LOCUS       sequence-217554-       12360 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 12360)
  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 12360)
  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..12360
                     /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..2654
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1704..2646
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2603..2623
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2685..2732
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(2733..2756,2757..2759,2760..5315,5316..5318,
                     5319..6833)
                     /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             6888..6908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6933..7082
                     /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             7113..7166
                     /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      7173..7182
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7182..7568
                     /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             7623..7643
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7659..8201
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             8226..8597
                     /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(8622..9196,9197..9199,9200..9208)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8789..8806)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     9016..9035
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9209..9604
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9608..9832
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(9853..9877)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   10059..10121
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10172..10192)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10172..10190)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10211..10227)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10211..10227)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10224..10246)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10235..10251
                     /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(10259..10265,10266..10283,10284..10289))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10304..10325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10442..10459)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10613..11201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10693..10712)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11372..12163,12164..12232))
                     /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     11995..12014
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12233..12337)
                     /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 agagaaagtg 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 gcctggcctg ggcgctgggg ccgccgcgtg
     1921 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     1981 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     2041 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
     2101 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     2161 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     2221 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     2281 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     2341 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     2401 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     2461 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     2521 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     2581 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     2641 tttcaggtgt cgtggaagct tggcgcgccc gtacggccac catgaaaagg ccggcggcca
     2701 cgaaaaaggc cggccaggca aaaaagaaaa aggacaagaa gtacagcatc ggcctggcca
     2761 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     2821 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     2881 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     2941 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3001 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3061 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3121 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3181 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3241 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3301 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     3361 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     3421 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     3481 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     3541 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     3601 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     3661 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     3721 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     3781 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     3841 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     3901 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     3961 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4021 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4081 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4141 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     4201 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4261 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     4321 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     4381 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     4441 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     4501 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     4561 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     4621 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     4681 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     4741 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     4801 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     4861 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     4921 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     4981 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     5041 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     5101 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     5161 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     5221 accggctgtc cgactacgat gtggaccaca tcgtgcctca gagctttctg aaggacgact
     5281 ccatcgacaa caaggtgctg accagaagcg acaaggcccg gggcaagagc gacaacgtgc
     5341 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     5401 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     5461 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     5521 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     5581 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     5641 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     5701 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     5761 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     5821 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     5881 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     5941 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     6001 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     6061 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6121 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6181 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6241 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6301 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6361 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     6421 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6481 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     6541 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     6601 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     6661 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     6721 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     6781 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcgctg
     6841 gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagcggacct aagaaaaaga
     6901 ggaaggtggc ggccgctgga tccggacggg ctgacgcatt ggacgatttt gatctggata
     6961 tgctgggaag tgacgccctc gatgattttg accttgacat gcttggttcg gatgcccttg
     7021 atgactttga cctcgacatg ctcggcagtg acgcccttga tgatttcgac ctggacatgc
     7081 tgattaactg tacgggcggc gggtccggag gagagggcag aggaagtctc ctaacatgcg
     7141 gtgacgtgga ggagaatcct ggcccatgta cggccaccat ggcttcaaac tttactcagt
     7201 tcgtgctcgt ggacaatggt gggacagggg atgtgacagt ggctccttct aatttcgcta
     7261 atggggtggc agagtggatc agctccaact cacggagcca ggcctacaag gtgacatgca
     7321 gcgtcaggca gtctagtgcc cagaagagaa agtataccat caaggtggag gtccccaaag
     7381 tggctaccca gacagtgggc ggagtcgaac tgcctgtcgc cgcttggagg tcctacctga
     7441 acatggagct cactatccca attttcgcta ccaattctga ctgtgaactc atcgtgaagg
     7501 caatgcaggg gctcctcaaa gacggtaatc ctatcccttc cgccatcgcc gctaactcag
     7561 gtatctacag cgctggagga ggtggaagcg gaggaggagg aagcggagga ggaggtagcg
     7621 gacctaagaa aaagaggaag gtggcggccg ctggatcccc ttcagggcag atcagcaacc
     7681 aggccctggc tctggcccct agctccgctc cagtgctggc ccagactatg gtgccctcta
     7741 gtgctatggt gcctctggcc cagccacctg ctccagcccc tgtgctgacc ccaggaccac
     7801 cccagtcact gagcgctcca gtgcccaagt ctacacaggc cggcgagggg actctgagtg
     7861 aagctctgct gcacctgcag ttcgacgctg atgaggacct gggagctctg ctggggaaca
     7921 gcaccgatcc cggagtgttc acagatctgg cctccgtgga caactctgag tttcagcagc
     7981 tgctgaatca gggcgtgtcc atgtctcata gtacagccga accaatgctg atggagtacc
     8041 ccgaagccat tacccggctg gtgaccggca gccagcggcc ccccgacccc gctccaactc
     8101 ccctgggaac cagcggcctg cctaatgggc tgtccggaga tgaagacttc tcaagcatcg
     8161 ctgatatgga ctttagtgcc ctgctgtcac agatttcctc tagtgggcag ggaggaggtg
     8221 gaagcggctt cagcgtggac accagtgccc tgctggacct gttcagcccc tcggtgaccg
     8281 tgcccgacat gagcctgcct gaccttgaca gcagcctggc cagtatccaa gagctcctgt
     8341 ctccccagga gccccccagg cctcccgagg cagagaacag cagcccggat tcagggaagc
     8401 agctggtgca ctacacagcg cagccgctgt tcctgctgga ccccggctcc gtggacaccg
     8461 ggagcaacga cctgccggtg ctgtttgagc tgggagaggg ctcctacttc tccgaagggg
     8521 acggcttcgc cgaggacccc accatctccc tgctgacagg ctcggagcct cccaaagcca
     8581 aggaccccac tgtctcctgt acatgagaat tccgcccccc ccccctctcc ctcccccccc
     8641 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     8701 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     8761 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     8821 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     8881 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     8941 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     9001 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     9061 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     9121 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     9181 aaaacacgat gataatatgg ccacaaccat ggccaagcct ttgtctcaag aagaatccac
     9241 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     9301 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     9361 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     9421 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     9481 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     9541 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     9601 gggccgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     9661 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     9721 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     9781 gaggattggg aagtcaatag caggcatgct ggggatgcgg tgggctctat ggactagtct
     9841 cgagatatct agacccagct ttcttgtaca aagtggtact agttaaaagt tttgttactt
     9901 tatagaagaa attttgagtt tttgtttttt tttaataaat aaataaacat aaataaattg
     9961 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
    10021 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacgcatga
    10081 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag tatacgcgta
    10141 tgcggccgct taattaatcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
    10201 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
    10261 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
    10321 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
    10381 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
    10441 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
    10501 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
    10561 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
    10621 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
    10681 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
    10741 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
    10801 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
    10861 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
    10921 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
    10981 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
    11041 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
    11101 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
    11161 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
    11221 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
    11281 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
    11341 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
    11401 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
    11461 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
    11521 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
    11581 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
    11641 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
    11701 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
    11761 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
    11821 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
    11881 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
    11941 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
    12001 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
    12061 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
    12121 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
    12181 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
    12241 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
    12301 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
