LOCUS       sequence-198249-       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"
     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             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             6644..7186
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             7211..7582
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             7604..7657
                     /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(7664..7666,7667..7669,7670..8383)
                     /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(7664..7666,7667..7669,7670..8380)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7970..7989)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8317..8338
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(8405..8979,8980..8982,8983..8991)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8572..8589)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     8799..8818
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             8992..9786
                     /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(9046..9065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9656..9675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(10088..10105)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10094..10318
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(10365..10381)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    10389..10405
                     /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(10413..10419,10420..10437,10438..10443))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10458..10479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(10538..10662)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   10952..10986
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11015..11034)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11016..11034)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11044..11061)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11044..11060)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11053..11075)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11202..11657
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11289..11308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11499..11520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11683..11787
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11788..11856,11857..12648)
                     /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(12006..12025)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12819..13407
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13308..13327
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13561..13578
                     /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 tcgagttcga aggcagcggc gggccttcag ggcagatcag
     6661 caaccaggcc ctggctctgg cccctagctc cgctccagtg ctggcccaga ctatggtgcc
     6721 ctctagtgct atggtgcctc tggcccagcc acctgctcca gcccctgtgc tgaccccagg
     6781 accaccccag tcactgagcg ctccagtgcc caagtctaca caggccggcg aggggactct
     6841 gagtgaagct ctgctgcacc tgcagttcga cgctgatgag gacctgggag ctctgctggg
     6901 gaacagcacc gatcccggag tgttcacaga tctggcctcc gtggacaact ctgagtttca
     6961 gcagctgctg aatcagggcg tgtccatgtc tcatagtaca gccgaaccaa tgctgatgga
     7021 gtaccccgaa gccattaccc ggctggtgac cggcagccag cggccccccg accccgctcc
     7081 aactcccctg ggaaccagcg gcctgcctaa tgggctgtcc ggagatgaag acttctcaag
     7141 catcgctgat atggacttta gtgccctgct gtcacagatt tcctctagtg ggcagggagg
     7201 aggtggaagc ggcttcagcg tggacaccag tgccctgctg gacctgttca gcccctcggt
     7261 gaccgtgccc gacatgagcc tgcctgacct tgacagcagc ctggccagta tccaagagct
     7321 cctgtctccc caggagcccc ccaggcctcc cgaggcagag aacagcagcc cggattcagg
     7381 gaagcagctg gtgcactaca cagcgcagcc gctgttcctg ctggaccccg gctccgtgga
     7441 caccgggagc aacgacctgc cggtgctgtt tgagctggga gagggctcct acttctccga
     7501 aggggacggc ttcgccgagg accccaccat ctccctgctg acaggctcgg agcctcccaa
     7561 agccaaggac cccactgtct cctctagcgt cgacggctcc ggagagggca gaggaagtct
     7621 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatc gatatggtga gcaagggcga
     7681 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     7741 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     7801 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     7861 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     7921 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     7981 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     8041 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     8101 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     8161 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     8221 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     8281 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     8341 cgccgccggg atcactctcg gcatggacga gctgtacaag taatttaaag aattccgccc
     8401 cccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     8461 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     8521 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     8581 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     8641 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     8701 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     8761 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     8821 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     8881 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     8941 acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac catggctgaa
     9001 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     9061 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     9121 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     9181 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     9241 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     9301 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     9361 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     9421 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     9481 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     9541 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     9601 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     9661 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     9721 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     9781 ttctgagcgg gatcggctag agtcgacccg gacgggacgc tcctgcgcct gatacagaac
     9841 gaattgcttg caggcatctc atgagtgtgt cttcccgttt tccgcctgag gtcactgcgt
     9901 ggatggagcg ctggcgcctg ctgcgcgacg gcgagctgct caccacccac tcgagggctg
     9961 cagcgctgca gaggccgagt gcagaactgc tccaaaggga cctcaaggct ttccgaggga
    10021 cactaggctg actccatcga gccagtgtag agataagctt atcgatgatc tagagctcgc
    10081 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
    10141 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
    10201 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
    10261 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
    10321 tctgaggcgg aaagaacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct
    10381 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
    10441 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
    10501 gctttccagt cgggaaacct gtcgtgccag cggatcccat agtgactgga tatgttgtgt
    10561 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
    10621 atcattttac gtttctcgtt cagctttttt gtacaaactt gtgctagcat tctagttgat
    10681 atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca
    10741 atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt
    10801 tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc
    10861 tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta
    10921 tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt
    10981 ctagggttaa tctagaatgg cgcgccccaa ttcgccctat agtgagtcgt attacgcgcg
    11041 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
    11101 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
    11161 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
    11221 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
    11281 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
    11341 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
    11401 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
    11461 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
    11521 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    11581 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
    11641 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
    11701 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
    11761 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
    11821 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
    11881 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
    11941 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
    12001 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
    12061 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
    12121 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
    12181 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
    12241 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
    12301 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
    12361 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
    12421 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
    12481 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
    12541 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
    12601 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
    12661 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
    12721 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
    12781 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
    12841 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
    12901 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
    12961 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
    13021 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
    13081 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
    13141 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
    13201 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
    13261 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
    13321 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
    13381 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
    13441 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
    13501 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
    13561 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
    13621 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
    13681 gagcgcaacg caat
//
