LOCUS       sequence-470314-       13021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible expression of dCas9-GCN4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13021)
  AUTHORS   Policarpi C, Munafo M, Tsagkris S, Carlini V, Hackett JA
  TITLE     Systematic epigenome editing captures the context-dependent 
            instructive function of chromatin modifications.
  JOURNAL   Nat Genet. 2024 Jun;56(6):1168-1180. doi: 
            10.1038/s41588-024-01706-w. Epub 2024 May 9.
  PUBMED    38724747
REFERENCE   2  (bases 1 to 13021)
  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"; doi: 
            "10.1038/s41588-024-01706-w"; journalName: "Nat Genet"; date: 
            "2024-06"; volume: "56"; issue: "6"; pages: "1168-1180"
COMMENT     Sequence Label: pPB_TRE3G_dCas9-5xGCN4_Hygro
FEATURES             Location/Qualifiers
     source          1..13021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1385..1763
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1393..1411
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1429..1447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1465..1483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1501..1519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1537..1555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1573..1591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1609..1627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1672..1696
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1784..1804
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1853..1862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1859..1885,1886..1888,1889..4375,4376..4378,
                     4379..5962)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1859..5962
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5966..5992
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5972..5998
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             6011..6031
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6038..6058
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6122..6178
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6245..6301
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6368..6424
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6491..6547
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             6614..6670
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     primer_bind     complement(6793..6812)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6837..6885
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7116..7327
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             7340..7608
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             7642..8385
                     /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             8386..8439
                     /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             8461..9480
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             8461..9480
                     /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(9537..9557)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    9591..9712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9628..9647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9682..9701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10475..10509
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11000..11016)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11000..11016)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11013..11035)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11024..11040
                     /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(11048..11054,11055..11072,11073..11078))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11093..11114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11231..11248)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11402..11990)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11482..11501)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12161..12952,12953..13021))
                     /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     12784..12803
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtctttcgtc ttcaagaatt
     1381 cctcgagttt actccctatc agtgatagag aacgtatgaa gagtttactc cctatcagtg
     1441 atagagaacg tatgcagact ttactcccta tcagtgatag agaacgtata aggagtttac
     1501 tccctatcag tgatagagaa cgtatgacca gtttactccc tatcagtgat agagaacgta
     1561 tctacagttt actccctatc agtgatagag aacgtatatc cagtttactc cctatcagtg
     1621 atagagaacg tataagcttt aggcgtgtac ggtgggcgcc tataaaagca gagctcgttt
     1681 agtgaaccgt cagatcgcct ggagcaattc cacaacactt ttgtcttata ccaactttcc
     1741 gtaccacttc ctaccctcgt aaaggtctag agctagcatg gcgcccaaga agaagcgcaa
     1801 ggtgggacgc gtctgcagga tatcaagctt gcggtaccgc gggcccggga tcgccaccat
     1861 ggacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat
     1921 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     1981 cagcatcaag aagaacctga tcggcgccct gctgttcgac agcggagaaa cagccgaggc
     2041 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     2101 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     2161 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     2221 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     2281 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     2341 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     2401 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     2461 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     2521 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct
     2581 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     2641 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     2701 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     2761 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     2821 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     2881 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     2941 ctacatcgat ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     3001 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     3061 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     3121 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     3181 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     3241 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     3301 ggtggacaag ggcgccagcg cccagagctt catcgagcgg atgaccaact tcgataagaa
     3361 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     3421 caacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     3481 cggcgagcag aaaaaagcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     3541 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     3601 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     3661 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     3721 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     3781 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     3841 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     3901 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     3961 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     4021 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     4081 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     4141 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     4201 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     4261 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     4321 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggacgctat
     4381 cgtgcctcag agctttctga aggacgactc catcgataac aaagtgctga ctcggagcga
     4441 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     4501 ctactggcgc cagctgctga atgccaagct gattacccag aggaagttcg acaatctgac
     4561 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     4621 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     4681 taagtacgac gagaacgaca aactgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     4741 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     4801 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     4861 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     4921 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     4981 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     5041 tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg gataagggcc gggactttgc
     5101 caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     5161 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgaca agctgatcgc
     5221 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     5281 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     5341 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     5401 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     5461 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     5521 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     5581 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     5641 gcacaaacac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     5701 cctggccgac gctaatctgg acaaggtgct gagcgcctac aacaagcaca gagacaagcc
     5761 tatcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     5821 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     5881 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     5941 cctgtctcag ctgggaggcg acgcctatcc ctatgacgtg cccgattatg ccagcctggg
     6001 cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgga
     6061 cggcattggt agtgggagca acggcagcag cggatccaac ggtccgactg acgccgcgga
     6121 agaggagctt ctgagcaaaa actatcacct cgaaaacgag gttgcgcgac tgaagaaagg
     6181 aagcgggtcc ggtggaagtg gctccggatc tggaggttct ggcagcggag gtagcggcag
     6241 tggcgaagag ctccttagta agaactatca tctggaaaat gaggtagcgc gcttaaagaa
     6301 agggtcggga agtggcggca gcggaagtgg gagtggaggg agcggttctg gcggttccgg
     6361 cagtggagag gagttgctgt ctaagaacta ccacttagaa aacgaagtcg cacggctaaa
     6421 aaaaggttcc ggctccggcg gctccggttc tggaagcggg ggctcgggat caggtggatc
     6481 tggatcagga gaggaattgc tttccaaaaa ctaccacctt gagaatgagg tggccaggtt
     6541 aaagaagggg agcggctcgg ggggtagtgg atcggggtcg ggcgggtcag gaagcggtgg
     6601 tagcggatct ggggaggagc tgctctcgaa gaattaccat ttggagaacg aagtggcgag
     6661 actaaagaag ggaagcggta gtggtggttc agggtctggt tcaggtggca gtgggtctgg
     6721 gggctcaggg tccgggacgg ccggccacta gccggtggcg gtggcggagg ggtttaaacg
     6781 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     6841 aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc
     6901 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     6961 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     7021 ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg aaatcggatc cagctaaatc
     7081 taagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
     7141 ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg
     7201 ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga
     7261 accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag
     7321 aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg
     7381 aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg
     7441 aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc
     7501 gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc
     7561 tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcta agctgtgacc
     7621 ggcgcctact ctagagccgc catgtctaga ctggacaaga gcaaagtcat aaactctgct
     7681 ctggaattac tcaatggagt cggtatcgaa ggcctgacga caaggaaact cgctcaaaag
     7741 ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc cctgctcgat
     7801 gccctgccaa tcgagatgct ggacaggcat catacccact cctgccccct ggaaggcgag
     7861 tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct cctctcacat
     7921 cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta cgaaaccctg
     7981 gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc actgtacgct
     8041 ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga gcatcaagta
     8101 gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct gaaacaagca
     8161 attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct ggaactaatc
     8221 atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac
     8281 gattttgact tagacatgct cccagccgat gcccttgacg actttgacct tgatatgctg
     8341 cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccggggaggg cagaggaagt
     8401 cttctaacat gcggtgacgt ggaggagaat cccggccctg aattcatgga tagatccgga
     8461 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     8521 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     8581 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     8641 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     8701 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     8761 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     8821 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     8881 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     8941 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     9001 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     9061 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     9121 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     9181 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     9241 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     9301 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     9361 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     9421 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa
     9481 gccaaggaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     9541 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcagtt aacttgttta
     9601 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     9661 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     9721 ggaattgact caaatgatgt caattagtct atcagaagct catctggtct cccttccggg
     9781 ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc
     9841 ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa
     9901 gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt
     9961 ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc
    10021 gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc
    10081 cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg
    10141 ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct
    10201 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
    10261 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
    10321 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
    10381 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
    10441 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
    10501 tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt
    10561 catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc
    10621 gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag
    10681 cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt
    10741 gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc
    10801 cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc
    10861 cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac
    10921 gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg
    10981 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
    11041 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
    11101 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
    11161 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
    11221 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
    11281 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
    11341 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
    11401 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
    11461 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
    11521 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
    11581 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
    11641 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
    11701 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
    11761 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
    11821 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
    11881 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
    11941 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
    12001 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
    12061 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
    12121 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
    12181 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
    12241 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
    12301 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
    12361 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
    12421 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
    12481 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
    12541 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
    12601 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
    12661 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
    12721 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
    12781 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
    12841 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
    12901 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
    12961 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
    13021 t
//
