LOCUS       sequence-198394-       12026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac construct with doxycyline inducible TRE-tight promoter 
            expression of dCas9-GFP followed by IRES-Neomycin selection 
            cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12026)
  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 12026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-07-6"; volume: "9(1):2643. doi"; pages: " 
            10.1038/s41467-018-05067-x"
COMMENT     Sequence Label: PB-tight-DDdCas9GFP-IRES-Neo
FEATURES             Location/Qualifiers
     source          1..12026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        join(37..42,43..60,61..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..1753
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     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             join(1838..1861,1862..1864,1865..4420,4421..4423,
                     4424..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 N863A 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             join(1838..1861,1862..1864,1865..4351,4352..4354,
                     4355..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             join(5987..5989,5990..5992,5993..6706)
                     /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             join(5987..5989,5990..5992,5993..6703)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6293..6312)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6640..6661
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    6739..7310
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6904..6921)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     7131..7150
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             7324..8118
                     /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(7378..7397)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7988..8007
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(8420..8437)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8426..8650
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8697..8713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8721..8737
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8745..8751,8752..8769,8770..8775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8790..8811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(8870..8994)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   9284..9318
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9347..9366)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9348..9366)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9376..9393)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9376..9392)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9385..9407)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9534..9989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9621..9640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9831..9852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10015..10119
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10120..10188,10189..10980)
                     /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(10338..10357)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11151..11739
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11640..11659
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11893..11910
                     /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 cctcgaggga atcgatatgg tgagcaaggg
     6001 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     6061 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     6121 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     6181 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     6241 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     6301 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     6361 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     6421 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     6481 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     6541 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     6601 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     6661 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaattta aagaattccb
     6721 ccccccccsc cccaccgccc cctccctccc ccccccctaa cgttactggc cgaagccgct
     6781 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     6841 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     6901 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     6961 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     7021 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     7081 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     7141 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     7201 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     7261 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa gcttgccaca
     7321 accatggctg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     7381 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     7441 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     7501 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     7561 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     7621 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     7681 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     7741 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     7801 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     7861 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     7921 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     7981 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     8041 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     8101 cttgacgagt tcttctgagc gggatcggct agagtcgacc cggacgggac gctcctgcgc
     8161 ctgatacaga acgaattgct tgcaggcatc tcatgagtgt gtcttcccgt tttccgcctg
     8221 aggtcactgc gtggatggag cgctggcgcc tgctgcgcga cggcgagctg ctcaccaccc
     8281 actcgagggc tgcagcgctg cagaggccga gtgcagaact gctccaaagg gacctcaagg
     8341 ctttccgagg gacactaggc tgactccatc gagccagtgt agagataagc ttatcgatga
     8401 tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     8461 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     8521 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     8581 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     8641 ggctctatgg cttctgaggc ggaaagaacc tgcagcccaa gcttggcgta atcatggtca
     8701 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     8761 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     8821 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccc atagtgactg
     8881 gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     8941 attgatattt atatcatttt acgtttctcg ttcagctttt ttgtacaaac ttgtgctagc
     9001 attctagttg atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa
     9061 catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat
     9121 catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt
     9181 gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac
     9241 ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg
     9301 cagactatct ttctagggtt aatctagaat ggcgcgcccc aattcgccct atagtgagtc
     9361 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     9421 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     9481 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     9541 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     9601 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     9661 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     9721 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     9781 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     9841 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     9901 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     9961 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
    10021 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
    10081 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
    10141 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
    10201 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
    10261 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
    10321 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
    10381 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
    10441 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
    10501 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
    10561 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
    10621 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
    10681 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
    10741 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
    10801 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
    10861 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
    10921 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
    10981 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
    11041 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
    11101 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
    11161 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
    11221 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
    11281 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
    11341 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
    11401 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
    11461 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
    11521 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
    11581 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
    11641 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
    11701 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
    11761 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
    11821 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
    11881 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
    11941 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
    12001 aaagcgggca gtgagcgcaa cgcaat
//
