LOCUS       sequence-256129-       11403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation, expression of dCas9-huTET1CD-T2A-mCherry
            and cloning backbone for sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11403)
  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 11403)
  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)
FEATURES             Location/Qualifiers
     source          1..11403
                     /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"
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1253..1279
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1283..1303
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1313..1336,1337..1339,1340..3895,3896..3898,
                     3899..5413)
                     /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(1313..1336,1337..1339,1340..3826,3827..3829,
                     3830..5413)
                     /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             7655..7708
                     /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             7709..8416
                     /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(7851..7869)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8444..8461)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8450..8657
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8666..8806
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8666..8795
                     /label=AAV2 ITR
     rep_origin      8881..9336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8968..8987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9178..9199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9353..9372)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9472..9494
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9532..9550)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9618..9722
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9723..9791,9792..10583)
                     /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(9941..9960)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10754..11342
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11243..11262
                     /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 cgcgccrggg gggggggggg gggggggggg ggggggsggg gsgrggsggr
      901 grggkgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgtacccata
     1261 cgatgttcca gattacgctt cgccgaagaa aaagcgcaag gtcgaagcgt ccgacaagaa
     1321 gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1381 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1441 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1501 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1561 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1621 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1681 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1741 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1801 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1861 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1921 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1981 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct
     2041 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     2101 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     2161 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2221 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2281 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2341 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2401 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2461 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2521 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2581 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2641 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2701 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2761 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2821 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2881 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     2941 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     3001 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     3061 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     3121 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3181 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3241 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3301 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3361 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3421 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3481 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3541 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3601 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3661 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3721 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3781 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggacgcca tcgtgcctca
     3841 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3901 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3961 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     4021 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     4081 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     4141 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4201 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4261 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4321 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4381 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4441 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4501 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4561 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4621 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4681 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4741 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4801 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4861 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4921 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4981 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     5041 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     5101 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     5161 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5221 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5281 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5341 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5401 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccagggag gaggatccct
     5461 gcccacctgc agctgtcttg atcgagttat acaaaaagac aaaggcccat attatacaca
     5521 ccttggggca ggaccaagtg ttgctgctgt cagggaaatc atggagaata ggtatggtca
     5581 aaaaggaaac gcaataagga tagaaatagt agtgtacacc ggtaaagaag ggaaaagctc
     5641 tcatgggtgt ccaattgcta agtgggtttt aagaagaagc agtgatgaag aaaaagttct
     5701 ttgtttggtc cggcagcgta caggccacca ctgtccaact gctgtgatgg tggtgctcat
     5761 catggtgtgg gatggcatcc ctcttccaat ggccgaccgg ctatacacag agctcacaga
     5821 gaatctaaag tcatacaatg ggcaccctac cgacagaaga tgcaccctca atgaaaatcg
     5881 tacctgtaca tgtcaaggaa ttgatccaga gacttgtgga gcttcattct cttttggctg
     5941 ttcatggagt atgtacttta atggctgtaa gtttggtaga agcccaagcc ccagaagatt
     6001 tagaattgat ccaagctctc ccttacatga aaaaaacctt gaagataact tacagagttt
     6061 ggctacacga ttagctccaa tttataagca gtatgctcca gtagcttacc aaaatcaggt
     6121 ggaatatgaa aatgttgccc gagaatgtcg gcttggcagc aaggaaggtc gacccttctc
     6181 tggggtcact gcttgcctgg acttctgtgc tcatccccac agggacattc acaacatgaa
     6241 taatggaagc actgtggttt gtaccttaac tcgagaagat aaccgctctt tgggtgttat
     6301 tcctcaagat gagcagctcc atgtgctacc tctttataag ctttcagaca cagatgagtt
     6361 tggctccaag gaaggaatgg aagccaagat caaatctggg gccatcgagg tcctggcacc
     6421 ccgccgcaaa aaaagaacgt gtttcactca gcctgttccc cgttctggaa agaagagggc
     6481 tgcgatgatg acagaggttc ttgcacataa gataagggca gtggaaaaga aacctattcc
     6541 ccgaatcaag cggaagaata actcaacaac aacaaacaac agtaagcctt cgtcactgcc
     6601 aaccttaggg agtaacactg agaccgtgca acctgaagta aaaagtgaaa ccgaacccca
     6661 ttttatctta aaaagttcag acaacactaa aacttattcg ctgatgccat ccgctcctca
     6721 cccagtgaaa gaggcatctc caggcttctc ctggtccccg aagactgctt cagccacacc
     6781 agctccactg aagaatgacg caacagcctc atgcgggttt tcagaaagaa gcagcactcc
     6841 ccactgtacg atgccttcgg gaagactcag tggtgccaat gctgcagctg ctgatggccc
     6901 tggcatttca cagcttggcg aagtggctcc tctccccacc ctgtctgctc ctgtgatgga
     6961 gcccctcatt aattctgagc cttccactgg tgtgactgag ccgctaacgc ctcatcagcc
     7021 aaaccaccag ccctccttcc tcacctctcc tcaagacctt gcctcttctc caatggaaga
     7081 agatgagcag cattctgaag cagatgagcc tccatcagac gaacccctat ctgatgaccc
     7141 cctgtcacct gctgaggaga aattgcccca cattgatgag tattggtcag acagtgagca
     7201 catctttttg gatgcaaata ttggtggggt ggccatcgca cctgctcacg gctcggtttt
     7261 gattgagtgt gcccggcgag agctgcacgc taccactcct gttgagcacc ccaaccgtaa
     7321 tcatccaacc cgcctctccc ttgtctttta ccagcacaaa aacctaaata agccccaaca
     7381 tggttttgaa ctaaacaaga ttaagtttga ggctaaagaa gctaagaata agaaaatgaa
     7441 ggcctcagag caaaaagacc aggcagctaa tgaaggtcca gaacagtcct ctgaagtaaa
     7501 tgaattgaac caaattcctt ctcataaagc attaacatta acccatgaca atgttgtcac
     7561 cgtgtcccct tatgctctca cacacgttgc ggggccctat aaccattggg tcatggccgg
     7621 ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg
     7681 cggtgacgtc gaggagaatc ctggcccagt gagcaagggc gaggaggata acatggccat
     7741 catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     7801 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     7861 ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta
     7921 cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt
     7981 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     8041 gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac
     8101 caacttcccc tccgacggcc ccgtaatgca gaagaaaacc atgggctggg aggcctcctc
     8161 cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct
     8221 gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt
     8281 gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga
     8341 ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga
     8401 cgagctgtac aagtaagaat tctaactaga gctcgctgat cagcctcgac tgtgccttct
     8461 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     8521 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     8581 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     8641 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     8701 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     8761 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     8821 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     8881 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     8941 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     9001 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     9061 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     9121 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     9181 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     9241 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     9301 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     9361 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     9421 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     9481 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     9541 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     9601 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     9661 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     9721 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     9781 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     9841 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     9901 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     9961 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
    10021 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
    10081 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
    10141 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
    10201 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
    10261 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
    10321 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
    10381 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
    10441 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
    10501 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
    10561 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
    10621 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
    10681 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
    10741 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
    10801 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
    10861 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
    10921 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
    10981 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
    11041 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
    11101 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
    11161 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
    11221 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
    11281 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
    11341 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
    11401 tgt
//
