LOCUS       sequence-247371-       11410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation, expression of dCas9-huTET1CD-T2A-EGFP 
            and cloning backbone for sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11410)
  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 11410)
  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-GFP (PX458)
FEATURES             Location/Qualifiers
     source          1..11410
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1254..1280
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1284..1304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1314..1337,1338..1340,1341..3896,3897..3899,
                     3900..5414)
                     /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(1314..1337,1338..1340,1341..3827,3828..3830,
                     3831..5414)
                     /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             7656..7709
                     /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             join(7710..7712,7713..8423)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7710..7712,7713..8423)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8013..8032)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8360..8381
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8451..8468)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8457..8664
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8673..8813
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8673..8802
                     /label=AAV2 ITR
     rep_origin      8888..9343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8975..8994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9185..9206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9360..9379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9479..9501
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9539..9557)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9625..9729
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9730..9798,9799..10590)
                     /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(9948..9967)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10761..11349
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11250..11269
                     /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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atgtacccat
     1261 acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga
     1321 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1381 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1441 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1501 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1561 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1621 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1681 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1741 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1801 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1861 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1921 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1981 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc
     2041 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2101 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2161 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2221 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2281 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2341 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2401 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2461 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2521 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2581 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2641 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2701 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2761 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2821 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2881 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2941 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3001 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3061 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3121 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3181 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3241 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3301 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3361 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3421 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3481 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3541 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3601 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3661 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3721 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3781 tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgcc atcgtgcctc
     3841 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3901 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3961 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4021 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4081 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4141 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4201 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4261 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4321 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4381 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4441 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4501 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4561 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4621 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4681 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4741 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4801 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4861 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4921 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4981 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5041 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5101 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5161 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5221 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5281 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5341 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5401 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggga ggaggatccc
     5461 tgcccacctg cagctgtctt gatcgagtta tacaaaaaga caaaggccca tattatacac
     5521 accttggggc aggaccaagt gttgctgctg tcagggaaat catggagaat aggtatggtc
     5581 aaaaaggaaa cgcaataagg atagaaatag tagtgtacac cggtaaagaa gggaaaagct
     5641 ctcatgggtg tccaattgct aagtgggttt taagaagaag cagtgatgaa gaaaaagttc
     5701 tttgtttggt ccggcagcgt acaggccacc actgtccaac tgctgtgatg gtggtgctca
     5761 tcatggtgtg ggatggcatc cctcttccaa tggccgaccg gctatacaca gagctcacag
     5821 agaatctaaa gtcatacaat gggcacccta ccgacagaag atgcaccctc aatgaaaatc
     5881 gtacctgtac atgtcaagga attgatccag agacttgtgg agcttcattc tcttttggct
     5941 gttcatggag tatgtacttt aatggctgta agtttggtag aagcccaagc cccagaagat
     6001 ttagaattga tccaagctct cccttacatg aaaaaaacct tgaagataac ttacagagtt
     6061 tggctacacg attagctcca atttataagc agtatgctcc agtagcttac caaaatcagg
     6121 tggaatatga aaatgttgcc cgagaatgtc ggcttggcag caaggaaggt cgacccttct
     6181 ctggggtcac tgcttgcctg gacttctgtg ctcatcccca cagggacatt cacaacatga
     6241 ataatggaag cactgtggtt tgtaccttaa ctcgagaaga taaccgctct ttgggtgtta
     6301 ttcctcaaga tgagcagctc catgtgctac ctctttataa gctttcagac acagatgagt
     6361 ttggctccaa ggaaggaatg gaagccaaga tcaaatctgg ggccatcgag gtcctggcac
     6421 cccgccgcaa aaaaagaacg tgtttcactc agcctgttcc ccgttctgga aagaagaggg
     6481 ctgcgatgat gacagaggtt cttgcacata agataagggc agtggaaaag aaacctattc
     6541 cccgaatcaa gcggaagaat aactcaacaa caacaaacaa cagtaagcct tcgtcactgc
     6601 caaccttagg gagtaacact gagaccgtgc aacctgaagt aaaaagtgaa accgaacccc
     6661 attttatctt aaaaagttca gacaacacta aaacttattc gctgatgcca tccgctcctc
     6721 acccagtgaa agaggcatct ccaggcttct cctggtcccc gaagactgct tcagccacac
     6781 cagctccact gaagaatgac gcaacagcct catgcgggtt ttcagaaaga agcagcactc
     6841 cccactgtac gatgccttcg ggaagactca gtggtgccaa tgctgcagct gctgatggcc
     6901 ctggcatttc acagcttggc gaagtggctc ctctccccac cctgtctgct cctgtgatgg
     6961 agcccctcat taattctgag ccttccactg gtgtgactga gccgctaacg cctcatcagc
     7021 caaaccacca gccctccttc ctcacctctc ctcaagacct tgcctcttct ccaatggaag
     7081 aagatgagca gcattctgaa gcagatgagc ctccatcaga cgaaccccta tctgatgacc
     7141 ccctgtcacc tgctgaggag aaattgcccc acattgatga gtattggtca gacagtgagc
     7201 acatcttttt ggatgcaaat attggtgggg tggccatcgc acctgctcac ggctcggttt
     7261 tgattgagtg tgcccggcga gagctgcacg ctaccactcc tgttgagcac cccaaccgta
     7321 atcatccaac ccgcctctcc cttgtctttt accagcacaa aaacctaaat aagccccaac
     7381 atggttttga actaaacaag attaagtttg aggctaaaga agctaagaat aagaaaatga
     7441 aggcctcaga gcaaaaagac caggcagcta atgaaggtcc agaacagtcc tctgaagtaa
     7501 atgaattgaa ccaaattcct tctcataaag cattaacatt aacccatgac aatgttgtca
     7561 ccgtgtcccc ttatgctctc acacacgttg cggggcccta taaccattgg gtcatggccg
     7621 gccaggcaaa aaagaaaaag gaattcggca gtggagaggg cagaggaagt ctgctaacat
     7681 gcggtgacgt cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg
     7741 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     7801 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     7861 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     7921 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     7981 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     8041 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     8101 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     8161 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     8221 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     8281 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     8341 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     8401 tcggcatgga cgagctgtac aaggaattct aactagagct cgctgatcag cctcgactgt
     8461 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     8521 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     8581 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     8641 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     8701 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     8761 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     8821 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     8881 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     8941 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     9001 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     9061 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     9121 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     9181 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     9241 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     9301 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     9361 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     9421 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     9481 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     9541 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     9601 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     9661 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     9721 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     9781 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     9841 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     9901 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     9961 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
    10021 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
    10081 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
    10141 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
    10201 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
    10261 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
    10321 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
    10381 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
    10441 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
    10501 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
    10561 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
    10621 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
    10681 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
    10741 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
    10801 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
    10861 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
    10921 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10981 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    11041 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    11101 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
    11161 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    11221 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    11281 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    11341 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
    11401 gctcacatgt
//
