LOCUS       sequence-256164-       11688 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_human_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA7 targeting human TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11688)
  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 11688)
  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-7
FEATURES             Location/Qualifiers
     source          1..11688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          297..524
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             545..571
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             575..595
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(605..628,629..631,632..3187,3188..3190,3191..4705)
                     /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(605..628,629..631,632..3118,3119..3121,3122..4705)
                     /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             6947..7000
                     /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             7001..7708
                     /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(7143..7161)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7736..7753)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7742..7949
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7958..8098
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7958..8087
                     /label=AAV2 ITR
     rep_origin      8173..8628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8260..8279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8470..8491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8645..8664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8764..8786
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8824..8842)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8910..9014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9015..9083,9084..9875)
                     /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(9233..9252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10046..10634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10535..10554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10696..10944
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10696..10716
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10867..10886
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10965..11040
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        11137..11422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
       61 gggggggggg gggggggggg gggggggggk gkgggkkggg gggggggggg gggggggggg
      121 gggggggggg gggggggggg cgcgcgcsgg gsggggsggg gcggggcgag gggcggggcg
      181 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
      241 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      301 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      361 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      421 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
      481 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
      541 catgtaccca tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc
      601 gtccgacaag aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt
      661 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
      721 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
      781 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
      841 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
      901 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
      961 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     1021 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     1081 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     1141 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     1201 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     1261 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa
     1321 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     1381 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     1441 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     1501 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     1561 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     1621 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     1681 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     1741 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     1801 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     1861 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     1921 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     1981 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     2041 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     2101 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     2161 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     2221 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     2281 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     2341 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     2401 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     2461 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     2521 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     2581 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     2641 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     2701 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     2761 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     2821 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     2881 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     2941 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     3001 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     3061 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc
     3121 catcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     3181 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     3241 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     3301 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     3361 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     3421 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     3481 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     3541 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     3601 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     3661 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     3721 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     3781 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     3841 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     3901 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     3961 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     4021 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     4081 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     4141 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     4201 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     4261 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     4321 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     4381 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     4441 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     4501 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     4561 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     4621 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     4681 cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggg
     4741 aggaggatcc ctgcccacct gcagctgtct tgatcgagtt atacaaaaag acaaaggccc
     4801 atattataca caccttgggg caggaccaag tgttgctgct gtcagggaaa tcatggagaa
     4861 taggtatggt caaaaaggaa acgcaataag gatagaaata gtagtgtaca ccggtaaaga
     4921 agggaaaagc tctcatgggt gtccaattgc taagtgggtt ttaagaagaa gcagtgatga
     4981 agaaaaagtt ctttgtttgg tccggcagcg tacaggccac cactgtccaa ctgctgtgat
     5041 ggtggtgctc atcatggtgt gggatggcat ccctcttcca atggccgacc ggctatacac
     5101 agagctcaca gagaatctaa agtcatacaa tgggcaccct accgacagaa gatgcaccct
     5161 caatgaaaat cgtacctgta catgtcaagg aattgatcca gagacttgtg gagcttcatt
     5221 ctcttttggc tgttcatgga gtatgtactt taatggctgt aagtttggta gaagcccaag
     5281 ccccagaaga tttagaattg atccaagctc tcccttacat gaaaaaaacc ttgaagataa
     5341 cttacagagt ttggctacac gattagctcc aatttataag cagtatgctc cagtagctta
     5401 ccaaaatcag gtggaatatg aaaatgttgc ccgagaatgt cggcttggca gcaaggaagg
     5461 tcgacccttc tctggggtca ctgcttgcct ggacttctgt gctcatcccc acagggacat
     5521 tcacaacatg aataatggaa gcactgtggt ttgtacctta actcgagaag ataaccgctc
     5581 tttgggtgtt attcctcaag atgagcagct ccatgtgcta cctctttata agctttcaga
     5641 cacagatgag tttggctcca aggaaggaat ggaagccaag atcaaatctg gggccatcga
     5701 ggtcctggca ccccgccgca aaaaaagaac gtgtttcact cagcctgttc cccgttctgg
     5761 aaagaagagg gctgcgatga tgacagaggt tcttgcacat aagataaggg cagtggaaaa
     5821 gaaacctatt ccccgaatca agcggaagaa taactcaaca acaacaaaca acagtaagcc
     5881 ttcgtcactg ccaaccttag ggagtaacac tgagaccgtg caacctgaag taaaaagtga
     5941 aaccgaaccc cattttatct taaaaagttc agacaacact aaaacttatt cgctgatgcc
     6001 atccgctcct cacccagtga aagaggcatc tccaggcttc tcctggtccc cgaagactgc
     6061 ttcagccaca ccagctccac tgaagaatga cgcaacagcc tcatgcgggt tttcagaaag
     6121 aagcagcact ccccactgta cgatgccttc gggaagactc agtggtgcca atgctgcagc
     6181 tgctgatggc cctggcattt cacagcttgg cgaagtggct cctctcccca ccctgtctgc
     6241 tcctgtgatg gagcccctca ttaattctga gccttccact ggtgtgactg agccgctaac
     6301 gcctcatcag ccaaaccacc agccctcctt cctcacctct cctcaagacc ttgcctcttc
     6361 tccaatggaa gaagatgagc agcattctga agcagatgag cctccatcag acgaacccct
     6421 atctgatgac cccctgtcac ctgctgagga gaaattgccc cacattgatg agtattggtc
     6481 agacagtgag cacatctttt tggatgcaaa tattggtggg gtggccatcg cacctgctca
     6541 cggctcggtt ttgattgagt gtgcccggcg agagctgcac gctaccactc ctgttgagca
     6601 ccccaaccgt aatcatccaa cccgcctctc ccttgtcttt taccagcaca aaaacctaaa
     6661 taagccccaa catggttttg aactaaacaa gattaagttt gaggctaaag aagctaagaa
     6721 taagaaaatg aaggcctcag agcaaaaaga ccaggcagct aatgaaggtc cagaacagtc
     6781 ctctgaagta aatgaattga accaaattcc ttctcataaa gcattaacat taacccatga
     6841 caatgttgtc accgtgtccc cttatgctct cacacacgtt gcggggccct ataaccattg
     6901 ggtcatggcc ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg gcagaggaag
     6961 tctgctaaca tgcggtgacg tcgaggagaa tcctggccca gtgagcaagg gcgaggagga
     7021 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
     7081 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     7141 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
     7201 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
     7261 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     7321 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
     7381 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaaa ccatgggctg
     7441 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
     7501 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
     7561 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
     7621 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
     7681 cggcggcatg gacgagctgt acaagtaaga attctaacta gagctcgctg atcagcctcg
     7741 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     7801 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     7861 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     7921 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg
     7981 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     8041 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     8101 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     8161 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     8221 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     8281 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     8341 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     8401 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     8461 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     8521 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     8581 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     8641 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     8701 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     8761 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     8821 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     8881 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     8941 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     9001 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     9061 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     9121 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     9181 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     9241 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     9301 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     9361 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     9421 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     9481 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     9541 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     9601 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     9661 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     9721 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     9781 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     9841 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     9901 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     9961 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    10021 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    10081 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    10141 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    10201 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    10261 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    10321 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    10381 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    10441 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    10501 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    10561 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    10621 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    10681 cttttgctca catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac
    10741 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa
    10801 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
    10861 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
    10921 atcttgtgga aaggacgaaa caccgttctc ggaacgaaac ctgtgtttta gagctagaaa
    10981 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
    11041 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
    11101 gcgccaattc tgcagacaaa tggctctaga ggtacccgtt acataactta cggtaaatgg
    11161 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa cgccaatagg
    11221 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
    11281 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
    11341 ctggcattgt gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
    11401 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
    11461 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
    11521 gatgggggcg gggggggggg gggggggsgc csgmgggggg gggggggggg gggggggggg
    11581 gggggggggg gggggggggg gggggggggg ggggnnnggg gggggggggg gggggggggg
    11641 gggggggggg nggggggggg gggggggggg gggggggggg gggggggg
//
