LOCUS       Exported               12733 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac transposon system construct with doxycyline inducible 
            TRE-tight promoter expression of TMP inducible DDdCas9VP192 
            activator followed by T2A-EGFP as a reporter and IRES-Neo as 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    PB-tight-DDdCas9VP192-T2A-GFP-IRES-Neo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12733)
  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 12733)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12733
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        37..67
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   205..267
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             557..667
                     /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"
     promoter        704..1018
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    712..730
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    748..766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    783..801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    819..837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    855..873
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    890..908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    926..944
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     991..1015
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1218..1237
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1277..1750
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER"
     CDS             1778..1804
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1808..1828
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1838..5938
                     /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             5942..5962
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5999..6109
                     /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             6161..6271
                     /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             6323..6433
                     /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             6485..6595
                     /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             6635..6688
                     /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             6695..7414
                     /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             6695..7411
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6740..6761)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7001..7020)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7348..7369
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7450..8017
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(7611..7628)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7838..7857
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8031..8825
                     /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(8085..8104)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8695..8714
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(9127..9144)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9133..9357
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(9404..9420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    9428..9444
                     /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(9452..9482)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9497..9518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(9577..9701)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   9991..10025
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10054..10073)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10055..10073)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10083..10100)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10083..10099)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10092..10114)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10241..10696
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10328..10347)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10538..10559
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10722..10826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             10827..11687
                     /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(11045..11064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11858..12446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12347..12366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12600..12617
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggatt aattaagcgg
      181 ccgcatacgc gtatactaga ttaaccctag aaagataatc atattgtgac gtacgttaaa
      241 gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta
      301 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
      361 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt
      421 ctataaagta acaaaacttt taactagaat tgagctgttc gaccggcagg gagccgaacc
      481 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
      541 aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc cagccgatgc
      601 ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt ttgaccttga
      661 catgctcccc gggtaactag aattgtaaat gcattcaaac gtcgagttta ctccctatca
      721 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacga tgtcgagttt
      781 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      841 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttat ccctatcagt
      901 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgaggtag
      961 gcgtgtacgg tgggaggcct atataagcag agctcgttta gtgaaccgtc agatcgcctg
     1021 gagaaggatc cgcggccgcg ccggctctag atcgcgaacg cgtgaattgt aaatgcattc
     1081 aaacgtcgac gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     1141 accccacctg taggtttggc aagctagagc ctctgctaac catgttcatg ccttcttctt
     1201 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     1261 attgggatcc gccaccatga tcagtctgat tgcggcgtta gcggtagatt acgttatcgg
     1321 catggaaaac gccatgccgt ggaacctgcc tgccgatctc gcctggttta aacgcaacac
     1381 cttaaataaa cccgtgatta tgggccgcca tacctgggaa tcaatcggtc gtccgttgcc
     1441 aggacgcaaa aatattatcc tcagcagtca accgagtacg gacgatcgcg taacgtgggt
     1501 gaagtcggtg gatgaagcca tcgcggcgtg tggtgacgta ccagaaatca tggtgattgg
     1561 cggcggtcgc gttattgaac agttcttgcc aaaagcgcaa aaactgtatc tgacgcatat
     1621 cgacgcagaa gtggaaggcg acacccattt cccggattac gagccggatg actgggaatc
     1681 ggtattcagc gaattccacg atgctgatgc gcagaactct cacagctatt gctttgagat
     1741 tctggagcgg cgaggctccg gaaccggtgc caccatgtac ccatacgatg ttccagatta
     1801 cgcttcgccg aagaaaaagc gcaaggtcga agcgtccgac aagaagtaca gcatcggcct
     1861 ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     1921 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     1981 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     2041 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     2101 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     2161 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     2221 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     2281 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     2341 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     2401 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     2461 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     2521 gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac
     2581 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     2641 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     2701 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     2761 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     2821 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     2881 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     2941 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     3001 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     3061 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     3121 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     3181 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     3241 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     3301 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     3361 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     3421 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     3481 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     3541 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     3601 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     3661 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     3721 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     3781 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     3841 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     3901 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     3961 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     4021 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     4081 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     4141 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     4201 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     4261 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     4321 catcaaccgg ctgtccgact acgatgtcga cgccatcgtg cctcagagct ttctgaagga
     4381 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
     4441 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     4501 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     4561 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     4621 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     4681 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     4741 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     4801 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     4861 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     4921 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     4981 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     5041 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     5101 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     5161 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     5221 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     5281 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     5341 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     5401 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     5461 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     5521 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     5581 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     5641 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     5701 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     5761 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     5821 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     5881 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacag
     5941 ccccaagaag aagagaaagg tggaggccag cctcgagtgc gaaagcggcg ggccggccga
     6001 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct
     6061 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccgggtcact
     6121 acccagcgct agcgtcgagt gcgaaagcgg cgggccggcc gacgcccttg acgattttga
     6181 cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga
     6241 cgctcttgac gattttgacc ttgacatgct ccccgggtca ctacccagcg ctagcgtcga
     6301 gtgcgaaagc ggcgggccgg ccgacgccct tgacgatttt gacttagaca tgctcccagc
     6361 cgatgccctt gacgactttg accttgatat gctgcctgct gacgctcttg acgattttga
     6421 ccttgacatg ctccccgggt cactacccag cgctagcgtc gagtgcgaaa gcggcgggcc
     6481 ggccgacgcc cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt
     6541 tgaccttgat atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg
     6601 gtcactaccc agcgctagcg tcgacggctc cggagagggc agaggaagtc tgctaacatg
     6661 cggtgacgtc gaggagaatc ctggcccaat cgatatggtg agcaagggcg aggagctgtt
     6721 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     6781 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     6841 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     6901 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     6961 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     7021 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     7081 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     7141 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     7201 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     7261 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     7321 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     7381 gatcactctc ggcatggacg agctgtacaa gtaatttaaa gaattccgcc ccccscccch
     7441 gbccacmtct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg
     7501 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc
     7561 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa
     7621 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag
     7681 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg
     7741 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg
     7801 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa
     7861 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg
     7921 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca
     7981 cggggacgtg gttttccttt gaaaaacacg atgataagct tgccacaacc atggctgaac
     8041 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     8101 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     8161 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     8221 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     8281 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     8341 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     8401 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     8461 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     8521 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     8581 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     8641 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     8701 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     8761 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     8821 tctgagcggg atcggctaga gtcgacccgg acgggacgct cctgcgcctg atacagaacg
     8881 aattgcttgc aggcatctca tgagtgtgtc ttcccgtttt ccgcctgagg tcactgcgtg
     8941 gatggagcgc tggcgcctgc tgcgcgacgg cgagctgctc accacccact cgagggctgc
     9001 agcgctgcag aggccgagtg cagaactgct ccaaagggac ctcaaggctt tccgagggac
     9061 actaggctga ctccatcgag ccagtgtaga gataagctta tcgatgatct agagctcgct
     9121 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     9181 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     9241 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     9301 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     9361 ctgaggcgga aagaacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg
     9421 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     9481 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     9541 ctttccagtc gggaaacctg tcgtgccagc ggatcccata gtgactggat atgttgtgtt
     9601 ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata
     9661 tcattttacg tttctcgttc agcttttttg tacaaacttg tgctagcatt ctagttgata
     9721 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa
     9781 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt
     9841 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct
     9901 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat
     9961 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc
    10021 tagggttaat ctagaatggc gcgccccaat tcgccctata gtgagtcgta ttacgcgcgc
    10081 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
    10141 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
    10201 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
    10261 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
    10321 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
    10381 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
    10441 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
    10501 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
    10561 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
    10621 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
    10681 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
    10741 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
    10801 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
    10861 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
    10921 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
    10981 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
    11041 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
    11101 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
    11161 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
    11221 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
    11281 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    11341 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    11401 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    11461 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    11521 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
    11581 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    11641 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    11701 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    11761 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    11821 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    11881 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    11941 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    12001 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    12061 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    12121 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    12181 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    12241 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    12301 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    12361 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    12421 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    12481 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    12541 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    12601 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
    12661 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
    12721 agcgcaacgc aat
//
