LOCUS       sequence-474756-       13066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac CRISPRi construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13066)
  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 13066)
  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-KRAB-dCas9-P2A-mCherry-pgk-hph
FEATURES             Location/Qualifiers
     source          1..13066
                     /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)"
     misc_feature    1455..2043
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1561..1572
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1970..1990
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2092..2802)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2370..2389)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2639..2657
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2803..2859)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2899..2919)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2926..2946)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2959..2985)
                     /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             complement(2965..2991)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(2995..4578,4579..4581,4582..7068,7069..7071,
                     7072..7098))
                     /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             complement(2995..7098)
                     /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             complement(7120..7305)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     primer_bind     complement(7386..7405)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          7413..8428
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(7467..7489)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        8738..9117
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(9133..9166)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        9189..9688
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(9224..9247)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     9621..9640
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9707..10732
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             9707..10732
                     /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   11214..11248
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11266..11285)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11267..11285)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11292..11309)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11292..11308)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11301..11323)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11450..11905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11537..11556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11747..11768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11931..12035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12036..12104,12105..12896)
                     /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(12254..12273)
                     /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 ggtagcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag
     1501 gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg
     1561 attgagggcc gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca
     1621 ggcgagcagc caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc
     1681 caacagccga gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc
     1741 aatagggagg gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca
     1801 accagtgggg gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg
     1861 ccacaactcc tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc
     1921 catacgggaa gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa
     1981 aggagcaaca tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg
     2041 attatcgata agcttgatat caagcttgca tgcctgcagg tcgactctag attacttgta
     2101 cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat
     2161 ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg
     2221 cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc
     2281 gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat
     2341 ccgctcggag gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg
     2401 gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc
     2461 ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
     2521 ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt
     2581 ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt
     2641 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
     2701 gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc
     2761 cttgatgatg gccatgttat cctcctcgcc cttgctcacc ataggtccag ggttctcctc
     2821 cacgtctcca gcctgcttca gcaggctgaa gttagtagcg gatccgctgc tgccgttgct
     2881 cccactacca atgccgtcca ctttccgctt tttcttagga tcttccacct tgcgtttttt
     2941 cttgggggag ccgctgccca ggctggcata atcgggcacg tcatagggat aggcgtcgcc
     3001 tcccagctga gacaggtcga tccgtgtctc gtacaggccg gtgatgctct ggtggatcag
     3061 ggtggcgtcc agcacctctt tggtgctggt gtacctcttc cggtcgatgg tggtgtcaaa
     3121 gtacttgaag gcggcagggg ctcccagatt ggtcagggta aacaggtgga tgatattctc
     3181 ggcctgctct ctgataggct tgtctctgtg cttgttgtag gcgctcagca ccttgtccag
     3241 attagcgtcg gccaggatca ctctcttgga gaactcgctg atctgctcga tgatctcgtc
     3301 caggtagtgt ttgtgctgtt ccacaaacag ctgtttctgc tcattatcct cgggggagcc
     3361 cttcagcttc tcatagtggc tggccaggta caggaagttc acatatttgg agggcagggc
     3421 cagttcgttt cccttctgca gttcgccggc agaggccagc attctcttcc ggccgttttc
     3481 cagctcgaac agggagtact taggcagctt gatgatcagg tcctttttca cttctttgta
     3541 gcccttggct tccagaaagt cgatgggatt cttctcgaag ctgcttcttt ccatgatggt
     3601 gatccccagc agctctttca cactcttcag tttcttggac ttgccctttt ccactttggc
     3661 caccaccagc acagaatagg ccacggtggg gctgtcgaag ccgccgtact tcttagggtc
     3721 ccagtccttc tttctggcga tcagcttgtc gctgttcctc ttgggcagga tagactcttt
     3781 gctgaagccg cctgtctgca cctcggtctt tttcacgata ttcacttggg gcatagacag
     3841 cactttccgc acggtggcaa agtcccggcc cttatcccac acgatctcgc ctgtttcgcc
     3901 gtttgtctcg atcagaggcc gcttccggat ctcgccgttg gccagggtaa tctcggtctt
     3961 gaaaaagttc atgatgttgc tgtagaagaa gtacttggcg gtagccttgc cgatttcctg
     4021 ctcgctcttg gcgatcatct tccgcacgtc gtacaccttg tagtcgccgt acacgaactc
     4081 gctttccagc ttagggtact ttttgatcag ggcggttccc acgacggcgt tcaggtaggc
     4141 gtcgtgggcg tggtggtagt tgttgatctc gcgcactttg taaaactgga aatccttccg
     4201 gaaatcggac accagcttgg acttcagggt gatcactttc acttcccgga tcagtttgtc
     4261 gttctcgtcg tacttagtgt tcatccggga gtccaggatc tgtgccacgt gctttgtgat
     4321 ctgccgggtt tccaccagct gtctcttgat gaagccggcc ttatccagtt cgctcaggcc
     4381 gcctctctcg gccttggtca gattgtcgaa cttcctctgg gtaatcagct tggcattcag
     4441 cagctggcgc cagtagttct tcatcttctt cacgacctct tcggagggca cgttgtcgct
     4501 cttgccccgg ttcttgtcgc tccgagtcag cactttgtta tcgatggagt cgtccttcag
     4561 aaagctctga ggcacgatag cgtccacatc gtagtcggac agccggttga tgtccagttc
     4621 ctggtccacg tacatatccc gcccattctg caggtagtac aggtacagct tctcgttctg
     4681 cagctgggtg ttttccacgg ggtgttcttt caggatctgg ctgcccagct ctttgatgcc
     4741 ctcttcgatc cgcttcattc tctcgcggct gttcttctgt cccttctggg tggtctggtt
     4801 ctctctggcc atttcgatca cgatgttctc gggcttgtgc cggcccatca ctttcacgag
     4861 ctcgtccacc accttcactg tctgcaggat gcccttctta atggcggggc tgccggccag
     4921 attggcaatg tgctcgtgca ggctatcgcc ctggccggac acctgggctt tctggatgtc
     4981 ctctttaaag gtcaggctgt cgtcgtggat cagctgcatg aagtttctgt tggcgaagcc
     5041 gtcggacttc aggaaatcca ggattgtctt gccggactgc ttgtcccgga tgccgttgat
     5101 cagcttccgg ctcagcctgc cccagccggt gtatctccgc cgcttcagct gcttcatcac
     5161 tttgtcgtcg aacaggtggg cataggtttt cagccgttcc tcgatcatct ctctgtcctc
     5221 aaacagtgtc agggtcagca cgatatcttc cagaatgtcc tcgttttcct cattgtccag
     5281 gaagtccttg tccttgataa ttttcagcag atcgtggtat gtgcccaggg aggcgttgaa
     5341 ccgatcttcc acgccggaga tttccacgga gtcgaagcac tcgattttct tgaagtagtc
     5401 ctctttcagc tgcttcacgg tcactttccg gttggtcttg aacagcaggt ccacgatggc
     5461 ttttttctgc tcgccgctca ggaaggcggg ctttctcatt ccctcggtca cgtatttcac
     5521 tttggtcagc tcgttgtaca cggtgaagta ctcgtacagc aggctgtgct tgggcagcac
     5581 cttctcgttg ggcaggttct tatcgaagtt ggtcatccgc tcgatgaagc tctgggcgct
     5641 ggcgcccttg tccaccactt cctcgaagtt ccagggggtg atggtttcct cgctctttct
     5701 ggtcatccag gcgaatctgc tgtttcccct ggccagaggg cccacgtagt aggggatgcg
     5761 gaaggtcagg atcttctcga tcttttcccg gttgtccttc aggaatgggt aaaaatcttc
     5821 ctgccgccgc agaatggcgt gcagctctcc caggtggatc tggtggggga tgctgccgtt
     5881 gtcgaaggtc cgctgcttcc gcagcaggtc ctctctgttc agcttcacga gcagttcctc
     5941 ggtgccgtcc atcttttcca ggatgggctt gatgaacttg tagaactctt cctggctggc
     6001 tccgccatcg atgtagccgg cgtagccgtt cttgctctgg tcgaagaaaa tctctttgta
     6061 cttctcaggc agctgctgcc gcacgagagc tttcagcagg gtcaggtcct ggtggtgctc
     6121 gtcgtatctc ttgatcatag aggcgctcag gggggccttg gtgatctcgg tgttcactct
     6181 caggatgtcg ctcagcagga tggcgtcgga caggttcttg gcggccagaa acaggtcggc
     6241 gtactggtcg ccgatctggg ccagcaggtt gtccaggtcg tcgtcgtagg tgtccttgct
     6301 cagctgcagt ttggcatcct cggccaggtc gaagttgctc ttgaagttgg gggtcaggcc
     6361 caggctcagg gcaatcaggt tgccgaacag gccattcttc ttctcgccgg gcagctgggc
     6421 gatcagattt tccagccgtc tgctcttgct cagtctggca gacaggatgg ccttggcgtc
     6481 cacgccgctg gcgttgatgg ggttttcctc gaacagctgg ttgtaggtct gcaccagctg
     6541 gatgaacagc ttgtccacgt cgctgttgtc ggggttcagg tcgccctcga tcaggaagtg
     6601 gccccggaac ttgatcatgt gggccagggc cagatagatc agccgcaggt cggccttgtc
     6661 ggtgctgtcc accagtttct ttctcaggtg gtagatggtg gggtacttct cgtggtaggc
     6721 cacctcgtcc acgatgttgc cgaagatggg gtgccgctcg tgcttcttat cctcttccac
     6781 caggaaggac tcttccagtc tgtggaagaa gctgtcgtcc accttggcca tctcgttgct
     6841 gaagatctct tgcagatagc agatccggtt cttccgtctg gtgtatcttc ttctggcggt
     6901 tctcttcagc cgggtggcct cggctgtttc tccgctgtcg aacagcaggg cgccgatcag
     6961 gttcttcttg atgctgtgcc ggtcggtgtt gcccagcacc ttgaatttct tgctgggcac
     7021 cttgtactcg tcggtgatca cggcccagcc cacagagttg gtgccgatgg ccaggccgat
     7081 gctgtacttc ttgtccatcg accctccgcc ggaaccgccg ggctcttctc ccttctccaa
     7141 ccggaggatc acatctggct tagtaagctg ataacccaag gaaaccaggt tcttatagtt
     7201 ctccagcatc acatttctgt acacgatctg ctgagcagtg tccagcagct tccactcctc
     7261 cctggtgaag tccacaaata catccttgaa ggtcaccagt gtccgggacc aggcagttag
     7321 tgacttagca tccatggaag cttatcgata ccgtcgagga attctttgcc aaaatgatga
     7381 gacagcacaa taaccagcac gttgcccagg agctgtagga aaaagaagaa ggcatgaaca
     7441 tggttagcag aggcgctaga gccgccggtc acacgccaga agccgaaccc cgccctgccc
     7501 cgtccccccc gaaggcagcc gtccccccgc ggacagcccc gaggctggag agggagaagg
     7561 ggacggcggc gcggcgacgc acgaaggccc tccccgccca tttccttcct gccggcgccg
     7621 caccgcttcg ccccgcgccc gctagagggg gtgcggcggc gcctcccaga tttcggctcc
     7681 gcacagattt gggacaaagg aagtccctgc gccctctcgc acgattacca taaaaggcaa
     7741 tggctgcggc tcgccgcgcc tcgacagccg ccggcgctcc gggggccgcc gcgcccctcc
     7801 cccgagccct ccccggcccg aggcggcccc gccccgcccg gcacccccac ctgccgccac
     7861 cccccgcccg gcacggcgag ccccgcgcca cgccccgtac ggagccccgc acccgaagcc
     7921 gggccgtgct cagcaactcg gggagggggg tgcagggggg gttgcagccc gaccgacgcg
     7981 cccacacccc ctgctcaccc ccccacgcac acaccccgca cgcagccttt gttcccctcg
     8041 cagccccccc gcaccgcggg gcaccgcccc cggccgcgct cccctcgcgc acactgcgga
     8101 gcgcacaaag ccccgcgccg cgcccgcagc gctcacagcc gccgggcagc gcggagccgc
     8161 acgcggcgct ccccacgcac acacacacgc acgcaccccc cgagccgctc cccccgcaca
     8221 aagggccctc ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa acaagccgtc
     8281 attaaaccaa gcgctaatta cagcccggag gagaagggcc gtcccgcccg ctcacctgtg
     8341 ggagtaacgc ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg gagcggggca
     8401 cggggcgaag gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt ttatagggcc
     8461 gccgccgccg ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc tgattggctg
     8521 ccgccgcacc tctccgcctc gccccgcccc gccccccgcc ccgccccgcc cccccccgcc
     8581 cgcgcccccc cccccccccc ccccccccgc ggcgcacccc cccccccccc gcccccatcg
     8641 ctgcacaaaa taattaaaaa ataaataaat acaaaattgg gggtggggag gggggggaga
     8701 tggggagagt gaagcagaac gtggggctca cctcgaccat ggtaatagcg atgactaata
     8761 cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg
     8821 cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat
     8881 gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc
     8941 ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg
     9001 gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc
     9061 tatgaactaa tgaccccgta attgattact attaataact agtcaataat caatgtcgac
     9121 ctcgagggcc gcataacttc gtatagcata cattatacga agttatgata tcatcgatga
     9181 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc
     9241 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac
     9301 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc
     9361 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
     9421 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt
     9481 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct
     9541 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg
     9601 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt
     9661 ctcctcttcc tcatctccgg gcctttcgac cgatccagcc gccaccatga aaaagcctga
     9721 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     9781 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     9841 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     9901 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     9961 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
    10021 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
    10081 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
    10141 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
    10201 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
    10261 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
    10321 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
    10381 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
    10441 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
    10501 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
    10561 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
    10621 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
    10681 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agtcgagaaa
    10741 ttgatgatct attaaacaat aaagatgtcc acatggaagt ttttcctgtc atactttgtt
    10801 aagaagggtg agaacagagt acctacattt tgaatggaag gattggagct acgggggtgg
    10861 gggtggggtg ggattagata aatgcctgct ctttactgaa ggctctttac tattgcttta
    10921 tgataatgtt tcatagttgg atatctataa caagaaaata tatatataat aagttatcac
    10981 gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt
    11041 aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac
    11101 ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat
    11161 gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt
    11221 caattttacg cagactatct ttctagggtt aaggtaccca attcgcccta tagtgagtcg
    11281 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
    11341 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
    11401 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
    11461 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
    11521 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
    11581 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
    11641 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
    11701 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
    11761 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
    11821 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
    11881 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
    11941 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
    12001 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
    12061 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
    12121 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
    12181 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
    12241 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
    12301 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
    12361 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
    12421 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
    12481 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
    12541 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
    12601 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
    12661 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
    12721 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
    12781 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
    12841 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
    12901 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
    12961 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
    13021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
