LOCUS       sequence-247382-       11406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_human_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA8 targeting human TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11406)
  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 11406)
  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-8
FEATURES             Location/Qualifiers
     source          1..11406
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        729..1006
                     /label=chicken beta-actin promoter
     intron          1007..1235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1256..1282
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1286..1306
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1316..1339,1340..1342,1343..3898,3899..3901,
                     3902..5416)
                     /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(1316..1339,1340..1342,1343..3829,3830..3832,
                     3833..5416)
                     /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             7658..7711
                     /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             7712..8419
                     /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(7854..7872)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8447..8464)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8453..8660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8669..8809
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8669..8798
                     /label=AAV2 ITR
     rep_origin      8884..9339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8971..8990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9181..9202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9356..9375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9475..9497
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9535..9553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9621..9725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9726..9794,9795..10586)
                     /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(9944..9963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10757..11345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11246..11265
                     /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 ggcttcatcg acaccacggg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg
      901 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatgtaccc
     1261 atacgatgtt ccagattacg cttcgccgaa gaaaaagcgc aaggtcgaag cgtccgacaa
     1321 gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga
     1381 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     1441 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     1501 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     1561 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     1621 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     1681 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     1741 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     1801 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     1861 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     1921 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     1981 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca acctgattgc
     2041 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     2101 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     2161 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     2221 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     2281 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     2341 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     2401 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     2461 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     2521 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     2581 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     2641 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     2701 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     2761 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     2821 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     2881 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     2941 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     3001 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     3061 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     3121 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     3181 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     3241 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     3301 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     3361 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     3421 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     3481 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     3541 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     3601 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     3661 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     3721 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     3781 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacg ccatcgtgcc
     3841 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     3901 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     3961 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     4021 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     4081 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     4141 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     4201 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     4261 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     4321 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     4381 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     4441 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     4501 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     4561 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     4621 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     4681 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     4741 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     4801 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     4861 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     4921 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     4981 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     5041 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     5101 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     5161 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     5221 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     5281 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     5341 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     5401 tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg gaggaggatc
     5461 cctgcccacc tgcagctgtc ttgatcgagt tatacaaaaa gacaaaggcc catattatac
     5521 acaccttggg gcaggaccaa gtgttgctgc tgtcagggaa atcatggaga ataggtatgg
     5581 tcaaaaagga aacgcaataa ggatagaaat agtagtgtac accggtaaag aagggaaaag
     5641 ctctcatggg tgtccaattg ctaagtgggt tttaagaaga agcagtgatg aagaaaaagt
     5701 tctttgtttg gtccggcagc gtacaggcca ccactgtcca actgctgtga tggtggtgct
     5761 catcatggtg tgggatggca tccctcttcc aatggccgac cggctataca cagagctcac
     5821 agagaatcta aagtcataca atgggcaccc taccgacaga agatgcaccc tcaatgaaaa
     5881 tcgtacctgt acatgtcaag gaattgatcc agagacttgt ggagcttcat tctcttttgg
     5941 ctgttcatgg agtatgtact ttaatggctg taagtttggt agaagcccaa gccccagaag
     6001 atttagaatt gatccaagct ctcccttaca tgaaaaaaac cttgaagata acttacagag
     6061 tttggctaca cgattagctc caatttataa gcagtatgct ccagtagctt accaaaatca
     6121 ggtggaatat gaaaatgttg cccgagaatg tcggcttggc agcaaggaag gtcgaccctt
     6181 ctctggggtc actgcttgcc tggacttctg tgctcatccc cacagggaca ttcacaacat
     6241 gaataatgga agcactgtgg tttgtacctt aactcgagaa gataaccgct ctttgggtgt
     6301 tattcctcaa gatgagcagc tccatgtgct acctctttat aagctttcag acacagatga
     6361 gtttggctcc aaggaaggaa tggaagccaa gatcaaatct ggggccatcg aggtcctggc
     6421 accccgccgc aaaaaaagaa cgtgtttcac tcagcctgtt ccccgttctg gaaagaagag
     6481 ggctgcgatg atgacagagg ttcttgcaca taagataagg gcagtggaaa agaaacctat
     6541 tccccgaatc aagcggaaga ataactcaac aacaacaaac aacagtaagc cttcgtcact
     6601 gccaacctta gggagtaaca ctgagaccgt gcaacctgaa gtaaaaagtg aaaccgaacc
     6661 ccattttatc ttaaaaagtt cagacaacac taaaacttat tcgctgatgc catccgctcc
     6721 tcacccagtg aaagaggcat ctccaggctt ctcctggtcc ccgaagactg cttcagccac
     6781 accagctcca ctgaagaatg acgcaacagc ctcatgcggg ttttcagaaa gaagcagcac
     6841 tccccactgt acgatgcctt cgggaagact cagtggtgcc aatgctgcag ctgctgatgg
     6901 ccctggcatt tcacagcttg gcgaagtggc tcctctcccc accctgtctg ctcctgtgat
     6961 ggagcccctc attaattctg agccttccac tggtgtgact gagccgctaa cgcctcatca
     7021 gccaaaccac cagccctcct tcctcacctc tcctcaagac cttgcctctt ctccaatgga
     7081 agaagatgag cagcattctg aagcagatga gcctccatca gacgaacccc tatctgatga
     7141 ccccctgtca cctgctgagg agaaattgcc ccacattgat gagtattggt cagacagtga
     7201 gcacatcttt ttggatgcaa atattggtgg ggtggccatc gcacctgctc acggctcggt
     7261 tttgattgag tgtgcccggc gagagctgca cgctaccact cctgttgagc accccaaccg
     7321 taatcatcca acccgcctct cccttgtctt ttaccagcac aaaaacctaa ataagcccca
     7381 acatggtttt gaactaaaca agattaagtt tgaggctaaa gaagctaaga ataagaaaat
     7441 gaaggcctca gagcaaaaag accaggcagc taatgaaggt ccagaacagt cctctgaagt
     7501 aaatgaattg aaccaaattc cttctcataa agcattaaca ttaacccatg acaatgttgt
     7561 caccgtgtcc ccttatgctc tcacacacgt tgcggggccc tataaccatt gggtcatggc
     7621 cggccaggca aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa gtctgctaac
     7681 atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagg ataacatggc
     7741 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     7801 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     7861 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     7921 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     7981 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     8041 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     8101 caccaacttc ccctccgacg gccccgtaat gcagaagaaa accatgggct gggaggcctc
     8161 ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa
     8221 gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc
     8281 cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga
     8341 ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat
     8401 ggacgagctg tacaagtaag aattctaact agagctcgct gatcagcctc gactgtgcct
     8461 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     8521 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     8581 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     8641 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc
     8701 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     8761 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     8821 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     8881 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     8941 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     9001 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     9061 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     9121 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     9181 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
     9241 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     9301 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     9361 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     9421 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     9481 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     9541 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     9601 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     9661 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     9721 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     9781 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     9841 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     9901 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     9961 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
    10021 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
    10081 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
    10141 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    10201 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    10261 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    10321 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    10381 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    10441 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    10501 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    10561 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    10621 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    10681 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    10741 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    10801 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    10861 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
    10921 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    10981 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    11041 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    11101 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    11161 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    11221 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    11281 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    11341 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    11401 acatgt
//
