138-dCas9-Dnmt3a

LOCUS       138-dCas9-Dnmt3a          14783 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac transposon system construct with dCas9-Dnmt3a.
ACCESSION   .
VERSION     .
KEYWORDS    138-dCas9-Dnmt3a
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14783)
  AUTHORS   Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, 
            Young RA, Jaenisch R
  TITLE     Editing DNA Methylation in the Mammalian Genome.
  JOURNAL   Cell. 2016 Sep 22;167(1):233-247.e17. doi: 
            10.1016/j.cell.2016.08.056.
  PUBMED    27662091
REFERENCE   2  (bases 1 to 14783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14783
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     46..68
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     60..77
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     61..77
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   207..269
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        718..1096
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    726..744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    762..780
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    798..816
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    834..852
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    870..888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    906..924
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    942..960
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1005..1029
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1112..5215
                     /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             5231..5251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5255..5275
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5279..5299
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5306..8044
                     /codon_start=1
                     /product="DNA methyltransferase 3A"
                     /label=DNMT3A
                     /translation="MPAMPSSGPGDTSSSAAEREEDRKDGEEQEEPRGKEERQEPSTTA
                     RKVGRPGRKRKHPPVESGDTPKDPAVISKSPSMAQDSGASELLPNGDLEKRSEPQPEEG
                     SPAGGQKGGAPAEGEGAAETLPEASRAVENGCCTPKEGRGAPAEAGKEQKETNIESMKM
                     EGSRGRLRGGLGWESSLRQRPMPRLTFQAGDPYYISKRKRDEWLARWKREAEKKAKVIA
                     GMNAVEENQGPGESQKVEEASPPAVQQPTDPASPTVATTPEPVGSDAGDKNATKAGDDE
                     PEYEDGRGFGIGELVWGKLRGFSWWPGRIVSWWMTGRSRAAEGTRWVMWFGDGKFSVVC
                     VEKLMPLSSFCSAFHQATYNKQPMYRKAIYEVLQVASSRAGKLFPVCHDSDESDTAKAV
                     EVQNKPMIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAPPPPAKKPR
                     KSTAEKPKVKEIIDERTRERLVYEVRQKCRNIEDICISCGSLNVTLEHPLFVGGMCQNC
                     KNCFLECAYQYDDDGYQSYCTICCGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAI
                     KEDPWNCYMCGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIR
                     VLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKH
                     IQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFW
                     LFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDK
                     LELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFP
                     VHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV"
     primer_bind     complement(8095..8114)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8139..8187
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8418..8629
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8642..8910
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8944..9687
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             9688..9741
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(9742..9761)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             9790..10593
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(9853..9872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     10463..10482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    10686..10807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10723..10742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10777..10796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11570..11604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12095..12111)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12095..12111)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12108..12130)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12119..12135
                     /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(12143..12173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12188..12209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12326..12343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12497..13085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12577..12596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13256..14116)
                     /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     13879..13898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14117..14221)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(14247..14702)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(14384..14405)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     14596..14615
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
       61 gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg
      121 ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag atgctcgaca
      181 cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta
      241 aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
      301 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
      361 caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact caaaatttct
      421 tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc cagggatgta
      481 attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg
      541 gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt
      601 ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg
      661 ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga attcctcgag
      721 tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga
      781 acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat
      841 cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag
      901 tttactccct atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga
      961 acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg tttagtgaac
     1021 cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
     1081 ttcctaccct cgtaaaggtc tagagctagc catggacaag aagtattcta tcggactggc
     1141 catcgggact aatagcgtcg ggtgggccgt gatcactgac gagtacaagg tgccctctaa
     1201 gaagttcaag gtgctcggga acaccgaccg gcattccatc aagaaaaatc tgatcggagc
     1261 tctcctcttt gattcagggg agaccgctga agcaacccgc ctcaagcgga ctgctagacg
     1321 gcggtacacc aggaggaaga accggatttg ttaccttcaa gagatattct ccaacgaaat
     1381 ggcaaaggtc gacgacagct tcttccatag gctggaagaa tcattcctcg tggaagagga
     1441 taagaagcat gaacggcatc ccatcttcgg taatatcgtc gacgaggtgg cctatcacga
     1501 gaaataccca accatctacc atcttcgcaa aaagctggtg gactcaaccg acaaggcaga
     1561 cctccggctt atctacctgg ccctggccca catgatcaag ttcagaggcc acttcctgat
     1621 cgagggcgac ctcaatcctg acaatagcga tgtggataaa ctgttcatcc agctggtgca
     1681 gacttacaac cagctctttg aagagaaccc catcaatgca agcggagtcg atgccaaggc
     1741 cattctgtca gcccggctgt caaagagccg cagacttgag aatcttatcg ctcagctgcc
     1801 gggtgaaaag aaaaatggac tgttcgggaa cctgattgct ctttcacttg ggctgactcc
     1861 caatttcaag tctaatttcg acctggcaga ggatgccaag ctgcaactgt ccaaggacac
     1921 ctatgatgac gatctcgaca acctcctggc ccagatcggt gaccaatacg ccgacctttt
     1981 ccttgctgct aagaatcttt ctgacgccat cctgctgtct gacattctcc gcgtgaacac
     2041 tgaaatcacc aaggcccctc tttcagcttc aatgattaag cggtatgatg agcaccacca
     2101 ggacctgacc ctgcttaagg cactcgtccg gcagcagctt ccggagaagt acaaggaaat
     2161 cttctttgac cagtcaaaga atggatacgc cggctacatc gacggaggtg cctcccaaga
     2221 ggaattttat aagtttatca aacctatcct tgagaagatg gacggcaccg aagagctcct
     2281 cgtgaaactg aatcgggagg atctgctgcg gaagcagcgc actttcgaca atgggagcat
     2341 tccccaccag atccatcttg gggagcttca cgccatcctt cggcgccaag aggacttcta
     2401 cccctttctt aaggacaaca gggagaagat tgagaaaatt ctcactttcc gcatccccta
     2461 ctacgtggga cccctcgcca gaggaaatag ccggtttgct tggatgacca gaaagtcaga
     2521 agaaactatc actccctgga acttcgaaga ggtggtggac aagggagcca gcgctcagtc
     2581 attcatcgaa cggatgacta acttcgataa gaacctcccc aatgagaagg tcctgccgaa
     2641 acattccctg ctctacgagt actttaccgt gtacaacgag ctgaccaagg tgaaatatgt
     2701 caccgaaggg atgaggaagc ccgcattcct gtcaggcgaa caaaagaagg caattgtgga
     2761 ccttctgttc aagaccaata gaaaggtgac cgtgaagcag ctgaaggagg actatttcaa
     2821 gaaaattgaa tgcttcgact ctgtggagat tagcggggtc gaagatcggt tcaacgcaag
     2881 cctgggtacc taccatgatc tgcttaagat catcaaggac aaggattttc tggacaatga
     2941 ggagaacgag gacatccttg aggacattgt cctgactctc actctgttcg aggaccggga
     3001 aatgatcgag gagaggctta agacctacgc ccatctgttc gacgataaag tgatgaagca
     3061 acttaaacgg agaagatata ccggatgggg acgccttagc cgcaaactca tcaacggaat
     3121 ccgggacaaa cagagcggaa agaccattct tgatttcctt aagagcgacg gattcgctaa
     3181 tcgcaacttc atgcaactta tccatgatga ttccctgacc tttaaggagg acatccagaa
     3241 ggcccaagtg tctggacaag gtgactcact gcacgagcat atcgcaaatc tggctggttc
     3301 acccgctatt aagaagggta ttctccagac cgtgaaagtc gtggacgagc tggtcaaggt
     3361 gatgggtcgc cataaaccag agaacattgt catcgagatg gccagggaaa accagactac
     3421 ccagaaggga cagaagaaca gcagggagcg gatgaaaaga attgaggaag ggattaagga
     3481 gctcgggtca cagatcctta aagagcaccc ggtggaaaac acccagcttc agaatgagaa
     3541 gctctatctg tactaccttc aaaatggacg cgatatgtat gtggaccaag agcttgatat
     3601 caacaggctc tcagactacg acgtggacgc catcgtccct cagagcttcc tcaaagacga
     3661 ctcaattgac aataaggtgc tgactcgctc agacaagaac cggggaaagt cagataacgt
     3721 gccctcagag gaagtcgtga aaaagatgaa gaactattgg cgccagcttc tgaacgcaaa
     3781 gctgatcact cagcggaagt tcgacaatct cactaaggct gagaggggcg gactgagcga
     3841 actggacaaa gcaggattca ttaaacggca acttgtggag actcggcaga ttactaaaca
     3901 tgtcgcccaa atccttgact cacgcatgaa taccaagtac gacgaaaacg acaaacttat
     3961 ccgcgaggtg aaggtgatta ccctgaagtc caagctggtc agcgatttca gaaaggactt
     4021 tcaattctac aaagtgcggg agatcaataa ctatcatcat gctcatgacg catatctgaa
     4081 tgccgtggtg ggaaccgccc tgatcaagaa gtacccaaag ctggaaagcg agttcgtgta
     4141 cggagactac aaggtctacg acgtgcgcaa gatgattgcc aaatctgagc aggagatcgg
     4201 aaaggccacc gcaaagtact tcttctacag caacatcatg aatttcttca agaccgaaat
     4261 cacccttgca aacggtgaga tccggaagag gccgctcatc gagactaatg gggagactgg
     4321 cgaaatcgtg tgggacaagg gcagagattt cgctaccgtg cgcaaagtgc tttctatgcc
     4381 tcaagtgaac atcgtgaaga aaaccgaggt gcaaaccgga ggcttttcta aggaatcaat
     4441 cctccccaag cgcaactccg acaagctcat tgcaaggaag aaggattggg accctaagaa
     4501 gtacggcgga ttcgattcac caactgtggc ttattctgtc ctggtcgtgg ctaaggtgga
     4561 aaaaggaaag tctaagaagc tcaagagcgt gaaggaactg ctgggtatca ccattatgga
     4621 gcgcagctcc ttcgagaaga acccaattga ctttctcgaa gccaaaggtt acaaggaagt
     4681 caagaaggac cttatcatca agctcccaaa gtatagcctg ttcgaactgg agaatgggcg
     4741 gaagcggatg ctcgcctccg ctggcgaact tcagaagggt aatgagctgg ctctcccctc
     4801 caagtacgtg aatttcctct accttgcaag ccattacgag aagctgaagg ggagccccga
     4861 ggacaacgag caaaagcaac tgtttgtgga gcagcataag cattatctgg acgagatcat
     4921 tgagcagatt tccgagtttt ctaaacgcgt cattctcgct gatgccaacc tcgataaagt
     4981 ccttagcgca tacaataagc acagagacaa accaattcgg gagcaggctg agaatatcat
     5041 ccacctgttc accctcacca atcttggtgc ccctgccgca ttcaagtact tcgacaccac
     5101 catcgaccgg aaacgctata cctccaccaa agaagtgctg gacgccaccc tcatccacca
     5161 gagcatcacc ggactttacg aaactcggat tgacctctca cagctcggag gggatggtgg
     5221 cggaggctcg ccaaaaaaga agagaaaggt agacccaaag aaaaaacgaa aagtagatcc
     5281 gaaaaagaag aggaaggtgg gatccatgcc cgccatgccc tccagcggcc ccggggacac
     5341 cagcagctct gctgcggagc gggaggagga ccgaaaggac ggagaggagc aggaggagcc
     5401 gcgtggcaag gaggagcgcc aagagcccag caccacggca cggaaggtgg ggcggcctgg
     5461 gaggaagcgc aagcaccccc cggtggaaag cggtgacacg ccaaaggacc ctgcggtgat
     5521 ctccaagtcc ccatccatgg cccaggactc aggcgcctca gagctattac ccaatgggga
     5581 cttggagaag cggagtgagc cccagccaga ggaggggagc cctgctgggg ggcagaaggg
     5641 cggggcccca gcagagggag agggtgcagc tgagaccctg cctgaagcct caagagcagt
     5701 ggaaaatggc tgctgcaccc ccaaggaggg ccgaggagcc cctgcagaag cgggcaaaga
     5761 acagaaggag accaacatcg aatccatgaa aatggagggc tcccggggcc ggctgcgggg
     5821 tggcttgggc tgggagtcca gcctccgtca gcggcccatg ccgaggctca ccttccaggc
     5881 gggggacccc tactacatca gcaagcgcaa gcgggacgag tggctggcac gctggaaaag
     5941 ggaggctgag aagaaagcca aggtcattgc aggaatgaat gctgtggaag aaaaccaggg
     6001 gcccggggag tctcagaagg tggaggaggc cagccctcct gctgtgcagc agcccactga
     6061 ccccgcatcc cccactgtgg ctaccacgcc tgagcccgtg gggtccgatg ctggggacaa
     6121 gaatgccacc aaagcaggcg atgacgagcc agagtacgag gacggccggg gctttggcat
     6181 tggggagctg gtgtggggga aactgcgggg cttctcctgg tggccaggcc gcattgtgtc
     6241 ttggtggatg acgggccgga gccgagcagc tgaaggcacc cgctgggtca tgtggttcgg
     6301 agacggcaaa ttctcagtgg tgtgtgttga gaagctgatg ccgctgagct cgttttgcag
     6361 tgcgttccac caggccacgt acaacaagca gcccatgtac cgcaaagcca tctacgaggt
     6421 cctgcaggtg gccagcagcc gcgcggggaa gctgttcccg gtgtgccacg acagcgatga
     6481 gagtgacact gccaaggccg tggaggtgca gaacaagccc atgattgaat gggccctggg
     6541 gggcttccag ccttctggcc ctaagggcct ggagccacca gaagaagaga agaatcccta
     6601 caaagaagtg tacacggaca tgtgggtgga acctgaggca gctgcctacg caccacctcc
     6661 accagccaaa aagccccgga agagcacagc ggagaagccc aaggtcaagg agattattga
     6721 tgagcgcaca agagagcggc tggtgtacga ggtgcggcag aagtgccgga acattgagga
     6781 catctgcatc tcctgtggga gcctcaatgt taccctggaa caccccctct tcgttggagg
     6841 aatgtgccaa aactgcaaga actgctttct ggagtgtgcg taccagtacg acgacgacgg
     6901 ctaccagtcc tactgcacca tctgctgtgg gggccgtgag gtgctcatgt gcggaaacaa
     6961 caactgctgc aggtgctttt gcgtggagtg tgtggacctc ttggtggggc cgggggctgc
     7021 ccaggcagcc attaaggaag acccctggaa ctgctacatg tgcgggcaca agggtaccta
     7081 cgggctgctg cggcggcgag aggactggcc ctcccggctc cagatgttct tcgctaataa
     7141 ccacgaccag gaatttgacc ctccaaaggt ttacccacct gtcccagctg agaagaggaa
     7201 gcccatccgg gtgctgtctc tctttgatgg aatcgctaca gggctcctgg tgctgaagga
     7261 cttgggcatt caggtggacc gctacattgc ctcggaggtg tgtgaggact ccatcacggt
     7321 gggcatggtg cggcaccagg ggaagatcat gtacgtcggg gacgtccgca gcgtcacaca
     7381 gaagcatatc caggagtggg gcccattcga tctggtgatt gggggcagtc cctgcaatga
     7441 cctctccatc gtcaaccctg ctcgcaaggg cctctacgag ggcactggcc ggctcttctt
     7501 tgagttctac cgcctcctgc atgatgcgcg gcccaaggag ggagatgatc gccccttctt
     7561 ctggctcttt gagaatgtgg tggccatggg cgttagtgac aagagggaca tctcgcgatt
     7621 tctcgagtcc aaccctgtga tgattgatgc caaagaagtg tcagctgcac acagggcccg
     7681 ctacttctgg ggtaaccttc ccggtatgaa caggccgttg gcatccactg tgaatgataa
     7741 gctggagctg caggagtgtc tggagcatgg caggatagcc aagttcagca aagtgaggac
     7801 cattactacg aggtcaaact ccataaagca gggcaaagac cagcattttc ctgtcttcat
     7861 gaatgagaaa gaggacatct tatggtgcac tgaaatggaa agggtatttg gtttcccagt
     7921 ccactatact gacgtctcca acatgagccg cttggcgagg cagagactgc tgggccggtc
     7981 atggagcgtg ccagtcatcc gccacctctt cgctccgctg aaggagtatt ttgcgtgtgt
     8041 gtaagcggcc gcatatatac cggttagtaa tgagtttaaa cgggggaggc taactgaaac
     8101 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     8161 aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     8221 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     8281 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     8341 gccctgccat agcagatctg cgaaatcgga tccagctgaa tctaagtgga tctgcgatcg
     8401 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     8461 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     8521 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     8581 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
     8641 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
     8701 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
     8761 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
     8821 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
     8881 ttcgttttct gttctgcgcc gttacagatc taagctgtga ccggcgccta ctctagagcc
     8941 gccatgtcta gactggacaa gagcaaagtc ataaactctg ctctggaatt actcaatgga
     9001 gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt tgagcagcct
     9061 accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctgcc aatcgagatg
     9121 ctggacaggc atcataccca ctcctgcccc ctggaaggcg agtcatggca agactttctg
     9181 cggaacaacg ccaagtcata ccgctgtgct ctcctctcac atcgcgacgg ggctaaagtg
     9241 catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca gctcgcgttc
     9301 ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc cgtgggccac
     9361 tttacactgg gctgcgtatt ggaggaacag gagcatcaag tagcaaaaga ggaaagagag
     9421 acacctacca ccgattctat gcccccactt ctgaaacaag caattgagct gttcgaccgg
     9481 cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg cctggagaaa
     9541 cagctaaagt gcgaaagcgg cgggccgacc gacgcccttg acgattttga cttagacatg
     9601 ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga cgctcttgac
     9661 gattttgacc ttgacatgct ccccggggag ggcagaggaa gtcttctaac atgcggtgac
     9721 gtggaggaga atcccggccc tatgaccgag tacaagccca cggtgcgcct cgccacccgc
     9781 gacgacgtca tgggatcggc cattgaacaa gatggattgc acgcaggttc tccggccgct
     9841 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     9901 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     9961 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
    10021 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
    10081 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
    10141 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
    10201 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
    10261 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
    10321 aaggcgcgca tgcccgacgg cgatgatctc gtcgtgaccc atggcgatgc ctgcttgccg
    10381 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
    10441 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
    10501 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
    10561 gccttctatc gccttcttga cgagttcttc tgagacgtcg aggtgcccga aggaccgcgc
    10621 acctggtgca tgacccgcaa gcccggtgcc tgaaatcaac ctctggatta caaaatttgt
    10681 gggtaaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
    10741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
    10801 tatcttatca tgtctggaat tgactcaaat gatgtcaatt agtctatcag aagctcatct
    10861 ggtctccctt ccgggggaca agacatccct gtttaatatt taaacagcag tgttcccaaa
    10921 ctgggttctt atatcccttg ctctggtcaa ccaggttgca gggtttcctg tcctcacagg
    10981 aacgaagtcc ctaaagaaac agtggcagcc aggtttagcc ccggaattga ctggattcct
    11041 tttttagggc ccattggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa
    11101 agagcagcga gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct
    11161 gtccccgcac gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg
    11221 ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg
    11281 gggggggctg tccctgatat ctataacaag aaaatatata tataataagt tatcacgtaa
    11341 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
    11401 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
    11461 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
    11521 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
    11581 tttacgcaga ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct
    11641 tttttccggc tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc
    11701 cgtgcctttt cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc
    11761 tgcattgacg ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg
    11821 cacgccttta acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt
    11881 ttccggacac agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc
    11941 gacagccgga actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat
    12001 tacgtcagcg aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt
    12061 gagttacccg gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
    12121 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
    12181 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
    12241 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    12301 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
    12361 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
    12421 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
    12481 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
    12541 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
    12601 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
    12661 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
    12721 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
    12781 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
    12841 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
    12901 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
    12961 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
    13021 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
    13081 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
    13141 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
    13201 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
    13261 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
    13321 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
    13381 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
    13441 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
    13501 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
    13561 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
    13621 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
    13681 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
    13741 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
    13801 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
    13861 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
    13921 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
    13981 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
    14041 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
    14101 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
    14161 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
    14221 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
    14281 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
    14341 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
    14401 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
    14461 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
    14521 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
    14581 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
    14641 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
    14701 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
    14761 gctattacgc cagctggcga aag
//