LOCUS       sequence-256165-       11405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted DNA demethylation_human_TSDR, expression of 
            dCas9-huTET1CD-T2A-mCherry and sgRNA8 targeting human TSDR .
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-mCherry(PX458)-SghuTSDR-8
FEATURES             Location/Qualifiers
     source          1..11405
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1007..1234
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1255..1281
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1285..1305
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1315..1338,1339..1341,1342..3897,3898..3900,
                     3901..5415)
                     /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(1315..1338,1339..1341,1342..3828,3829..3831,
                     3832..5415)
                     /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             7657..7710
                     /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             7711..8418
                     /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(7853..7871)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8446..8463)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8452..8659
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8668..8808
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8668..8797
                     /label=AAV2 ITR
     rep_origin      8883..9338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8970..8989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9180..9201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9355..9374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9474..9496
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9534..9552)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9620..9724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9725..9793,9794..10585)
                     /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(9943..9962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10756..11344
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11245..11264
                     /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 ggcttcatcg acaccacggg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccgg gggggggggg gggggggggg ggggggggsg gggsgrggsg
      901 grgrggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catgtaccca
     1261 tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag
     1321 aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac
     1381 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     1441 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     1501 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     1561 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     1621 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     1681 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     1741 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     1801 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     1861 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     1921 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     1981 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa cctgattgcc
     2041 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     2101 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     2161 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     2221 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     2281 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     2341 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     2401 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     2461 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     2521 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     2581 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     2641 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     2701 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     2761 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     2821 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     2881 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     2941 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     3001 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     3061 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     3121 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     3181 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     3241 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     3301 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     3361 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     3421 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     3481 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     3541 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     3601 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     3661 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     3721 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     3781 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc catcgtgcct
     3841 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     3901 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     3961 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     4021 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     4081 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     4141 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     4201 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     4261 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     4321 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     4381 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     4441 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     4501 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     4561 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     4621 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     4681 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     4741 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     4801 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     4861 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     4921 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     4981 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     5041 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     5101 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     5161 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     5221 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     5281 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     5341 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     5401 cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggg aggaggatcc
     5461 ctgcccacct gcagctgtct tgatcgagtt atacaaaaag acaaaggccc atattataca
     5521 caccttgggg caggaccaag tgttgctgct gtcagggaaa tcatggagaa taggtatggt
     5581 caaaaaggaa acgcaataag gatagaaata gtagtgtaca ccggtaaaga agggaaaagc
     5641 tctcatgggt gtccaattgc taagtgggtt ttaagaagaa gcagtgatga agaaaaagtt
     5701 ctttgtttgg tccggcagcg tacaggccac cactgtccaa ctgctgtgat ggtggtgctc
     5761 atcatggtgt gggatggcat ccctcttcca atggccgacc ggctatacac agagctcaca
     5821 gagaatctaa agtcatacaa tgggcaccct accgacagaa gatgcaccct caatgaaaat
     5881 cgtacctgta catgtcaagg aattgatcca gagacttgtg gagcttcatt ctcttttggc
     5941 tgttcatgga gtatgtactt taatggctgt aagtttggta gaagcccaag ccccagaaga
     6001 tttagaattg atccaagctc tcccttacat gaaaaaaacc ttgaagataa cttacagagt
     6061 ttggctacac gattagctcc aatttataag cagtatgctc cagtagctta ccaaaatcag
     6121 gtggaatatg aaaatgttgc ccgagaatgt cggcttggca gcaaggaagg tcgacccttc
     6181 tctggggtca ctgcttgcct ggacttctgt gctcatcccc acagggacat tcacaacatg
     6241 aataatggaa gcactgtggt ttgtacctta actcgagaag ataaccgctc tttgggtgtt
     6301 attcctcaag atgagcagct ccatgtgcta cctctttata agctttcaga cacagatgag
     6361 tttggctcca aggaaggaat ggaagccaag atcaaatctg gggccatcga ggtcctggca
     6421 ccccgccgca aaaaaagaac gtgtttcact cagcctgttc cccgttctgg aaagaagagg
     6481 gctgcgatga tgacagaggt tcttgcacat aagataaggg cagtggaaaa gaaacctatt
     6541 ccccgaatca agcggaagaa taactcaaca acaacaaaca acagtaagcc ttcgtcactg
     6601 ccaaccttag ggagtaacac tgagaccgtg caacctgaag taaaaagtga aaccgaaccc
     6661 cattttatct taaaaagttc agacaacact aaaacttatt cgctgatgcc atccgctcct
     6721 cacccagtga aagaggcatc tccaggcttc tcctggtccc cgaagactgc ttcagccaca
     6781 ccagctccac tgaagaatga cgcaacagcc tcatgcgggt tttcagaaag aagcagcact
     6841 ccccactgta cgatgccttc gggaagactc agtggtgcca atgctgcagc tgctgatggc
     6901 cctggcattt cacagcttgg cgaagtggct cctctcccca ccctgtctgc tcctgtgatg
     6961 gagcccctca ttaattctga gccttccact ggtgtgactg agccgctaac gcctcatcag
     7021 ccaaaccacc agccctcctt cctcacctct cctcaagacc ttgcctcttc tccaatggaa
     7081 gaagatgagc agcattctga agcagatgag cctccatcag acgaacccct atctgatgac
     7141 cccctgtcac ctgctgagga gaaattgccc cacattgatg agtattggtc agacagtgag
     7201 cacatctttt tggatgcaaa tattggtggg gtggccatcg cacctgctca cggctcggtt
     7261 ttgattgagt gtgcccggcg agagctgcac gctaccactc ctgttgagca ccccaaccgt
     7321 aatcatccaa cccgcctctc ccttgtcttt taccagcaca aaaacctaaa taagccccaa
     7381 catggttttg aactaaacaa gattaagttt gaggctaaag aagctaagaa taagaaaatg
     7441 aaggcctcag agcaaaaaga ccaggcagct aatgaaggtc cagaacagtc ctctgaagta
     7501 aatgaattga accaaattcc ttctcataaa gcattaacat taacccatga caatgttgtc
     7561 accgtgtccc cttatgctct cacacacgtt gcggggccct ataaccattg ggtcatggcc
     7621 ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg gcagaggaag tctgctaaca
     7681 tgcggtgacg tcgaggagaa tcctggccca gtgagcaagg gcgaggagga taacatggcc
     7741 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     7801 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     7861 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     7921 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     7981 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     8041 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     8101 accaacttcc cctccgacgg ccccgtaatg cagaagaaaa ccatgggctg ggaggcctcc
     8161 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     8221 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     8281 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     8341 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     8401 gacgagctgt acaagtaaga attctaacta gagctcgctg atcagcctcg actgtgcctt
     8461 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     8521 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     8581 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga
     8641 atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct
     8701 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     8761 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     8821 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     8881 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     8941 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     9001 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     9061 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     9121 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     9181 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     9241 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     9301 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     9361 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     9421 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     9481 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     9541 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     9601 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     9661 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     9721 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     9781 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     9841 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     9901 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     9961 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
    10021 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
    10081 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
    10141 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
    10201 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
    10261 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
    10321 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
    10381 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
    10441 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
    10501 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
    10561 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
    10621 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
    10681 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
    10741 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
    10801 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
    10861 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
    10921 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
    10981 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
    11041 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
    11101 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
    11161 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    11221 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    11281 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    11341 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
    11401 catgt
//
