LOCUS       sequence-212992-       12367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac transposon vector carrying human EF1a promoter-driven 
            dCas9VP64-T2A-MS2p65HSF1 with Blasticidin resistant marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12367)
  AUTHORS   Chong ZS, Ohnishi S, Yusa K, Wright GJ
  TITLE     Pooled extracellular receptor-ligand interaction screening using 
            CRISPR activation.
  JOURNAL   Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3.
  PUBMED    30477585
REFERENCE   2  (bases 1 to 12367)
  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: "Genome 
            Biol."; date: "2018-11-26"; volume: "19(1):205. doi"; pages: " 
            10.1186/s13059-018-1581-3"
COMMENT     Sequence Label: pPB-R1R2_EF1adCas9VP64_T2A_MS2p65HSF1-IRESbsdpA
FEATURES             Location/Qualifiers
     source          1..12367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    998..1022
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1473..2658
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1704..2650
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2607..2627
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2690..2737
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(2738..2761,2762..2764,2765..5320,5321..5323,
                     5324..6838)
                     /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             6893..6913
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6938..7087
                     /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             7118..7171
                     /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"
     regulatory      7178..7187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7187..7573
                     /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             7628..7648
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7664..8206
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             8231..8602
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     misc_feature    join(8627..9200,9201..9203,9204..9212)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8794..8811)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     9021..9040
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9213..9611
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9615..9839
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(9860..9884)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   10066..10128
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10179..10199)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10179..10197)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10218..10234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10218..10234)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10231..10253)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10242..10258
                     /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(10266..10272,10273..10290,10291..10296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10311..10332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10449..10466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10620..11208)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10700..10719)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11379..12170,12171..12239))
                     /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     12002..12021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12240..12344)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcaca agtttgtaca aaaaagcagg
     1021 ctggcgccgg aaccaattca gtcgactgga tccacgcgtg aggggcgcgg aagccggggc
     1081 ggaggaggag ccaaccccgg gctgggctga gacccgcaga ggaagacgct ctagggattt
     1141 gtcccggact agcgagatgg caaggctgag gacgggaggc tgattgagag gcgaaggtac
     1201 accctaatct caatacaacc tttggagcta agccagcaat ggtagaggga agattctgca
     1261 cgtcccttcc aggcggcctc cccgtcacca ccccccccaa cccgccccga ccggagctga
     1321 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa
     1381 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc
     1441 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc
     1501 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     1561 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     1621 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     1681 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
     1741 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg
     1801 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
     1861 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg
     1921 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     1981 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     2041 caagatcgat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     2101 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     2161 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     2221 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     2281 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     2341 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     2401 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     2461 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     2521 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     2581 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     2641 tccatttcag gtgtcgtggg aagcttggcg cgcccgtacg gccaccatga aaaggccggc
     2701 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggac aagaagtaca gcatcggcct
     2761 ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     2821 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     2881 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     2941 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     3001 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     3061 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     3121 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     3181 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     3241 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     3301 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     3361 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     3421 gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac
     3481 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     3541 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     3601 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     3661 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     3721 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     3781 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     3841 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     3901 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     3961 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     4021 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     4081 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     4141 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     4201 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     4261 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     4321 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     4381 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     4441 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     4501 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     4561 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     4621 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     4681 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     4741 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     4801 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     4861 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     4921 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     4981 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     5041 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     5101 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     5161 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     5221 catcaaccgg ctgtccgact acgatgtgga ccacatcgtg cctcagagct ttctgaagga
     5281 cgactccatc gacaacaagg tgctgaccag aagcgacaag gcccggggca agagcgacaa
     5341 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     5401 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     5461 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     5521 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     5581 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     5641 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     5701 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     5761 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     5821 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     5881 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     5941 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     6001 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     6061 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     6121 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     6181 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     6241 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     6301 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     6361 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     6421 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     6481 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     6541 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     6601 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     6661 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     6721 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     6781 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacag
     6841 cgctggagga ggtggaagcg gaggaggagg aagcggagga ggaggtagcg gacctaagaa
     6901 aaagaggaag gtggcggccg ctggatccgg acgggctgac gcattggacg attttgatct
     6961 ggatatgctg ggaagtgacg ccctcgatga ttttgacctt gacatgcttg gttcggatgc
     7021 ccttgatgac tttgacctcg acatgctcgg cagtgacgcc cttgatgatt tcgacctgga
     7081 catgctgatt aactgtacgg gcggcgggtc cggaggagag ggcagaggaa gtctcctaac
     7141 atgcggtgac gtggaggaga atcctggccc atgtacggcc accatggctt caaactttac
     7201 tcagttcgtg ctcgtggaca atggtgggac aggggatgtg acagtggctc cttctaattt
     7261 cgctaatggg gtggcagagt ggatcagctc caactcacgg agccaggcct acaaggtgac
     7321 atgcagcgtc aggcagtcta gtgcccagaa gagaaagtat accatcaagg tggaggtccc
     7381 caaagtggct acccagacag tgggcggagt cgaactgcct gtcgccgctt ggaggtccta
     7441 cctgaacatg gagctcacta tcccaatttt cgctaccaat tctgactgtg aactcatcgt
     7501 gaaggcaatg caggggctcc tcaaagacgg taatcctatc ccttccgcca tcgccgctaa
     7561 ctcaggtatc tacagcgctg gaggaggtgg aagcggagga ggaggaagcg gaggaggagg
     7621 tagcggacct aagaaaaaga ggaaggtggc ggccgctgga tccccttcag ggcagatcag
     7681 caaccaggcc ctggctctgg cccctagctc cgctccagtg ctggcccaga ctatggtgcc
     7741 ctctagtgct atggtgcctc tggcccagcc acctgctcca gcccctgtgc tgaccccagg
     7801 accaccccag tcactgagcg ctccagtgcc caagtctaca caggccggcg aggggactct
     7861 gagtgaagct ctgctgcacc tgcagttcga cgctgatgag gacctgggag ctctgctggg
     7921 gaacagcacc gatcccggag tgttcacaga tctggcctcc gtggacaact ctgagtttca
     7981 gcagctgctg aatcagggcg tgtccatgtc tcatagtaca gccgaaccaa tgctgatgga
     8041 gtaccccgaa gccattaccc ggctggtgac cggcagccag cggccccccg accccgctcc
     8101 aactcccctg ggaaccagcg gcctgcctaa tgggctgtcc ggagatgaag acttctcaag
     8161 catcgctgat atggacttta gtgccctgct gtcacagatt tcctctagtg ggcagggagg
     8221 aggtggaagc ggcttcagcg tggacaccag tgccctgctg gacctgttca gcccctcggt
     8281 gaccgtgccc gacatgagcc tgcctgacct tgacagcagc ctggccagta tccaagagct
     8341 cctgtctccc caggagcccc ccaggcctcc cgaggcagag aacagcagcc cggattcagg
     8401 gaagcagctg gtgcactaca cagcgcagcc gctgttcctg ctggaccccg gctccgtgga
     8461 caccgggagc aacgacctgc cggtgctgtt tgagctggga gagggctcct acttctccga
     8521 aggggacggc ttcgccgagg accccaccat ctccctgctg acaggctcgg agcctcccaa
     8581 agccaaggac cccactgtct cctgtacatg agaattccgc cccccccccc tctccctccc
     8641 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     8701 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     8761 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     8821 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     8881 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     8941 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     9001 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     9061 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     9121 gtgtttagtc gaggttaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
     9181 ttgaaaaaca cgatgataat atggccacaa ccatggccaa gcctttgtct caagaagaat
     9241 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     9301 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     9361 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     9421 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     9481 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     9541 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     9601 gggagggctg acgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     9661 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     9721 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     9781 caagggggag gattgggaag tcaatagcag gcatgctggg gatgcggtgg gctctatgga
     9841 ctagtctcga gatatctaga cccagctttc ttgtacaaag tggtactagt taaaagtttt
     9901 gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa taaacataaa
     9961 taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa acttaatatc
    10021 tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc gtcaatttta
    10081 cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt aatctagtat
    10141 acgcgtatgc ggccgcttaa ttaatccagc ttttgttccc tttagtgagg gttaattgcg
    10201 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
    10261 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
    10321 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    10381 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
    10441 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
    10501 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
    10561 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
    10621 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
    10681 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
    10741 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
    10801 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
    10861 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
    10921 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
    10981 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
    11041 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
    11101 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
    11161 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
    11221 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
    11281 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
    11341 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
    11401 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
    11461 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
    11521 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
    11581 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
    11641 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
    11701 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
    11761 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
    11821 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
    11881 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
    11941 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
    12001 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
    12061 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
    12121 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
    12181 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
    12241 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
    12301 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
    12361 gtgccac
//
