LOCUS       sequence-247385-       11407 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 11407)
  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 11407)
  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..11407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1236
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1257..1283
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1287..1307
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1317..1340,1341..1343,1344..3899,3900..3902,
                     3903..5417)
                     /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(1317..1340,1341..1343,1344..3830,3831..3833,
                     3834..5417)
                     /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             7659..7712
                     /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             7713..8420
                     /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(7855..7873)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8448..8465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8454..8661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8670..8810
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8670..8799
                     /label=AAV2 ITR
     rep_origin      8885..9340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8972..8991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9182..9203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9357..9376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9476..9498
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9536..9554)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9622..9726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9727..9795,9796..10587)
                     /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(9945..9964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10758..11346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11247..11266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg agtcgggggc tgtgacaaca gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
     1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatgtacc
     1261 catacgatgt tccagattac gcttcgccga agaaaaagcg caaggtcgaa gcgtccgaca
     1321 agaagtacag catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg
     1381 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     1441 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     1501 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     1561 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     1621 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     1681 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     1741 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     1801 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     1861 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     1921 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     1981 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcggc aacctgattg
     2041 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     2101 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     2161 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     2221 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     2281 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     2341 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     2401 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     2461 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     2521 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     2581 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     2641 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     2701 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     2761 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     2821 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     2881 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     2941 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     3001 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     3061 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     3121 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     3181 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     3241 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     3301 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     3361 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     3421 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     3481 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     3541 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     3601 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     3661 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     3721 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     3781 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac gccatcgtgc
     3841 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     3901 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     3961 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     4021 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     4081 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     4141 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     4201 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     4261 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     4321 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     4381 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     4441 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     4501 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     4561 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     4621 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     4681 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     4741 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     4801 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     4861 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     4921 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     4981 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     5041 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     5101 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     5161 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     5221 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     5281 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     5341 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     5401 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag ggaggaggat
     5461 ccctgcccac ctgcagctgt cttgatcgag ttatacaaaa agacaaaggc ccatattata
     5521 cacaccttgg ggcaggacca agtgttgctg ctgtcaggga aatcatggag aataggtatg
     5581 gtcaaaaagg aaacgcaata aggatagaaa tagtagtgta caccggtaaa gaagggaaaa
     5641 gctctcatgg gtgtccaatt gctaagtggg ttttaagaag aagcagtgat gaagaaaaag
     5701 ttctttgttt ggtccggcag cgtacaggcc accactgtcc aactgctgtg atggtggtgc
     5761 tcatcatggt gtgggatggc atccctcttc caatggccga ccggctatac acagagctca
     5821 cagagaatct aaagtcatac aatgggcacc ctaccgacag aagatgcacc ctcaatgaaa
     5881 atcgtacctg tacatgtcaa ggaattgatc cagagacttg tggagcttca ttctcttttg
     5941 gctgttcatg gagtatgtac tttaatggct gtaagtttgg tagaagccca agccccagaa
     6001 gatttagaat tgatccaagc tctcccttac atgaaaaaaa ccttgaagat aacttacaga
     6061 gtttggctac acgattagct ccaatttata agcagtatgc tccagtagct taccaaaatc
     6121 aggtggaata tgaaaatgtt gcccgagaat gtcggcttgg cagcaaggaa ggtcgaccct
     6181 tctctggggt cactgcttgc ctggacttct gtgctcatcc ccacagggac attcacaaca
     6241 tgaataatgg aagcactgtg gtttgtacct taactcgaga agataaccgc tctttgggtg
     6301 ttattcctca agatgagcag ctccatgtgc tacctcttta taagctttca gacacagatg
     6361 agtttggctc caaggaagga atggaagcca agatcaaatc tggggccatc gaggtcctgg
     6421 caccccgccg caaaaaaaga acgtgtttca ctcagcctgt tccccgttct ggaaagaaga
     6481 gggctgcgat gatgacagag gttcttgcac ataagataag ggcagtggaa aagaaaccta
     6541 ttccccgaat caagcggaag aataactcaa caacaacaaa caacagtaag ccttcgtcac
     6601 tgccaacctt agggagtaac actgagaccg tgcaacctga agtaaaaagt gaaaccgaac
     6661 cccattttat cttaaaaagt tcagacaaca ctaaaactta ttcgctgatg ccatccgctc
     6721 ctcacccagt gaaagaggca tctccaggct tctcctggtc cccgaagact gcttcagcca
     6781 caccagctcc actgaagaat gacgcaacag cctcatgcgg gttttcagaa agaagcagca
     6841 ctccccactg tacgatgcct tcgggaagac tcagtggtgc caatgctgca gctgctgatg
     6901 gccctggcat ttcacagctt ggcgaagtgg ctcctctccc caccctgtct gctcctgtga
     6961 tggagcccct cattaattct gagccttcca ctggtgtgac tgagccgcta acgcctcatc
     7021 agccaaacca ccagccctcc ttcctcacct ctcctcaaga ccttgcctct tctccaatgg
     7081 aagaagatga gcagcattct gaagcagatg agcctccatc agacgaaccc ctatctgatg
     7141 accccctgtc acctgctgag gagaaattgc cccacattga tgagtattgg tcagacagtg
     7201 agcacatctt tttggatgca aatattggtg gggtggccat cgcacctgct cacggctcgg
     7261 ttttgattga gtgtgcccgg cgagagctgc acgctaccac tcctgttgag caccccaacc
     7321 gtaatcatcc aacccgcctc tcccttgtct tttaccagca caaaaaccta aataagcccc
     7381 aacatggttt tgaactaaac aagattaagt ttgaggctaa agaagctaag aataagaaaa
     7441 tgaaggcctc agagcaaaaa gaccaggcag ctaatgaagg tccagaacag tcctctgaag
     7501 taaatgaatt gaaccaaatt ccttctcata aagcattaac attaacccat gacaatgttg
     7561 tcaccgtgtc cccttatgct ctcacacacg ttgcggggcc ctataaccat tgggtcatgg
     7621 ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga agtctgctaa
     7681 catgcggtga cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag gataacatgg
     7741 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     7801 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     7861 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     7921 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     7981 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     8041 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     8101 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa aaccatgggc tgggaggcct
     8161 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     8221 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     8281 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     8341 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     8401 tggacgagct gtacaagtaa gaattctaac tagagctcgc tgatcagcct cgactgtgcc
     8461 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     8521 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     8581 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     8641 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     8701 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     8761 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     8821 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     8881 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     8941 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     9001 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     9061 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     9121 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     9181 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     9241 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     9301 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     9361 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     9421 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     9481 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     9541 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     9601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     9661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     9721 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     9781 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     9841 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     9901 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     9961 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
    10021 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
    10081 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
    10141 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
    10201 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
    10261 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
    10321 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
    10381 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
    10441 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
    10501 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
    10561 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
    10621 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
    10681 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
    10741 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
    10801 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
    10861 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
    10921 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
    10981 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
    11041 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
    11101 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
    11161 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
    11221 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
    11281 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
    11341 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
    11401 cacatgt
//
