9541 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     9601 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     9661 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     9721 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     9781 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     9841 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     9901 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     9961 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
    10021 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
    10081 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
    10141 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
    10201 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
    10261 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
    10321 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
    10381 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
    10441 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
    10501 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
    10561 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
    10621 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
    10681 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
    10741 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
    10801 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
    10861 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
    10921 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
    10981 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
    11041 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
    11101 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc
    11161 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
    11221 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
    11281 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
    11341 atttcgatt
//
