LOCUS       sequence-193893-       16156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac transposon DEST vector encoding dCAS9-VP64 and MS2-P65-HSF1
            activator helper complex. It is used for insertion of multiple 
            U6-sgRNA expression cassettes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16156)
  AUTHORS   Li S, Zhang A, Xue H, Li D, Liu Y
  TITLE     One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of 
            Multiple Genes.
  JOURNAL   Mol Ther Nucleic Acids. 2017 Sep 15;8:64-76. doi: 
            10.1016/j.omtn.2017.06.007. Epub 2017 Jun 15.
  PUBMED    28918057
REFERENCE   2  (bases 1 to 16156)
  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: "Mol Ther 
            Nucleic Acids."; date: "2017-09-15"; volume: "8:64-76. doi"; pages: 
            " 10.1016/j.omtn.2017.06.007. Epub 2017 Jun 15"
COMMENT     Sequence Label: PB-SAM-Dest
FEATURES             Location/Qualifiers
     source          1..16156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     173..191
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(229..251)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     351..370
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        451..469
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     451..468
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    535..556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(571..576,577..594,595..601)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    609..625
                     /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     614..636
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     633..649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     633..649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        670..688
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   871..933
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1236..2414
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1467..2405
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2362..2382
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2427..2813
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2868..2888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2904..3446
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             3471..3842
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             3858..3911
                     /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             3915..4934
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     "
     polyA_signal    5057..5178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        5179..5558
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5561..5836
                     /label=chicken beta-actin promoter
     intron          5837..6845
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6769..6791
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6853..6872
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             6905..6952
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(6953..6976,6977..6979,6980..9535,9536..9538,
                     9539..11053)
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             11108..11128
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11153..11302
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             11324..11377
                     /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             11378..11776
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    11918..12039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    12048..12172
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(12197..12202,12203..12220,12221..12227)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             12281..12940
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(12347..12366)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             13282..13587
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     13466..13485
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(13628..13752)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     repeat_region   14102..14136
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(14260..14279)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(14261..14279)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(14366..14383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14537..15125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14617..14636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15296..16087,16088..16156))
                     /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     15919..15938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
      181 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      241 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      301 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      361 gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta
      421 atacataacc ttatgtatca tacacatacg atttaggtga cactatagaa ctcgacctcg
      481 aggctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      541 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      601 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      661 caagctcgaa attaaccctc actaaaggga acaaaagctg gagctcgtct ttgatcaaaa
      721 cgcaaatcga cgaaaatgtg tcggacaata tcaagtcgat gagcgaaaaa ctaaaaaggc
      781 tagaatacga caatctcaca gacagcgttg agatatacgg tattcacgac agcaggctga
      841 ataataaaaa aattagaaac tattatttaa ccctagaaag ataatcatat tgtgacgtac
      901 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcgatctgta tatcgaggtt
      961 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     1021 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     1081 tttcttctat aaagtaacaa aacttttaaa cattctctct tttacaaaaa taaacttatt
     1141 ttgtacttta aaaacagtca tgttgtatta taaaataagt aattagctta acttatacat
     1201 aatagaaaca aattatactt attaattaat cgtgaggctc cggtgcccgt cagtgggcag
     1261 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     1321 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     1381 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     1441 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
     1501 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga
     1561 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag
     1621 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc
     1681 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa
     1741 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc
     1801 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt
     1861 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg
     1921 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc
     1981 cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc
     2041 ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg
     2101 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact
     2161 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt
     2221 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg
     2281 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg
     2341 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat
     2401 ttcaggtgtc gtgaaggttt accatggctt caaactttac tcagttcgtg ctcgtggaca
     2461 atggtgggac aggggatgtg acagtggctc cttctaattt cgctaatggg gtggcagagt
     2521 ggatcagctc caactcacgg agccaggcct acaaggtgac atgcagcgtc aggcagtcta
     2581 gtgcccagaa gagaaagtat accatcaagg tggaggtccc caaagtggct acccagacag
     2641 tgggcggagt cgaactgcct gtcgccgctt ggaggtccta cctgaacatg gagctcacta
     2701 tcccaatttt cgctaccaat tctgactgtg aactcatcgt gaaggcaatg caggggctcc
     2761 tcaaagacgg taatcctatc ccttccgcca tcgccgctaa ctcaggtatc tacagcgctg
     2821 gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagcggacct aagaaaaaga
     2881 ggaaggtggc ggccgctgga tccccttcag ggcagatcag caaccaggcc ctggctctgg
     2941 cccctagctc cgctccagtg ctggcccaga ctatggtgcc ctctagtgct atggtgcctc
     3001 tggcccagcc acctgctcca gcccctgtgc tgaccccagg accaccccag tcactgagcg
     3061 ctccagtgcc caagtctaca caggccggcg aggggactct gagtgaagct ctgctgcacc
     3121 tgcagttcga cgctgatgag gacctgggag ctctgctggg gaacagcacc gatcccggag
     3181 tgttcacaga tctggcctcc gtggacaact ctgagtttca gcagctgctg aatcagggcg
     3241 tgtccatgtc tcatagtaca gccgaaccaa tgctgatgga gtaccccgaa gccattaccc
     3301 ggctggtgac cggcagccag cggccccccg accccgctcc aactcccctg ggaaccagcg
     3361 gcctgcctaa tgggctgtcc ggagatgaag acttctcaag catcgctgat atggacttta
     3421 gtgccctgct gtcacagatt tcctctagtg ggcagggagg aggtggaagc ggcttcagcg
     3481 tggacaccag tgccctgctg gacctgttca gcccctcggt gaccgtgccc gacatgagcc
     3541 tgcctgacct tgacagcagc ctggccagta tccaagagct cctgtctccc caggagcccc
     3601 ccaggcctcc cgaggcagag aacagcagcc cggattcagg gaagcagctg gtgcactaca
     3661 cagcgcagcc gctgttcctg ctggaccccg gctccgtgga caccgggagc aacgacctgc
     3721 cggtgctgtt tgagctggga gagggctcct acttctccga aggggacggc ttcgccgagg
     3781 accccaccat ctccctgctg acaggctcgg agcctcccaa agccaaggac cccactgtct
     3841 cctgtacagg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     3901 atcctggccc aaccatgaaa aagcctgaac tcaccgctac ctctgtcgag aagtttctga
     3961 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc cgagggcgaa gaatctcggg
     4021 ctttcagctt cgatgtggga gggcgtggat atgtcctgcg ggtgaatagc tgcgccgatg
     4081 gtttctacaa agatcgctat gtttatcggc actttgcatc cgccgctctc cctattcccg
     4141 aagtgcttga cattggggag ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     4201 agggtgtcac cttgcaagac ctgcctgaaa ccgaactgcc cgctgttctc cagcccgtcg
     4261 ccgaggccat ggatgccatc gctgccgccg atcttagcca gaccagcggg ttcggcccat
     4321 tcggacctca aggaatcggt caatacacta catggcgcga tttcatctgc gctattgctg
     4381 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcc tccgtcgccc
     4441 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4501 acgccgattt cggctccaac aatgtcctga ccgacaatgg ccgcataaca gccgtcattg
     4561 actggagcga ggccatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4621 ggccctggtt ggcttgtatg gagcagcaga cccgctactt cgagcggagg catcccgagc
     4681 ttgcaggatc tcctcggctc cgggcttata tgctccgcat tggtcttgac caactctatc
     4741 agagcttggt tgacggcaat ttcgatgatg cagcttgggc tcagggtcgc tgcgacgcaa
     4801 tcgtccggtc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgctgccg
     4861 tctggaccga tggctgtgtg gaagtgctcg ccgatagtgg aaacagacgc cccagcactc
     4921 gtcctagggc aaaggatctg cagtaaaaac gatcataatc agccatacca catttgtaga
     4981 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     5041 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5101 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5161 actcatcaat gtatcttaga cattgattat tgactagtta ttaatagtaa tcaattacgg
     5221 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     5281 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     5341 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg
     5401 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     5461 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     5521 ggcagtacat ctacgtatta gtcatcgcta ttaccatggg tcgaggtgag ccccacgttc
     5581 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     5641 taattatttt gtgcagcgat gggggcgggg gggggggggg cgcgcgccag gcggggcggg
     5701 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     5761 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     5821 aagcgcgcgg cgggcgggag tcgctgcgtt gccttcgccc cgtgccccgc tccgcgccgc
     5881 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     5941 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggctcg tttcttttct
     6001 gtggctgcgt gaaagcctta aagggctccg ggagggccct ttgtgcgggg gggagcggct
     6061 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggcccg cgctgcccgg
     6121 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcgtgt gcgcgagggg
     6181 agcgcggccg ggggcggtgc cccgcggtgc gggggggctg cgaggggaac aaaggctgcg
     6241 tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac
     6301 ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc
     6361 cgtgcggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg
     6421 ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc
     6481 ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt
     6541 gcgagagggc gcagggactt cctttgtccc aaatctggcg gagccgaaat ctgggaggcg
     6601 ccgccgcacc ccctctagcg ggcgcgggcg aagcggtgcg gcgccggcag gaaggaaatg
     6661 ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccatct ccagcctcgg
     6721 ggctgccgca gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     6781 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     6841 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagatttac
     6901 catgaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggacaagaa
     6961 gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     7021 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     7081 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     7141 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     7201 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     7261 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     7321 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     7381 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     7441 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     7501 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     7561 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     7621 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct
     7681 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     7741 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     7801 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     7861 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     7921 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     7981 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     8041 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     8101 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     8161 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     8221 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     8281 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     8341 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     8401 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     8461 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     8521 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     8581 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     8641 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     8701 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     8761 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     8821 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     8881 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     8941 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     9001 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     9061 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     9121 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     9181 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     9241 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     9301 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     9361 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     9421 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccaca tcgtgcctca
     9481 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaaggcccg
     9541 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     9601 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     9661 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     9721 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     9781 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     9841 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     9901 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     9961 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
    10021 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
    10081 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
    10141 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
    10201 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
    10261 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
    10321 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
    10381 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
    10441 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
    10501 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
    10561 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
    10621 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
    10681 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
    10741 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
    10801 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
    10861 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
    10921 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
    10981 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
    11041 gctgggaggc gacagcgctg gaggaggtgg aagcggagga ggaggaagcg gaggaggagg
    11101 tagcggacct aagaaaaaga ggaaggtggc ggccgctgga tccggacggg ctgacgcatt
    11161 ggacgatttt gatctggata tgctgggaag tgacgccctc gatgattttg accttgacat
    11221 gcttggttcg gatgcccttg atgactttga cctcgacatg ctcggcagtg acgcccttga
    11281 tgatttcgac ctggacatgc tgattaactg tacaggcagt ggagagggca gaggaagtct
    11341 gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg gccaagcctt tgtctcaaga
    11401 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
    11461 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
    11521 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
    11581 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
    11641 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
    11701 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
    11761 tgtgtgggag ggctaaaaat gtcgacggta ccgcgggccc gggatcctct agatcataat
    11821 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
    11881 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
    11941 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
    12001 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa tttatcaaca agtttgtaca
    12061 aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc
    12121 ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc
    12181 attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag
    12241 ttaggatccg gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
    12301 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
    12361 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
    12421 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
    12481 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag
    12541 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
    12601 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
    12661 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
    12721 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
    12781 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
    12841 ggcgattcag gttcatcatg ccgtctgtga tggcttccat gtcggcagaa tgcttaatga
    12901 attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggat ccggcttact
    12961 aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac
    13021 tgatatgtat acccgaagta tgtcaaaaag aggtgtgcta tgaagcagcg tattacagtg
    13081 acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc
    13141 tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg
    13201 aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg
    13261 acgagaacag ggactggtga aatgcagttt aaggtttaca cctataaaag agagagccgt
    13321 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
    13381 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
    13441 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
    13501 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
    13561 attaacctga tgttctgggg aatataaatg tcaggctccg ttatacacag ccagtctgca
    13621 ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg
    13681 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt
    13741 gtacaaagtg gttgataaat ccatcgaagc agctccagcc tacacaatcg ctcaagacgt
    13801 gtaatgcttt tattatatat tagtcacgat atctataaca agaaaatata tatataataa
    13861 gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa
    13921 agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc
    13981 agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg
    14041 tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat
    14101 gcatgcgtca attttacgca gactatcttt ctagggttaa aaaagatttg cgctttactc
    14161 gacctaaact ttaaacacgt catagaatct tcgtttgaca aaaaccacat tgtggggtac
    14221 cgagctcgaa ttcatcgatg atatcagatc tgccggtctc cctatagtga gtcgtattaa
    14281 tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
    14341 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
    14401 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
    14461 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
    14521 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
    14581 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
    14641 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
    14701 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
    14761 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
    14821 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
    14881 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
    14941 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
    15001 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
    15061 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
    15121 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
    15181 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
    15241 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
    15301 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
    15361 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
    15421 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
    15481 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
    15541 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
    15601 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
    15661 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
    15721 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
    15781 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
    15841 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
    15901 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
    15961 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
    16021 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
    16081 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
    16141 aatgttgaat actcat
//
