LOCUS       Exported               15506 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac transposon system construct with constitutive CAG promoter 
            expression of TMP inducible DDdCas9VP192-P300core activator followed
            by T2A-EGFP as a reporter and IRES-Neo as selection.
ACCESSION   .
VERSION     .
KEYWORDS    PB-CAG-DDdCas9VPP300-T2A-GFP-IRES-Neo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15506)
  AUTHORS   Weltner J, Balboa D, Katayama S, Bespalov M, Krjutskov K, Jouhilahti
            EM, Trokovic R, Kere J, Otonkoski T
  TITLE     Human pluripotent reprogramming with CRISPR activators.
  JOURNAL   Nat Commun. 2018 Jul 6;9(1):2643. doi: 10.1038/s41467-018-05067-x.
  PUBMED    29980666
REFERENCE   2  (bases 1 to 15506)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15506
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             129..989
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2072..2102
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   2240..2302
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2540..2843
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2845..3122
                     /label=chicken beta-actin promoter
     intron          3123..4139
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4063..4085
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4147..4166
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4206..4679
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER"
     CDS             4707..4733
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4737..4757
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4767..8867
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8871..8891
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8928..9038
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             9090..9200
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             9252..9362
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             9414..9524
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     CDS             9573..11423
                     /codon_start=1
                     /gene="EP300"
                     /product="catalytic histone acetyltransferase core of the 
                     human E1A-associated protein p300 (Delvecchio et al., 
                     2013)"
                     /label=p300 core
                     /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI
                     VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ
                     EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG
                     ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG
                     FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA
                     SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP
                     PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY
                     IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE
                     GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS
                     RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL
                     MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD"
     regulatory      11187..11196
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             11451..11504
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             11511..12230
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             11511..12227
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(11556..11577)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(11817..11836)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     12164..12185
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    12252..12825
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(12419..12436)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     12646..12665
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             12839..13633
                     /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="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(12893..12912)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     13503..13522
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(13935..13952)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    13941..14165
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(14212..14228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    14236..14252
                     /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(14260..14290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14305..14326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(14385..14509)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   14799..14833
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(14862..14881)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(14863..14881)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(14891..14908)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(14891..14907)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14900..14922)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      15049..15504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(15136..15155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     15346..15367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggattaatta agcggccgca
     2221 tacgcgtata ctagattaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa
     2281 tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat
     2341 ttgaatagat attaagtttt attatattta cacttacata ctaataataa attcaacaaa
     2401 caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata
     2461 aagtaacaaa acttttaact agttattaat agtaatcaat tacggggtca ttagttcata
     2521 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2581 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2641 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2701 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2761 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2821 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
     2881 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     2941 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
     3001 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     3061 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     3121 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     3181 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     3241 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     3301 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     3361 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     3421 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     3481 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     3541 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     3601 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     3661 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     3721 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     3781 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     3841 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     3901 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     3961 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     4021 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     4081 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcntacagc
     4141 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgggatccg
     4201 ccaccatgat cagtctgatt gcggcgttag cggtagatta cgttatcggc atggaaaacg
     4261 ccatgccgtg gaacctgcct gccgatctcg cctggtttaa acgcaacacc ttaaataaac
     4321 ccgtgattat gggccgccat acctgggaat caatcggtcg tccgttgcca ggacgcaaaa
     4381 atattatcct cagcagtcaa ccgagtacgg acgatcgcgt aacgtgggtg aagtcggtgg
     4441 atgaagccat cgcggcgtgt ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg
     4501 ttattgaaca gttcttgcca aaagcgcaaa aactgtatct gacgcatatc gacgcagaag
     4561 tggaaggcga cacccatttc ccggattacg agccggatga ctgggaatcg gtattcagcg
     4621 aattccacga tgctgatgcg cagaactctc acagctattg ctttgagatt ctggagcggc
     4681 gaggctccgg aaccggtgcc accatgtacc catacgatgt tccagattac gcttcgccga
     4741 agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg gccatcggca
     4801 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     4861 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     4921 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     4981 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     5041 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     5101 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     5161 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     5221 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     5281 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     5341 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     5401 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     5461 agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca
     5521 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     5581 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     5641 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     5701 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     5761 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     5821 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     5881 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     5941 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     6001 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     6061 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     6121 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     6181 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     6241 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     6301 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     6361 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     6421 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     6481 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     6541 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     6601 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     6661 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     6721 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     6781 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     6841 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     6901 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     6961 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     7021 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     7081 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     7141 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     7201 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     7261 tgtccgacta cgatgtcgac gccatcgtgc ctcagagctt tctgaaggac gactccatcg
     7321 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     7381 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     7441 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     7501 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     7561 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     7621 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     7681 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     7741 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     7801 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     7861 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     7921 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     7981 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     8041 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     8101 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     8161 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     8221 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     8281 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     8341 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     8401 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     8461 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     8521 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     8581 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     8641 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     8701 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     8761 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     8821 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc cccaagaaga
     8881 agagaaaggt ggaggccagc ctcgagtgcg aaagcggcgg gccggccgac gcccttgacg
     8941 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     9001 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtcacta cccagcgcta
     9061 gcgtcgagtg cgaaagcggc gggccggccg acgcccttga cgattttgac ttagacatgc
     9121 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
     9181 attttgacct tgacatgctc cccgggtcac tacccagcgc tagcgtcgag tgcgaaagcg
     9241 gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     9301 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     9361 tccccgggtc actacccagc gctagcgtcg agtgcgaaag cggcgggccg gccgacgccc
     9421 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata
     9481 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg tcactaccca
     9541 gcgctagcgt cgagttcgaa ggcagcggcg ggattttcaa accagaagaa ctacgacagg
     9601 cactgatgcc aactttggag gcactttacc gtcaggatcc agaatccctt ccctttcgtc
     9661 aacctgtgga ccctcagctt ttaggaatcc ctgattactt tgatattgtg aagagcccca
     9721 tggatctttc taccattaag aggaagttag acactggaca gtatcaggag ccctggcagt
     9781 atgtcgatga tatttggctt atgttcaata atgcctggtt atataaccgg aaaacatcac
     9841 gggtatacaa atactgctcc aagctctctg aggtctttga acaagaaatt gacccagtga
     9901 tgcaaagcct tggatactgt tgtggcagaa agttggagtt ctctccacag acactgtgtt
     9961 gctacggcaa acagttgtgc acaatacctc gtgatgccac ttattacagt taccagaaca
    10021 ggtatcattt ctgtgagaag tgtttcaatg agatccaagg ggagagcgtt tctttggggg
    10081 atgacccttc ccagcctcaa actacaataa ataaagaaca attttccaag agaaaaaatg
    10141 acacactgga tcctgaactg tttgttgaat gtacagagtg cggaagaaag atgcatcaga
    10201 tctgtgtcct tcaccatgag atcatctggc ctgctggatt cgtctgtgat ggctgtttaa
    10261 agaaaagtgc acgaactagg aaagaaaata agttttctgc taaaaggttg ccatctacca
    10321 gacttggcac ctttctagag aatcgtgtga atgactttct gaggcgacag aatcaccctg
    10381 agtcaggaga ggtcactgtt agagtagttc atgcttctga caaaaccgtg gaagtaaaac
    10441 caggcatgaa agcaaggttt gtggacagtg gagagatggc agaatccttt ccataccgaa
    10501 ccaaagccct ctttgccttt gaagaaattg atggtgttga cctgtgcttc tttggcatgc
    10561 atgttcaaga gtatggctct gactgccctc cacccaacca gaggagagta tacatatctt
    10621 acctcgatag tgttcatttc ttccgtccta aatgcttgag gactgcagtc tatcatgaaa
    10681 tcctaattgg atatttagaa tatgtcaaga aattaggtta cacaacaggg catatttggg
    10741 catgtccacc aagtgaggga gatgattata tcttccattg ccatcctcct gaccagaaga
    10801 tacccaagcc caagcgactg caggaatggt acaaaaaaat gcttgacaag gctgtatcag
    10861 agcgtattgt ccatgactac aaggatattt ttaaacaagc tactgaagat agattaacaa
    10921 gtgcaaagga attgccttat ttcgagggtg atttctggcc caatgttctg gaagaaagca
    10981 ttaaggaact ggaacaggag gaagaagaga gaaaacgaga ggaaaacacc agcaatgaaa
    11041 gcacagatgt gaccaaggga gacagcaaaa atgctaaaaa gaagaataat aagaaaacca
    11101 gcaaaaataa gagcagcctg agtaggggca acaagaagaa acccgggatg cccaatgtat
    11161 ctaacgacct ctcacagaaa ctatatgcca ccatggagaa gcataaagag gtcttctttg
    11221 tgatccgcct cattgctggc cctgctgcca actccctgcc tcccattgtt gatcctgatc
    11281 ctctcatccc ctgcgatctg atggatggtc gggatgcgtt tctcacgctg gcaagggaca
    11341 agcacctgga gttctcttca ctccgaagag cccagtggtc caccatgtgc atgctggtgg
    11401 agctgcacac gcagagccag gacgtcgaca ctagcgtcga cggctccgga gagggcagag
    11461 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaatcgat atggtgagca
    11521 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
    11581 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
    11641 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
    11701 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
    11761 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
    11821 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
    11881 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
    11941 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
    12001 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
    12061 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
    12121 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
    12181 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa tttaaagaat
    12241 tccgcccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa
    12301 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
    12361 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
    12421 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
    12481 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
    12541 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
    12601 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
    12661 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
    12721 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
    12781 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaaccat
    12841 ggctgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
    12901 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
    12961 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
    13021 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
    13081 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
    13141 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
    13201 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
    13261 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
    13321 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
    13381 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
    13441 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
    13501 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
    13561 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
    13621 cgagttcttc tgagcgggat cggctagagt cgacccggac gggacgctcc tgcgcctgat
    13681 acagaacgaa ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc
    13741 actgcgtgga tggagcgctg gcgcctgctg cgcgacggcg agctgctcac cacccactcg
    13801 agggctgcag cgctgcagag gccgagtgca gaactgctcc aaagggacct caaggctttc
    13861 cgagggacac taggctgact ccatcgagcc agtgtagaga taagcttatc gatgatctag
    13921 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
    13981 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
    14041 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
    14101 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
    14161 tatggcttct gaggcggaaa gaacctgcag cccaagcttg gcgtaatcat ggtcatagct
    14221 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    14281 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    14341 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atcccatagt gactggatat
    14401 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
    14461 tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtg ctagcattct
    14521 agttgatatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
    14581 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
    14641 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
    14701 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
    14761 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
    14821 tatctttcta gggttaatct agaatggcgc gccccaattc gccctatagt gagtcgtatt
    14881 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
    14941 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
    15001 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
    15061 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    15121 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    15181 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    15241 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
    15301 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
    15361 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
    15421 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
    15481 acaaaatatt aacgcttaca atttag
//
