LOCUS       sequence-194725-       13694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac construct with doxycyline inducible TRE-tight promoter 
            expression of TMP inducible DDdCas9VP192-p65-HSF1 activator followed
            by T2A-EGFP as a reporter and IRES-Neo as selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13694)
  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 13694)
  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-DDdCas9VPH-T2A-GFP-IRES-Neo
FEATURES             Location/Qualifiers
     source          1..13694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..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        join(2072..2077,2078..2095,2096..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)"
     CDS             2592..2702
                     /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        2739..3053
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    2747..2765
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2783..2801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2818..2836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2854..2872
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2890..2908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2925..2943
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2961..2979
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3026..3050
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     3253..3272
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3312..3788
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             3813..3839
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3843..3863
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3873..3896,3897..3899,3900..6455,6456..6458,
                     6459..7973)
                     /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(3873..3896,3897..3899,3900..6386,6387..6389,
                     6390..7973)
                     /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             7977..7997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8034..8144
                     /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             8196..8306
                     /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             8358..8468
                     /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             8520..8630
                     /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             8679..9221
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             9246..9617
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             9639..9692
                     /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             join(9699..9701,9702..9704,9705..10418)
                     /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(9699..9701,9702..9704,9705..10415)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(10005..10024)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10352..10373
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(10440..11014,11015..11017,11018..11026)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10607..10624)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10834..10853
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             11027..11821
                     /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(11081..11100)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     11691..11710
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(12123..12140)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12129..12353
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(12400..12416)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    12424..12440
                     /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(12448..12454,12455..12472,12473..12478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12493..12514
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(12573..12697)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   12987..13021
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(13050..13069)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(13051..13069)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(13079..13096)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(13079..13095)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13088..13110)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      13237..13692
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(13324..13343)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     13534..13555
                     /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 agaattgagc tgttcgaccg gcagggagcc gaacctgcct
     2521 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg
     2581 gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     2641 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     2701 tccccgggta actagaattg taaatgcatt caaacgtcga gtttactccc tatcagtgat
     2761 agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc
     2821 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
     2881 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta tcagtgatag
     2941 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga ggtaggcgtg
     3001 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagaa
     3061 ggatccgcgg ccgcgccggc tctagatcgc gaacgcgtga attgtaaatg cattcaaacg
     3121 tcgacgataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc
     3181 acctgtaggt ttggcaagct agagcctctg ctaaccatgt tcatgccttc ttctttttcn
     3241 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg
     3301 gatccgccac catgatcagt ctgattgcgg cgttagcggt agattacgtt atcggcatgg
     3361 aaaacgccat gccgtggaac ctgcctgccg atctcgcctg gtttaaacgc aacaccttaa
     3421 ataaacccgt gattatgggc cgccatacct gggaatcaat cggtcgtccg ttgccaggac
     3481 gcaaaaatat tatcctcagc agtcaaccga gtacggacga tcgcgtaacg tgggtgaagt
     3541 cggtggatga agccatcgcg gcgtgtggtg acgtaccaga aatcatggtg attggcggcg
     3601 gtcgcgttat tgaacagttc ttgccaaaag cgcaaaaact gtatctgacg catatcgacg
     3661 cagaagtgga aggcgacacc catttcccgg attacgagcc ggatgactgg gaatcggtat
     3721 tcagcgaatt ccacgatgct gatgcgcaga actctcacag ctattgcttt gagattctgg
     3781 agcggcgagg ctccggaacc ggtgccacca tgtacccata cgatgttcca gattacgctt
     3841 cgccgaagaa aaagcgcaag gtcgaagcgt ccgacaagaa gtacagcatc ggcctggcca
     3901 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     3961 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     4021 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     4081 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     4141 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     4201 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     4261 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     4321 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     4381 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     4441 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     4501 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     4561 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     4621 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     4681 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     4741 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     4801 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     4861 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     4921 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     4981 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     5041 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     5101 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     5161 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     5221 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     5281 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     5341 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     5401 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     5461 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     5521 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     5581 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     5641 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     5701 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     5761 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     5821 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     5881 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     5941 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     6001 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     6061 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     6121 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     6181 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     6241 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     6301 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     6361 accggctgtc cgactacgat gtcgacgcca tcgtgcctca gagctttctg aaggacgact
     6421 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     6481 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     6541 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     6601 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     6661 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     6721 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     6781 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     6841 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     6901 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     6961 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     7021 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     7081 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     7141 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     7201 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     7261 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     7321 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     7381 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     7441 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     7501 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     7561 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     7621 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     7681 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     7741 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     7801 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     7861 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     7921 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcccca
     7981 agaagaagag aaaggtggag gccagcctcg agtgcgaaag cggcgggccg gccgacgccc
     8041 ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata
     8101 tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg tcactaccca
     8161 gcgctagcgt cgagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag
     8221 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc
     8281 ttgacgattt tgaccttgac atgctccccg ggtcactacc cagcgctagc gtcgagtgcg
     8341 aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc ccagccgatg
     8401 cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg
     8461 acatgctccc cgggtcacta cccagcgcta gcgtcgagtg cgaaagcggc gggccggccg
     8521 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc
     8581 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtcac
     8641 tacccagcgc tagcgtcgag ttcgaaggca gcggcgggcc ttcagggcag atcagcaacc
     8701 aggccctggc tctggcccct agctccgctc cagtgctggc ccagactatg gtgccctcta
     8761 gtgctatggt gcctctggcc cagccacctg ctccagcccc tgtgctgacc ccaggaccac
     8821 cccagtcact gagcgctcca gtgcccaagt ctacacaggc cggcgagggg actctgagtg
     8881 aagctctgct gcacctgcag ttcgacgctg atgaggacct gggagctctg ctggggaaca
     8941 gcaccgatcc cggagtgttc acagatctgg cctccgtgga caactctgag tttcagcagc
     9001 tgctgaatca gggcgtgtcc atgtctcata gtacagccga accaatgctg atggagtacc
     9061 ccgaagccat tacccggctg gtgaccggca gccagcggcc ccccgacccc gctccaactc
     9121 ccctgggaac cagcggcctg cctaatgggc tgtccggaga tgaagacttc tcaagcatcg
     9181 ctgatatgga ctttagtgcc ctgctgtcac agatttcctc tagtgggcag ggaggaggtg
     9241 gaagcggctt cagcgtggac accagtgccc tgctggacct gttcagcccc tcggtgaccg
     9301 tgcccgacat gagcctgcct gaccttgaca gcagcctggc cagtatccaa gagctcctgt
     9361 ctccccagga gccccccagg cctcccgagg cagagaacag cagcccggat tcagggaagc
     9421 agctggtgca ctacacagcg cagccgctgt tcctgctgga ccccggctcc gtggacaccg
     9481 ggagcaacga cctgccggtg ctgtttgagc tgggagaggg ctcctacttc tccgaagggg
     9541 acggcttcgc cgaggacccc accatctccc tgctgacagg ctcggagcct cccaaagcca
     9601 aggaccccac tgtctcctct agcgtcgacg gctccggaga gggcagagga agtctgctaa
     9661 catgcggtga cgtcgaggag aatcctggcc caatcgatat ggtgagcaag ggcgaggagc
     9721 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     9781 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     9841 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     9901 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     9961 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
    10021 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
    10081 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
    10141 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
    10201 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
    10261 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
    10321 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
    10381 ccgggatcac tctcggcatg gacgagctgt acaagtaatt taaagaattc cgcccccccc
    10441 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
    10501 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
    10561 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
    10621 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
    10681 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
    10741 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
    10801 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
    10861 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
    10921 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
    10981 gacgtggttt tcctttgaaa aacacgatga taagcttgcc acaaccatgg ctgaacaaga
    11041 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
    11101 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
    11161 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
    11221 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
    11281 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
    11341 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
    11401 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
    11461 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
    11521 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
    11581 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
    11641 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
    11701 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
    11761 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
    11821 agcgggatcg gctagagtcg acccggacgg gacgctcctg cgcctgatac agaacgaatt
    11881 gcttgcaggc atctcatgag tgtgtcttcc cgttttccgc ctgaggtcac tgcgtggatg
    11941 gagcgctggc gcctgctgcg cgacggcgag ctgctcacca cccactcgag ggctgcagcg
    12001 ctgcagaggc cgagtgcaga actgctccaa agggacctca aggctttccg agggacacta
    12061 ggctgactcc atcgagccag tgtagagata agcttatcga tgatctagag ctcgctgatc
    12121 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
    12181 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
    12241 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
    12301 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
    12361 ggcggaaaga acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    12421 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    12481 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    12541 ccagtcggga aacctgtcgt gccagcggat cccatagtga ctggatatgt tgtgttttac
    12601 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
    12661 tttacgtttc tcgttcagct tttttgtaca aacttgtgct agcattctag ttgatatcta
    12721 taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata
    12781 attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact
    12841 cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg
    12901 ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag
    12961 aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg
    13021 gttaatctag aatggcgcgc cccaattcgc cctatagtga gtcgtattac gcgcgctcac
    13081 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
    13141 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
    13201 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
    13261 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
    13321 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
    13381 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
    13441 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
    13501 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
    13561 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
    13621 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
    13681 cgcttacaat ttag
//
