LOCUS       sequence-194745-       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"
     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             join(8022..8024,8025..8027,8028..8741)
                     /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(8022..8024,8025..8027,8028..8738)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8328..8347)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8675..8696
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(8772..9346,9347..9349,9350..9358)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8939..8956)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     9166..9185
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9359..10153
                     /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(9413..9432)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     10023..10042
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(10455..10472)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    10461..10685
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(10732..10748)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    10756..10772
                     /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(10780..10786,10787..10804,10805..10810))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10825..10846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(10905..11029)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   11319..11353
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11382..11401)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11383..11401)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11411..11428)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11411..11427)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11420..11442)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11569..12024
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11656..11675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11866..11887
                     /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 agggaatcga tatggtgagc aagggcgagg
     8041 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     8101 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     8161 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     8221 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     8281 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     8341 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     8401 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     8461 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     8521 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     8581 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     8641 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     8701 ccgccgggat cactctcggc atggacgagc tgtacaagta atttaaagaa ttcccgcccc
     8761 cccccccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa
     8821 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     8881 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     8941 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     9001 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     9061 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     9121 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     9181 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     9241 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     9301 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaaccat
     9361 ggctgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     9421 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     9481 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     9541 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     9601 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     9661 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     9721 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     9781 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     9841 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     9901 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     9961 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
    10021 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
    10081 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
    10141 cgagttcttc tgagcgggat cggctagagt cgacccggac gggacgctcc tgcgcctgat
    10201 acagaacgaa ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc
    10261 actgcgtgga tggagcgctg gcgcctgctg cgcgacggcg agctgctcac cacccactcg
    10321 agggctgcag cgctgcagag gccgagtgca gaactgctcc aaagggacct caaggctttc
    10381 cgagggacac taggctgact ccatcgagcc agtgtagaga taagcttatc gatgatctag
    10441 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
    10501 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
    10561 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
    10621 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
    10681 tatggcttct gaggcggaaa gaacctgcag cccaagcttg gcgtaatcat ggtcatagct
    10741 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    10801 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    10861 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atcccatagt gactggatat
    10921 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
    10981 tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtg ctagcattct
    11041 agttgatatc tataacaaga aaatatatat ataataagtt atcacgtaag tagaacatga
    11101 aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc
    11161 gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc gcattgacaa
    11221 gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca gcgacggatt
    11281 cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt ttacgcagac
    11341 tatctttcta gggttaatct agaatggcgc gccccaattc gccctatagt gagtcgtatt
    11401 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
    11461 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
    11521 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
    11581 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    11641 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    11701 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    11761 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
    11821 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
    11881 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
    11941 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
    12001 acaaaatatt aacgcttaca atttag
//
