LOCUS       sequence-435331-       13391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  doxycycline-inducible dCas9-Zim3-MeCP2 expression for Epigenetic 
            repression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13391)
  AUTHORS   Ding L, Schmitt LT, Brux M, Surun D, Augsburg M, Lansing F, Mircetic
            J, Theis M, Buchholz F
  TITLE     DNA methylation-independent long-term epigenetic silencing with 
            dCRISPR/Cas9 fusion proteins.
  JOURNAL   Life Sci Alliance. 2022 Mar 14;5(6). pii: 5/6/e202101321. doi: 
            10.26508/lsa.202101321. Print 2022 Jun.
  PUBMED    35288457
REFERENCE   2  (bases 1 to 13391)
  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: "Life Sci 
            Alliance."; date: "2022-03-14"; volume: "5(6). pii: 5/6/e202101321. 
            doi"; pages: " 10.26508/lsa.202101321. Print 2022 Jun"
COMMENT     Sequence Label: PB-TRE-dCas9-Zim3-MeCP2
FEATURES             Location/Qualifiers
     source          1..13391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1162..1617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1511..1530
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1744..1766
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1758..1775
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1759..1775
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1905..1967
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2416..2794
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2424..2442
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2460..2478
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2496..2514
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2532..2550
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2568..2586
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2604..2622
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2640..2658
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2703..2727
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2821..2830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2827..2853,2854..2856,2857..5340,5341..5346,5347..5412,
                     5413..5415,5416..6930)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6943..6963
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7285..8142
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(8194..8213)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8238..8286
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8517..8728
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8741..9009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             9043..9786
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             9787..9840
                     /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             9862..10881
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             9862..10881
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(10938..10958)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    10992..11113
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11029..11048)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11083..11102
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11876..11910
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12401..12417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12401..12417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12414..12436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12425..12441
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(12449..12455,12456..12473,12474..12479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12494..12515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12632..12649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12803..13391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12883..12902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     1321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     1621 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1681 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     1741 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcctcgttc attcacgttt
     1801 ttgaacccgt ggaggacggg cagactcgcg gtgcaaatgt gttttacagc gtgatggagc
     1861 agatgaagat gctcgacacg ctgcagaaca cgcagctaga ttaaccctag aaagataatc
     1921 atattgtgac gtacgttaaa gataatcatg tgtaaaattg acgcatgtgt tttatcggtc
     1981 tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt
     2041 acatactaat aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa
     2101 caaaaactca aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc
     2161 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     2221 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     2281 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     2341 gagcctgcag acacctgggg ggatacgggg aaaaggcctc cacggccact agtctttcgt
     2401 cttcaagaat tcctcgagtt tactccctat cagtgataga gaacgtatga agagtttact
     2461 ccctatcagt gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat
     2521 aaggagttta ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga
     2581 tagagaacgt atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact
     2641 ccctatcagt gatagagaac gtataagctt taggcgtgta cggtgggcgc ctataaaagc
     2701 agagctcgtt tagtgaaccg tcagatcgcc tggagcaatt ccacaacact tttgtcttat
     2761 accaactttc cgtaccactt cctaccctcg taaaggtcta gagctagcga attcgaattt
     2821 gccaccatgg acaagaagta ctccattggg ctcgctatcg gcacaaacag cgtcggctgg
     2881 gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc
     2941 gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg
     3001 gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg
     3061 atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc
     3121 cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc
     3181 tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg
     3241 aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg
     3301 gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac
     3361 agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag
     3421 aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa
     3481 tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt
     3541 ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg
     3601 gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg
     3661 ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac
     3721 gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc
     3781 gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt
     3841 gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc
     3901 tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc
     3961 atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg
     4021 ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa
     4081 ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa
     4141 aagattgaga aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga
     4201 aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc
     4261 gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt
     4321 gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc
     4381 acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca
     4441 ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa
     4501 gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt
     4561 gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg
     4621 aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac
     4681 attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact
     4741 tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga
     4801 tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca
     4861 atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat
     4921 gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac
     4981 agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg
     5041 cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat
     5101 atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg
     5161 gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa
     5221 cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac
     5281 ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg
     5341 gctgctatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca
     5401 agatccgata aagctagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa
     5461 atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat
     5521 aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa
     5581 aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc
     5641 atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg
     5701 aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc
     5761 aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc
     5821 aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt
     5881 aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt
     5941 tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg
     6001 aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg
     6061 gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc
     6121 gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag
     6181 ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca
     6241 gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa
     6301 agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc
     6361 atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt
     6421 cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc
     6481 gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg
     6541 gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc
     6601 gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa
     6661 agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg
     6721 gataagccca tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg
     6781 ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
     6841 acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca
     6901 agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag
     6961 gtgggtacca tgaacaattc ccagggaaga gtgaccttcg aggatgtcac tgtgaacttc
     7021 acccaggggg agtggcagcg gctgaatccc gaacagagaa acttgtacag ggatgtgatg
     7081 ctggagaatt acagcaacct tgtctctgtg ggacaagggg aaaccaccaa acccgatgtg
     7141 atcttgaggt tggaacaagg aaaggagcca tggttggagg aagaggaagt gctgggaagt
     7201 ggccgtgcag aaaaaaatgg ggacattgga gggcagattt ggaagccaaa ggatgtgaaa
     7261 gagagtctca cgcgtgaagc ctcagtgcag gtgaaaaggg tgctggaaaa atcccccggc
     7321 aaactcctcg tgaagatgcc cttccaggct tcccctggcg gaaaaggtga agggggtggc
     7381 gcaaccacat ctgcccaggt catggtcatc aagcgacctg gaaggaaaag aaaggccgag
     7441 gctgaccctc aggccattcc aaagaaacgg ggacgcaagc cagggtccgt ggtcgcagct
     7501 gcagcagctg aggctaagaa aaaggcagtg aaggaaagct ccatccgcag tgtgcaggag
     7561 actgtcctgc ccatcaagaa gaggaagact agggagaccg tgtccatcga ggtcaaagaa
     7621 gtggtcaagc ccctgctcgt gtccaccctg ggcgaaaaat ctggaaaggg gctcaaaaca
     7681 tgcaagtcac ctggacggaa aagcaaggag tctagtccaa aggggcgctc aagctccgct
     7741 tctagtcccc ctaaaaagga acaccatcac catcaccatc acgccgagtc tcctaaggct
     7801 cctatgccac tgctcccacc acctccacca cctgagccac agtcaagcga agaccccatc
     7861 agcccacccg agcctcagga tctgtcctct agtatttgca aagaggaaaa gatgcccaga
     7921 gcaggcagcc tggagagtga tggctgtcca aaagaacccg ccaagaccca gcctatggtg
     7981 gcagccgctg caactaccac cacaaccaca actaccacag tggccgaaaa atacaagcat
     8041 cgcggcgagg gcgaacgaaa ggacattgtg tcaagctcca tgcccagacc taaccgggag
     8101 gaaccagtcg atagtaggac acccgtgact gagagagtct cataggcggc cgcctagacc
     8161 ggttagtaat gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga
     8221 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt
     8281 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     8341 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     8401 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcagatctgc
     8461 gaaatcggat ccagctaaat ctaagtggat ctgcgatcgc tccggtgccc gtcagtgggc
     8521 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
     8581 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     8641 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     8701 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
     8761 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
     8821 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
     8881 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
     8941 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
     9001 ttacagatct aagctgtgac cggcgcctac tctagagccg ccatgtctag actggacaag
     9061 agcaaagtca taaactctgc tctggaatta ctcaatggag tcggtatcga aggcctgacg
     9121 acaaggaaac tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag
     9181 aacaagcggg ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac
     9241 tcctgccccc tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac
     9301 cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca
     9361 gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc
     9421 ctggagaacg cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg
     9481 gaggaacagg agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg
     9541 cccccacttc tgaaacaagc aattgagctg ttcgaccggc agggagccga acctgccttc
     9601 cttttcggcc tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc
     9661 gggccgaccg acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac
     9721 gactttgacc ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc
     9781 cccggggagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     9841 gaattcatgg atagatccgg aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     9901 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     9961 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
    10021 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
    10081 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
    10141 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
    10201 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
    10261 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
    10321 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
    10381 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
    10441 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
    10501 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
    10561 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
    10621 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
    10681 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
    10741 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
    10801 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
    10861 cgtccgaggg caaaggaata agccaaggaa cctctggatt acaaaatttg tgaaagattg
    10921 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
    10981 ttgtatcagt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
    11041 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
    11101 tcaatgtatc ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc
    11161 tcatctggtc tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt
    11221 cccaaactgg gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct
    11281 cacaggaacg aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg
    11341 attccttttt tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag
    11401 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
    11461 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
    11521 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg
    11581 ctttgggggg gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc
    11641 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
    11701 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
    11761 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
    11821 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
    11881 gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc
    11941 gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa
    12001 gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac
    12061 tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg
    12121 gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg
    12181 cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat
    12241 accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg
    12301 ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata
    12361 ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
    12421 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    12481 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    12541 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    12601 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    12661 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    12721 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    12781 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    12841 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    12901 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    12961 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    13021 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    13081 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    13141 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    13201 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    13261 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    13321 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    13381 aaggatctca a
//
