LOCUS       sequence-474753-       12961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac CRISPRa construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12961)
  AUTHORS   Vucicevic D, Hsu C-W, Lopez Zepeda LS, Burkert M, Hirsekorn A, Bilic
            I, Kastelic N, Landthaler M, Lacadie SA, Ohler U
  TITLE     Sensitive dissection of a genomic regulatory landscape using bulk 
            and targeted single-cell activation
  JOURNAL   bioRxiv 2024.09.12.612198
REFERENCE   2  (bases 1 to 12961)
  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: "bioRxiv 
            2024.09.12.612198"
COMMENT     Sequence Label: pPB-CAG-dCas9-VPR-pgk-hph
FEATURES             Location/Qualifiers
     source          1..12961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   1054..1116
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    complement(1287..1320)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1382..1430
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1455..1474
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1529..2098)
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             complement(2117..2473)
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             complement(2903..2923)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2948..3097)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(3122..3142)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(3155..4669,4670..4672,4673..4738,4739..4744,
                     4745..7228,7229..7231,7232..7258))
                     /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"
     primer_bind     complement(7309..7328)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(7390..7412)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        8633..9012
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(9028..9061)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        9084..9583
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(9119..9142)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     9516..9535
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9602..10627
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9602..10627
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     repeat_region   11109..11143
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11161..11180)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11162..11180)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11187..11204)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11187..11203)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11196..11218)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11345..11800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11432..11451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11642..11663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11826..11930
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11931..11999,12000..12791)
                     /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(12149..12168)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     1021 tcactaaagg gaacaaaagc tggagctctt taaccctaga aagataatca tattgtgacg
     1081 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
     1141 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1201 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1261 aaatttcttc tataaagtaa caaaacataa cttcgtatag catacattat acgaagttat
     1321 ctcgacgcgg ccgctggggt atcgacagag tgccagccct gggaccgaac cccgcgttta
     1381 tgaacaaacg acccaacacc cgtgcgtttt attctgtctt tttattgccg tcatagcgcg
     1441 ggttccttcc ggtattgtct ccttccgtgt ttcagttagc ctcccccgtt taaactcatt
     1501 actaaccggt agggatcgaa ccctttcaaa acagagatgt gtcgaagatg gacagtcctg
     1561 tgctgatatg catggcatgc aagaggcact cgtcgttcag gaaggtatcc agaatctcgt
     1621 tcaattccgg ggtcaggggt gagtccaggt tcagatcctc ggtcatggac tcaagtgtgg
     1681 ttgtcagctc atccagatgg ccccttgggg gcggatggga aaggtccatt tggccacaga
     1741 ttgcagcctc ttccttctgg ggaatcacag tatcggccat ctcccgaagg gctttgacag
     1801 cctggctcgt ctcttcatcg ggatcctcca acaggtgact ggcctcggga gtcactgcgg
     1861 gcgctggatc cagtggctga gggactggtg ccggggtcag tgacccgact ggctcatgta
     1921 ctggaccggt tggtgttggt gcgaggctgg cggggagtgg gcggttggcc catggacttc
     1981 ctggaggatg aaatggccgg attcgttttg gctggcacac ctcgcggccc tcaaacacgt
     2041 cactaatagc ggagccggcc tcaggcttcg gcaaaaacat cccttccctg gaatcccgag
     2101 atcccgatcc gctacccagc agtgcggaga agtccatgtc ggcgatagag ctgaagtcct
     2161 cgtcgccaga cagcagtcca ttaggcaggc ctggtgctcc cagaggggca ggagctggat
     2221 caggaggcct ctgagcgcct gtcacgagcc gggtgatggc ctcggggtat tccatcagca
     2281 tgggctcggt ggtgtgaggg gccacaggga tgccctggtt cagcagctgc tggaactcgc
     2341 tgttgtccac gctggccagg tcggtgaaca cggcaggatc ggtgctgttt cccagcaggg
     2401 ctcccagatc ctcgtcgtcg aactgcagct gcagcagagc ttcagacagt gtgccctcgc
     2461 cggcctgtgt aggtttaggg gctggtggag ccacagcctg tggaggtcca ggagccagca
     2521 caggcacggg tgctggtgcc tgagccagtg cagacaccat ggctggagct ggtgcaggag
     2581 caggagcctg aggcagcacc tgaggagggg ctggagccag agcagaggcc tgagagatct
     2641 ggccgctggg gaacaccatg gtagggaact cgtcgtagtt gatggtgctc aggctgctgg
     2701 tgaaggggta aggctggggg gcaggttttg gcacgctggc gctggatctg ctgggcacgg
     2761 cgattcttct aggtggaggt ctagggtcgg tggggccgct gaagggggac ttcttcatga
     2821 tgctcttgaa tgtctcgtag gtccgcttcc gcttttcctc gatccggtgc cggtcgtcgg
     2881 tgtcgggcag gtactggcta ccaactttgc gtttcttttt cggagatccg gaacttctag
     2941 agttaatcag catgtccagg tcgaaatcat caagggcgtc actgccgagc atgtcgaggt
     3001 caaagtcatc aagggcatcc gaaccaagca tgtcaaggtc aaaatcatcg agggcgtcac
     3061 ttcccagcat atccagatca aaatcgtcca atgcgtcagc ccgtccggaa ccgctggcct
     3121 ccaccttcct cttcttcttg gggtcagccc tgctgtctcc accgagctga gagaggtcga
     3181 ttcttgtttc atagagcccc gtaattgact gatgaatcag tgtggcgtcc aggacctcct
     3241 ttgtagaggt gtaccgcttt ctgtctatgg tggtgtcgaa gtacttgaag gctgcaggcg
     3301 cgcccaagtt ggtcagagta aacaagtgga taatgttttc tgcctgctcc ctgatgggct
     3361 tatccctgtg cttattgtaa gcagaaagca ccttatcgag gttagcgtcg gcgaggatca
     3421 ctcttttgga gaattcgctt atttgctcga tgatctcatc aaggtagtgt ttgtgttgtt
     3481 ccacgaacag ctgcttctgc tcattatctt cgggagaccc tttgagcttt tcatagtggc
     3541 tggccagata caagaaatta acgtatttag agggcagtgc cagctcgtta cctttctgca
     3601 gctcgcccgc actagcgagc attcgtttcc ggccgttttc aagctcaaag agagagtact
     3661 tgggaagctt aatgatgagg tcttttttga cctctttata tcctttcgcc tcgagaaagt
     3721 cgatggggtt tttttcgaag cttgatcgct ccatgattgt gatgcccagc agttccttga
     3781 cgcttttgag ttttttagac ttccctttct ccactttggc cacaaccagt acactgtaag
     3841 cgactgtagg agaatcgaat ccgccgtatt tcttggggtc ccaatctttt ttgcgtgcga
     3901 tcagcttgtc gctgttcctt ttcgggagga tactttcctt ggagaagcct ccggtctgta
     3961 cttcggtctt tttaacgatg ttcacctgcg gcatggacag gaccttccgg actgtcgcga
     4021 aatccctacc cttgtcccac acgatttctc ctgtttctcc gtttgtttcg ataagtggtc
     4081 gcttccgaat ctctccattg gccagtgtaa tctcggtctt gaaaaaattc ataatattgc
     4141 tgtaaaagaa gtacttagcg gtggccttgc ctatttcctg ctcagacttt gcgatcattt
     4201 tcctaacatc gtacacttta tagtctccgt aaacaaattc agattcaagc ttgggatatt
     4261 ttttgataag tgcagtgcct accactgcat tcaggtaggc atcatgcgca tggtggtaat
     4321 tgttgatctc tctcacctta taaaactgaa agtcctttct gaaatctgag accagcttag
     4381 acttcagagt aataactttc acctctcgaa tcagtttgtc attttcatcg tacttggtgt
     4441 tcatgcgtga atcgagaatt tgggccacgt gcttggtgat ctggcgtgtc tcaacaagct
     4501 gccttttgat gaagccggct ttatccaact cagacaggcc acctcgttca gccttagtca
     4561 gattatcgaa cttccgttgt gtgatcagtt tggcgttcag cagctgccgc caataatttt
     4621 tcattttctt gacaacttct tctgagggga cgttatcact cttccctcta gctttatcgg
     4681 atcttgtcaa cactttatta tcaatagaat catctttgag aaaagactgg ggcacgatag
     4741 cagccacgtc gtagtcggag agccgattga tgtccagttc ctgatccacg tacatgtccc
     4801 tgccgttctg caggtagtac aggtagagct tctcattctg aagctgggtg ttttcaactg
     4861 ggtgttcctt aaggatttgg gaccccagtt cttttatacc ctcttcaatc ctcttcatcc
     4921 tttccctact gttcttctgt cccttctggg tagtttggtt ctctcgggcc atctcgataa
     4981 cgatattctc gggcttatgc cttcccatta ctttgacgag ttcatccacg accttaacgg
     5041 tctgcagtat tccctttttg atagctgggc tacctgcaag attagcgatg tgctcgtgaa
     5101 gactgtcccc ctggccagaa acttgtgctt tctggatgtc ctccttaaag gtgagagagt
     5161 catcatggat caactgcatg aagttccggt tggcaaatcc atcggactta agaaaatcca
     5221 ggattgtctt tccactctgc ttgtctcgga tcccattgat cagttttctt gacagccgcc
     5281 cccatcctgt atatcggcgc ctcttgagct gtttcatgac tttgtcgtcg aagagatgag
     5341 cgtaagtttt caagcgttct tcaatcatct ccctatcttc aaacaacgta agggtgagga
     5401 caatgtcctc aagaatgtcc tcgttctcct cattgtccag gaagtccttg tctttaatga
     5461 ttttcaggag atcgtgatac gttcccaggg atgcgttgaa gcgatcctcc actccgctga
     5521 tttcaacaga gtcgaaacat tcaatctttt tgaaatagtc ttctttgagc tgtttcacgg
     5581 taactttccg gttcgtcttg aagaggaggt ccacgatagc tttcttctgc tctccagaca
     5641 ggaatgctgg ctttctcatc ccttctgtga cgtatttgac cttggtgagc tcgttataaa
     5701 ctgtgaagta ctcgtacagc agagagtgtt taggaagcac cttttcgtta ggcagatttt
     5761 tatcaaagtt agtcatcctt tcgatgaagg actgggcaga ggccccctta tccacgactt
     5821 cctcgaagtt ccagggagtg atggtctctt ctgatttgcg agtcatccac gcgaatctgg
     5881 aatttccccg ggcgaggggg cctacatagt agggtatccg aaatgtgagg attttctcaa
     5941 tcttttccct gttatctttc aaaaaggggt agaaatcctc ttgccgcctg aggatagcgt
     6001 gcagttcgcc caggtgaatc tggtggggga tgcttccatt gtcgaaagtg cgctgtttgc
     6061 gcaacagatc ttctctgtta agctttacca gcagctcctc ggtgccgtcc attttttcca
     6121 agatgggctt aataaatttg taaaattcct cctggcttgc tccgccgtca atgtatccgg
     6181 cgtagccatt tttagactga tcgaagaaaa tttccttgta cttctcaggc agttgctgtc
     6241 tgacaagggc cttcagcaaa gtcaagtctt ggtggtgctc atcatagcgc ttgatcatac
     6301 tagcgctcag cggagctttg gtgatctccg tgttcactcg cagaatatca ctcagcagaa
     6361 tggcgtctga caggttcttt gccgccaaaa aaaggtctgc gtactggtcg ccgatctggg
     6421 ccagcagatt gtcgagatca tcatcgtagg tgtctttgct cagttgaagc ttggcatctt
     6481 cggccaggtc gaagttagat ttaaagttgg gggtcagccc gagtgacagg gcgataagat
     6541 taccaaacag gccgttcttc ttctccccag ggagctgtgc gatgaggttt tcgagccgcc
     6601 gggatttgga cagcctagcg ctcaggattg ctttggcgtc aactccggat gcgttgatcg
     6661 ggttctcttc gaaaagctga ttgtaagtct gaaccagttg gataaagagt ttgtcgacat
     6721 cgctgttgtc tgggttcagg tccccctcga tgaggaagtg tccccgaaat ttgatcatat
     6781 gcgccagcgc gagatagatc aaccgcaagt cagccttatc agtactgtct acaagcttct
     6841 tcctcagatg atatatggtt gggtactttt catggtacgc cacctcgtcc acgatattgc
     6901 caaagattgg gtggcgctcg tgctttttat cctcctccac caaaaaggac tcctccagcc
     6961 tatggaagaa agagtcatcc accttagcca tctcattact aaagatctcc tgcaggtagc
     7021 agatccgatt ctttctgcgg gtatatctgc gccgtgctgt tcttttgagc cgcgtggctt
     7081 cggccgtctc cccggagtcg aacaggaggg cgccaatgag gttcttcttt atgctgtggc
     7141 gatcggtatt gcccagaact ttgaattttt tgctcggcac cttgtactcg tccgtaatga
     7201 cggcccagcc gacgctgttt gtgccgatag cgagcccaat ggagtacttc ttgtccatgg
     7261 aagcttatcg ataccgtcga ggaattcttt gccaaaatga tgagacagca caataaccag
     7321 cacgttgccc aggagctgta ggaaaaagaa gaaggcatga acatggttag cagaggcgct
     7381 agagccgccg gtcacacgcc agaagccgaa ccccgccctg ccccgtcccc cccgaaggca
     7441 gccgtccccc cgcggacagc cccgaggctg gagagggaga aggggacggc ggcgcggcga
     7501 cgcacgaagg ccctccccgc ccatttcctt cctgccggcg ccgcaccgct tcgccccgcg
     7561 cccgctagag ggggtgcggc ggcgcctccc agatttcggc tccgcacaga tttgggacaa
     7621 aggaagtccc tgcgccctct cgcacgatta ccataaaagg caatggctgc ggctcgccgc
     7681 gcctcgacag ccgccggcgc tccgggggcc gccgcgcccc tcccccgagc cctccccggc
     7741 ccgaggcggc cccgccccgc ccggcacccc cacctgccgc caccccccgc ccggcacggc
     7801 gagccccgcg ccacgccccg tacggagccc cgcacccgaa gccgggccgt gctcagcaac
     7861 tcggggaggg gggtgcaggg ggggttgcag cccgaccgac gcgcccacac cccctgctca
     7921 cccccccacg cacacacccc gcacgcagcc tttgttcccc tcgcagcccc cccgcaccgc
     7981 ggggcaccgc ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg
     8041 ccgcgcccgc agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg
     8101 cacacacaca cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc
     8161 ccctcaaggc tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa
     8221 ttacagcccg gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt
     8281 cagagccggg gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagccg
     8341 cccaccaccc cccccccccc cgcgctgacc gtttttgatg ggcgccccgc cccccccccc
     8401 ccccctaaaa aaaaaccctc cccccccccc ccccccctgg ggcgcccccc cccccccccc
     8461 cccccccccc cccccccccc cccccccccc cccccccccc cggggcccgc cccccccccc
     8521 cccccgcccc catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg
     8581 gggagggggg ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa
     8641 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     8701 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     8761 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     8821 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     8881 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     8941 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     9001 ataatcaatg tcgacctcga gggccgcata acttcgtata gcatacatta tacgaagtta
     9061 tgatatcatc gatgaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     9121 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     9181 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     9241 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     9301 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     9361 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     9421 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     9481 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     9541 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgaccgatc cagccgccac
     9601 catgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     9661 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     9721 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     9781 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     9841 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     9901 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     9961 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
    10021 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
    10081 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
    10141 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
    10201 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
    10261 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
    10321 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
    10381 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
    10441 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
    10501 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
    10561 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
    10621 ggaatagtcg agaaattgat gatctattaa acaataaaga tgtccacatg gaagtttttc
    10681 ctgtcatact ttgttaagaa gggtgagaac agagtaccta cattttgaat ggaaggattg
    10741 gagctacggg ggtgggggtg gggtgggatt agataaatgc ctgctcttta ctgaaggctc
    10801 tttactattg ctttatgata atgtttcata gttggatatc tataacaaga aaatatatat
    10861 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
    10921 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
    10981 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
    11041 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
    11101 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaaggt acccaattcg
    11161 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
    11221 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
    11281 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
    11341 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
    11401 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
    11461 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
    11521 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
    11581 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
    11641 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
    11701 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
    11761 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
    11821 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
    11881 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
    11941 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
    12001 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
    12061 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
    12121 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
    12181 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
    12241 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
    12301 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
    12361 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
    12421 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
    12481 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
    12541 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
    12601 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
    12661 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
    12721 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
    12781 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
    12841 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
    12901 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
    12961 c
//
