LOCUS       sequence-256198-       11356 bp    DNA     circular SYN 15-APR-2025
DEFINITION  inactivated control (targeted DNA demethylation_human_TSDR), 
            expression of dCas9-hudTET1CD-T2A-mCherry sgRNA4 targeting human 
            TSDR .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11356)
  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 11356)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-hudTET1CD-T2A-mCherry(PX458)-SghuTSDR-4
FEATURES             Location/Qualifiers
     source          1..11356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          112..339
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             360..386
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             390..410
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(420..443,444..446,447..3002,3003..3005,3006..4520)
                     /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(420..443,444..446,447..2933,2934..2936,2937..4520)
                     /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             6762..6815
                     /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             6816..7523
                     /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(6958..6976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7551..7568)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7557..7764
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7773..7913
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7773..7902
                     /label=AAV2 ITR
     rep_origin      7988..8443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8075..8094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8285..8306
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8460..8479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8579..8601
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8639..8657)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8725..8829
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8830..8898,8899..9690)
                     /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(9048..9067)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9861..10449
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10350..10369
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10511..10759
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10511..10531
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10682..10701
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10781..10856
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        10953..11238
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
       61 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
      121 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
      181 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
      241 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
      301 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgt
      361 acccatacga tgttccagat tacgcttcgc cgaagaaaaa gcgcaaggtc gaagcgtccg
      421 acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca
      481 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
      541 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
      601 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
      661 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
      721 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
      781 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
      841 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
      901 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
      961 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     1021 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     1081 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
     1141 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     1201 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     1261 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     1321 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     1381 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     1441 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     1501 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     1561 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     1621 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     1681 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     1741 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     1801 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     1861 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     1921 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     1981 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     2041 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     2101 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     2161 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     2221 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     2281 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     2341 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     2401 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     2461 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     2521 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     2581 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     2641 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     2701 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     2761 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     2821 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     2881 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgccatcg
     2941 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     3001 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     3061 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     3121 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     3181 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     3241 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     3301 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     3361 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     3421 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     3481 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     3541 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     3601 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     3661 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     3721 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     3781 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     3841 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     3901 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     3961 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     4021 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     4081 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     4141 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     4201 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     4261 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     4321 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     4381 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     4441 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     4501 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc cagggaggag
     4561 gatccctgcc cacctgcagc tgtcttgatc gagttataca aaaagacaaa ggcccatatt
     4621 atacacacct tggggcagga ccaagtgttg ctgctgtcag ggaaatcatg gagaataggt
     4681 atggtcaaaa aggaaacgca ataaggatag aaatagtagt gtacaccggt aaagaaggga
     4741 aaagctctca tgggtgtcca attgctaagt gggttttaag aagaagcagt gatgaagaaa
     4801 aagttctttg tttggtccgg cagcgtacag gccaccactg tccaactgct gtgatggtgg
     4861 tgctcatcat ggtgtgggat ggcatccctc ttccaatggc cgaccggcta tacacagagc
     4921 tcacagagaa tctaaagtca tacaatgggc accctaccga cagaagatgc accctcaatg
     4981 aaaatcgtac ctgtacatgt caaggaattg atccagagac ttgtggagct tcattctctt
     5041 ttggctgttc atggagtatg tactttaatg gctgtaagtt tggtagaagc ccaagcccca
     5101 gaagatttag aattgatcca agctctccct tacatgaaaa aaaccttgaa gataacttac
     5161 agagtttggc tacacgatta gctccaattt ataagcagta tgctccagta gcttaccaaa
     5221 atcaggtgga atatgaaaat gttgcccgag aatgtcggct tggcagcaag gaaggtcgac
     5281 ccttctctgg ggtcactgct tgcctggact tctgtgctca tccctacagg gccattcaca
     5341 acatgaataa tggaagcact gtggtttgta ccttaactcg agaagataac cgctctttgg
     5401 gtgttattcc tcaagatgag cagctccatg tgctacctct ttataagctt tcagacacag
     5461 atgagtttgg ctccaaggaa ggaatggaag ccaagatcaa atctggggcc atcgaggtcc
     5521 tggcaccccg ccgcaaaaaa agaacgtgtt tcactcagcc tgttccccgt tctggaaaga
     5581 agagggctgc gatgatgaca gaggttcttg cacataagat aagggcagtg gaaaagaaac
     5641 ctattccccg aatcaagcgg aagaataact caacaacaac aaacaacagt aagccttcgt
     5701 cactgccaac cttagggagt aacactgaga ccgtgcaacc tgaagtaaaa agtgaaaccg
     5761 aaccccattt tatcttaaaa agttcagaca acactaaaac ttattcgctg atgccatccg
     5821 ctcctcaccc agtgaaagag gcatctccag gcttctcctg gtccccgaag actgcttcag
     5881 ccacaccagc tccactgaag aatgacgcaa cagcctcatg cgggttttca gaaagaagca
     5941 gcactcccca ctgtacgatg ccttcgggaa gactcagtgg tgccaatgct gcagctgctg
     6001 atggccctgg catttcacag cttggcgaag tggctcctct ccccaccctg tctgctcctg
     6061 tgatggagcc cctcattaat tctgagcctt ccactggtgt gactgagccg ctaacgcctc
     6121 atcagccaaa ccaccagccc tccttcctca cctctcctca agaccttgcc tcttctccaa
     6181 tggaagaaga tgagcagcat tctgaagcag atgagcctcc atcagacgaa cccctatctg
     6241 atgaccccct gtcacctgct gaggagaaat tgccccacat tgatgagtat tggtcagaca
     6301 gtgagcacat ctttttggat gcaaatattg gtggggtggc catcgcacct gctcacggct
     6361 cggttttgat tgagtgtgcc cggcgagagc tgcacgctac cactcctgtt gagcacccca
     6421 accgtaatca tccaacccgc ctctcccttg tcttttacca gcacaaaaac ctaaataagc
     6481 cccaacatgg ttttgaacta aacaagatta agtttgaggc taaagaagct aagaataaga
     6541 aaatgaaggc ctcagagcaa aaagaccagg cagctaatga aggtccagaa cagtcctctg
     6601 aagtaaatga attgaaccaa attccttctc ataaagcatt aacattaacc catgacaatg
     6661 ttgtcaccgt gtccccttat gctctcacac acgttgcggg gccctataac cattgggtca
     6721 tggccggcca ggcaaaaaag aaaaaggaat tcggcagtgg agagggcaga ggaagtctgc
     6781 taacatgcgg tgacgtcgag gagaatcctg gcccagtgag caagggcgag gaggataaca
     6841 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     6901 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     6961 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     7021 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     7081 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     7141 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     7201 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaaaaccatg ggctgggagg
     7261 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     7321 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     7381 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     7441 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     7501 gcatggacga gctgtacaag taagaattct aactagagct cgctgatcag cctcgactgt
     7561 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     7621 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     7681 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     7741 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     7801 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     7861 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     7921 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     7981 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     8041 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     8101 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     8161 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     8221 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     8281 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     8341 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     8401 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     8461 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     8521 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     8581 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     8641 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     8701 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     8761 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     8821 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     8881 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     8941 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     9001 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     9061 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     9121 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     9181 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     9241 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     9301 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     9361 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     9421 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     9481 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     9541 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     9601 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     9661 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     9721 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     9781 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     9841 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     9901 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     9961 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
    10021 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10081 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    10141 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    10201 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
    10261 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    10321 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    10381 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    10441 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
    10501 gctcacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
    10561 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
    10621 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
    10681 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
    10741 gtggaaagga cgaaacaccg tgtggtaggt gatgtccatc gttttagagc tagaaatagc
    10801 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
    10861 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
    10921 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
    10981 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
    11041 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
    11101 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
    11161 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
    11221 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
    11281 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
    11341 ggggcggggg gggggg
//
