LOCUS       sequence-372004-       13467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac compatible plasmid expressing dCas9-KRAB-MeCP2 expression 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13467)
  AUTHORS   Nghi Dang , Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J 
            Cecchi, Joanna Buchthal, Alejandro Chavez, Nan Cher Yeo
  TITLE     Generation and application of a versatile CRISPR toolkit for 
            mammalian cell engineering
  JOURNAL   Synthetic Biology, Volume 6, Issue 1, 2021
REFERENCE   2  (bases 1 to 13467)
  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: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: NAX_CAGGS_CAS9_m4_KRAB-MeCP2_Hygro
FEATURES             Location/Qualifiers
     source          1..13467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          110..1126
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1050..1072
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1134..1153
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1223..1232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1229..1255,1256..1258,1259..3742,3743..3748,3749..3814,
                     3815..3817,3818..5332)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5345..5365
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5426..5620
                     /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
                     QLTKPDVILRLEKGEEPWLV"
     CDS             5654..5674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5684..6541
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(6596..6615)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6640..6688
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        6919..7130
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7140..7149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7161..8180
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             7161..8180
                     /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"
     polyA_signal    8184..8305
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8221..8240)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8275..8294
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9068..9102
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9593..9609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9593..9609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9606..9628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9617..9633
                     /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(9641..9647,9648..9665,9666..9671))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9686..9707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9824..9841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9995..10583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10075..10094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10754..11545,11546..11614))
                     /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     11377..11396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11615..11719)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11745..12200)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11882..11903)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12094..12113
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12327..12349
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12341..12358
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12342..12358
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12488..12550
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        12971..13350
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
       61 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      121 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      181 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      241 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
      301 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
      361 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
      421 cgcggggctt tttgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
      481 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
      541 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
      601 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
      661 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
      721 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
      781 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
      841 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
      901 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
      961 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     1021 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     1081 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     1141 gctggttatt gtgctgtctc atcattttgg caaagaatct agataccctc gtaaaggtct
     1201 agagctagcg gatcgaaccc ttgccaccat ggacaagaag tactccattg ggctcgctat
     1261 cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa
     1321 attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct
     1381 cctgttcgac tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag
     1441 atatacccgc agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc
     1501 taaggtggat gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa
     1561 aaagcacgag cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa
     1621 gtacccaacc atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt
     1681 gcggttgatc tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga
     1741 gggggacctg aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac
     1801 ttacaatcag cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat
     1861 cctgagcgct aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg
     1921 ggagaagaag aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa
     1981 ctttaaatct aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta
     2041 cgatgatgat ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt
     2101 ggcggcaaag aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga
     2161 gatcaccaaa gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga
     2221 cttgactttg ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt
     2281 cttcgatcag tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga
     2341 attttacaaa tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt
     2401 aaagcttaac agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc
     2461 ccaccagatt cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc
     2521 ctttttgaaa gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta
     2581 tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga
     2641 gaccatcact ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt
     2701 catcgaaagg atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca
     2761 ctctctgctg tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac
     2821 agaagggatg agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct
     2881 cctcttcaag acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa
     2941 gattgaatgt ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct
     3001 gggaacgtat cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga
     3061 gaacgaggac attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat
     3121 gattgaagaa cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct
     3181 caagaggcgc cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg
     3241 agacaagcag agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg
     3301 gaacttcatg cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc
     3361 acaagtttct ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc
     3421 agctatcaaa aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat
     3481 gggaaggcat aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca
     3541 gaagggacag aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact
     3601 ggggtcccaa atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct
     3661 ctacctgtac tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa
     3721 tcggctctcc gactacgacg tggctgctat cgtgccccag tcttttctca aagatgattc
     3781 tattgataat aaagtgttga caagatccga taaagctaga gggaagagtg ataacgtccc
     3841 ctcagaagaa gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact
     3901 gatcacacaa cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt
     3961 ggataaagcc ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt
     4021 ggcccaaatt ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg
     4081 agaggtgaaa gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca
     4141 gttttataag gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc
     4201 agtggtaggc actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg
     4261 agactataaa gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa
     4321 ggccaccgct aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac
     4381 actggccaat ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga
     4441 aatcgtgtgg gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca
     4501 ggtgaacatc gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct
     4561 cccgaaaagg aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata
     4621 cggcggattc gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa
     4681 agggaagtct aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg
     4741 atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa
     4801 aaaagacctc atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa
     4861 acgaatgctc gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa
     4921 atacgttaat ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga
     4981 taatgagcag aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga
     5041 gcaaataagc gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct
     5101 ttctgcttac aataagcaca gggataagcc catcagggag caggcagaaa acattatcca
     5161 cttgtttact ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat
     5221 agacagaaag cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc
     5281 aattacgggg ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc
     5341 tgaccccaag aagaagagga aggtgagtgg tggaggaagt ggcgggtcag ggtcgatgga
     5401 cgcgaaatca cttacggcat ggtcgagaac actggttacg ttcaaggacg tgtttgtgga
     5461 ctttacacgt gaggagtgga aattgctgga tactgcgcaa caaattgtgt atcgaaatgt
     5521 catgcttgag aattacaaga acctcgtcag tctcggatac cagttgacga aaccggatgt
     5581 gatccttagg ctcgaaaagg gggaagaacc ttggctggta tcgggaggtg gttcgggtgg
     5641 ctctggatca agcccaaaga agaaacggaa ggtggaagcc tcagtgcagg tgaaaagggt
     5701 gctggaaaaa tcccccggca aactcctcgt gaagatgccc ttccaggctt cccctggcgg
     5761 aaaaggtgaa gggggtggcg caaccacatc tgcccaggtc atggtcatca agcgacctgg
     5821 aaggaaaaga aaggccgagg ctgaccctca ggccattcca aagaaacggg gacgcaagcc
     5881 agggtccgtg gtcgcagctg cagcagctga ggctaagaaa aaggcagtga aggaaagctc
     5941 catccgcagt gtgcaggaga ctgtcctgcc catcaagaag aggaagacta gggagaccgt
     6001 gtccatcgag gtcaaagaag tggtcaagcc cctgctcgtg tccaccctgg gcgaaaaatc
     6061 tggaaagggg ctcaaaacat gcaagtcacc tggacggaaa agcaaggagt ctagtccaaa
     6121 ggggcgctca agctccgctt ctagtccccc taaaaaggaa caccatcacc atcaccatca
     6181 cgccgagtct cctaaggctc ctatgccact gctcccacca cctccaccac ctgagccaca
     6241 gtcaagcgaa gaccccatca gcccacccga gcctcaggat ctgtcctcta gtatttgcaa
     6301 agaggaaaag atgcccagag caggcagcct ggagagtgat ggctgtccaa aagaacccgc
     6361 caagacccag cctatggtgg cagccgctgc aactaccacc acaaccacaa ctaccacagt
     6421 ggccgaaaaa tacaagcatc gcggcgaggg cgaacgaaag gacattgtgt caagctccat
     6481 gcccagacct aaccgggagg aaccagtcga tagtaggaca cccgtgactg agagagtctc
     6541 atagaagaat tcgatcccta ccggttagta atgagtttaa acgggggagg ctaactgaaa
     6601 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     6661 aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     6721 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     6781 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     6841 ggccctgcca tagcagatct gcgaaatcgg atccgcggcc gcactattct agctgcagag
     6901 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     6961 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     7021 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     7081 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gatcctcgag
     7141 ccaccatgga tagatccgga aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     7201 tcgaaaagtt cgacagcgtt tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     7261 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     7321 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     7381 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     7441 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     7501 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     7561 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     7621 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     7681 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     7741 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     7801 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     7861 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     7921 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     7981 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     8041 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     8101 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     8161 gtccgagggc aaaggaatag gttaacttgt ttattgcagc ttataatggt tacaaataaa
     8221 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     8281 tgtccaaact catcaatgta tcttatcatg tctggaattg actcaaatga tgtcaattag
     8341 tctatcagaa gctcatctgg tctcccttcc gggggacaag acatccctgt ttaatattta
     8401 aacagcagtg ttcccaaact gggttcttat atcccttgct ctggtcaacc aggttgcagg
     8461 gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag tggcagccag gtttagcccc
     8521 ggaattgact ggattccttt tttagggccc attggtatgg ctttttcccc gtatcccccc
     8581 aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac
     8641 cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg
     8701 gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta
     8761 catccctggg ggctttgggg gggggctgtc cctgatatct ataacaagaa aatatatata
     8821 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     8881 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     8941 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     9001 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     9061 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaatcta gctgcatcag
     9121 gatcatatcg tcgggtcttt tttccggctc agtcatcgcc caagctggcg ctatctgggc
     9181 atcggggagg aagaagcccg tgccttttcc cgcgaggttg aagcggcatg gaaagagttt
     9241 gccgaggatg actgctgctg cattgacgtt gagcgaaaac gcacgtttac catgatgatt
     9301 cgggaaggtg tggccatgca cgcctttaac ggtgaactgt tcgttcaggc cacctgggat
     9361 accagttcgt cgcggctttt ccggacacag ttccggatgg tcagcccgaa gcgcatcagc
     9421 aacccgaaca ataccggcga cagccggaac tgccgtgccg gtgtgcagat taatgacagc
     9481 ggtgcggcgc tgggatatta cgtcagcgag gacgggtatc ctggctggat gccgcagaaa
     9541 tggacatgga taccccgtga gttacccggc gggcgcgctt ggcgtaatca tggtcatagc
     9601 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     9661 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     9721 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     9781 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     9841 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     9901 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     9961 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    10021 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    10081 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    10141 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    10201 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    10261 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    10321 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    10381 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
    10441 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    10501 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    10561 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    10621 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    10681 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    10741 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    10801 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    10861 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    10921 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    10981 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    11041 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    11101 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    11161 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    11221 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    11281 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    11341 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    11401 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    11461 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    11521 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    11581 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
    11641 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
    11701 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
    11761 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
    11821 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
    11881 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
    11941 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
    12001 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
    12061 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
    12121 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
    12181 tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
    12241 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
    12301 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
    12361 gcgcgcctcg ttcattcacg tttttgaacc cgtggaggac gggcagactc gcggtgcaaa
    12421 tgtgttttac agcgtgatgg agcagatgaa gatgctcgac acgctgcaga acacgcagct
    12481 agattaaccc tagaaagata atcatattgt gacgtacgtt aaagataatc atgtgtaaaa
    12541 ttgacgcatg tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat
    12601 taagttttat tatatttaca cttacatact aataataaat tcaacaaaca atttatttat
    12661 gtttatttat ttattaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact
    12721 tttatgaggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
    12781 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
    12841 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
    12901 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaggc
    12961 ctccacggcc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
    13021 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
    13081 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
    13141 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
    13201 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
    13261 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
    13321 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
    13381 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
    13441 tgtgcagcga tgggggcggg ggggggg
//
