LOCUS       sequence-454365-       11405 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 11405)
  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 11405)
  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..11405
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          562..789
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             810..836
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             840..860
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(870..893,894..896,897..3452,3453..3455,3456..4970)
                     /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(870..893,894..896,897..3383,3384..3386,3387..4970)
                     /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             7212..7265
                     /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(7266..7268,7269..7979)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7266..7268,7269..7979)
                     /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(7569..7588)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7916..7937
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8007..8024)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8013..8220
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8229..8369
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8229..8358
                     /label=AAV2 ITR
     rep_origin      8444..8899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8531..8550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8741..8762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8916..8935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9035..9057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9095..9113)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9181..9285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9286..9354,9355..10146)
                     /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(9504..9523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10317..10905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10806..10825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10967..11215
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10967..10987
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     11138..11157
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        11234..11309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      361 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gccaggcggg
      421 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
      481 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
      541 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
      601 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
      661 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
      721 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
      781 ttttttcagg ttggaccggt gccaccatgt acccatacga tgttccagat tacgcttcgc
      841 cgaagaaaaa gcgcaaggtc gaagcgtccg acaagaagta cagcatcggc ctggccatcg
      901 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
      961 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     1021 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     1081 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     1141 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     1201 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     1261 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     1321 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     1381 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     1441 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     1501 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     1561 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     1621 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     1681 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     1741 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     1801 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     1861 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     1921 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     1981 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     2041 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     2101 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     2161 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     2221 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     2281 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     2341 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     2401 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     2461 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     2521 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     2581 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     2641 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     2701 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     2761 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     2821 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     2881 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     2941 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     3001 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     3061 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     3121 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     3181 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3241 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     3301 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3361 ggctgtccga ctacgatgtg gacgccatcg tgcctcagag ctttctgaag gacgactcca
     3421 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     3481 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     3541 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     3601 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     3661 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     3721 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     3781 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     3841 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     3901 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     3961 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     4021 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     4081 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     4141 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     4201 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     4261 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4321 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4381 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4441 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     4501 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     4561 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     4621 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     4681 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     4741 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     4801 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     4861 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     4921 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     4981 cggccacgaa aaaggccggc cagggaggag gatccctgcc cacctgcagc tgtcttgatc
     5041 gagttataca aaaagacaaa ggcccatatt atacacacct tggggcagga ccaagtgttg
     5101 ctgctgtcag ggaaatcatg gagaataggt atggtcaaaa aggaaacgca ataaggatag
     5161 aaatagtagt gtacaccggt aaagaaggga aaagctctca tgggtgtcca attgctaagt
     5221 gggttttaag aagaagcagt gatgaagaaa aagttctttg tttggtccgg cagcgtacag
     5281 gccaccactg tccaactgct gtgatggtgg tgctcatcat ggtgtgggat ggcatccctc
     5341 ttccaatggc cgaccggcta tacacagagc tcacagagaa tctaaagtca tacaatgggc
     5401 accctaccga cagaagatgc accctcaatg aaaatcgtac ctgtacatgt caaggaattg
     5461 atccagagac ttgtggagct tcattctctt ttggctgttc atggagtatg tactttaatg
     5521 gctgtaagtt tggtagaagc ccaagcccca gaagatttag aattgatcca agctctccct
     5581 tacatgaaaa aaaccttgaa gataacttac agagtttggc tacacgatta gctccaattt
     5641 ataagcagta tgctccagta gcttaccaaa atcaggtgga atatgaaaat gttgcccgag
     5701 aatgtcggct tggcagcaag gaaggtcgac ccttctctgg ggtcactgct tgcctggact
     5761 tctgtgctca tccccacagg gacattcaca acatgaataa tggaagcact gtggtttgta
     5821 ccttaactcg agaagataac cgctctttgg gtgttattcc tcaagatgag cagctccatg
     5881 tgctacctct ttataagctt tcagacacag atgagtttgg ctccaaggaa ggaatggaag
     5941 ccaagatcaa atctggggcc atcgaggtcc tggcaccccg ccgcaaaaaa agaacgtgtt
     6001 tcactcagcc tgttccccgt tctggaaaga agagggctgc gatgatgaca gaggttcttg
     6061 cacataagat aagggcagtg gaaaagaaac ctattccccg aatcaagcgg aagaataact
     6121 caacaacaac aaacaacagt aagccttcgt cactgccaac cttagggagt aacactgaga
     6181 ccgtgcaacc tgaagtaaaa agtgaaaccg aaccccattt tatcttaaaa agttcagaca
     6241 acactaaaac ttattcgctg atgccatccg ctcctcaccc agtgaaagag gcatctccag
     6301 gcttctcctg gtccccgaag actgcttcag ccacaccagc tccactgaag aatgacgcaa
     6361 cagcctcatg cgggttttca gaaagaagca gcactcccca ctgtacgatg ccttcgggaa
     6421 gactcagtgg tgccaatgct gcagctgctg atggccctgg catttcacag cttggcgaag
     6481 tggctcctct ccccaccctg tctgctcctg tgatggagcc cctcattaat tctgagcctt
     6541 ccactggtgt gactgagccg ctaacgcctc atcagccaaa ccaccagccc tccttcctca
     6601 cctctcctca agaccttgcc tcttctccaa tggaagaaga tgagcagcat tctgaagcag
     6661 atgagcctcc atcagacgaa cccctatctg atgaccccct gtcacctgct gaggagaaat
     6721 tgccccacat tgatgagtat tggtcagaca gtgagcacat ctttttggat gcaaatattg
     6781 gtggggtggc catcgcacct gctcacggct cggttttgat tgagtgtgcc cggcgagagc
     6841 tgcacgctac cactcctgtt gagcacccca accgtaatca tccaacccgc ctctcccttg
     6901 tcttttacca gcacaaaaac ctaaataagc cccaacatgg ttttgaacta aacaagatta
     6961 agtttgaggc taaagaagct aagaataaga aaatgaaggc ctcagagcaa aaagaccagg
     7021 cagctaatga aggtccagaa cagtcctctg aagtaaatga attgaaccaa attccttctc
     7081 ataaagcatt aacattaacc catgacaatg ttgtcaccgt gtccccttat gctctcacac
     7141 acgttgcggg gccctataac cattgggtca tggccggcca ggcaaaaaag aaaaaggaat
     7201 tcggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg
     7261 gcccagtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     7321 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     7381 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     7441 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     7501 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     7561 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     7621 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     7681 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     7741 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     7801 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     7861 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     7921 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     7981 aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     8041 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     8101 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     8161 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     8221 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     8281 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     8341 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     8401 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     8461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     8521 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     8581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     8641 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     8701 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     8761 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     8821 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     8881 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     8941 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     9001 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     9061 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     9121 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     9181 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     9241 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     9301 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     9361 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     9421 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     9481 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     9541 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     9601 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     9661 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     9721 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     9781 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     9841 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     9901 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     9961 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
    10021 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
    10081 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
    10141 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
    10201 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
    10261 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
    10321 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
    10381 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
    10441 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
    10501 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
    10561 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
    10621 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
    10681 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
    10741 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
    10801 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
    10861 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
    10921 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc
    10981 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
    11041 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
    11101 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
    11161 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc
    11221 ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
    11281 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
    11341 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag
    11401 gtacc
//
