LOCUS       sequence-227298-       14195 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses VP64-dCas9-p300 and MS2-p65-HSF1 fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14195)
  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 14195)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_EF1aVP64dCas9p300_T2A_MS2p65HSF1-IRES...
FEATURES             Location/Qualifiers
     source          1..14195
                     /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"
     regulatory      2665..2674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2674..2823
                     /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             2854..2901
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(2902..2925,2926..2928,2929..5484,5485..5487,
                     5488..7002)
                     /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             7057..7077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7099..8949
                     /codon_start=1
                     /gene="EP300"
                     /product="catalytic histone acetyltransferase core of the 
                     human E1A-associated protein p300 (Delvecchio et al., 
                     2013)"
                     /label=p300 core
                     /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI
                     VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ
                     EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG
                     ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG
                     FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA
                     SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP
                     PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY
                     IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE
                     GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS
                     RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL
                     MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD"
     regulatory      8713..8722
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8950..9003
                     /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             9013..9399
                     /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             9454..9474
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9490..10032
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             10057..10428
                     /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(10454..11028,11029..11031,11032..11040)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10621..10638)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10848..10867
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             11041..11439
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    11443..11667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(11688..11712)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   11894..11956
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12007..12027)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(12007..12025)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12046..12062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12046..12062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12059..12081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12070..12086
                     /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(12094..12100,12101..12118,12119..12124))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12139..12160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12277..12294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12448..13036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12528..12547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13207..13998,13999..14067))
                     /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     13830..13849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14068..14172)
                     /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 tggggccacc atggacgcat tggacgattt tgatctggat
     2701 atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt
     2761 gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg
     2821 ctgggtggag gttctggatg tacggccacc atgaaaaggc cggcggccac gaaaaaggcc
     2881 ggccaggcaa aaaagaaaaa ggacaagaag tacagcatcg gcctggccat cggcaccaac
     2941 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     3001 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     3061 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     3121 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     3181 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     3241 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     3301 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     3361 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     3421 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     3481 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     3541 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     3601 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     3661 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     3721 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     3781 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     3841 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     3901 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     3961 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     4021 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     4081 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     4141 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     4201 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     4261 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     4321 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     4381 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     4441 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     4501 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     4561 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     4621 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     4681 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     4741 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     4801 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     4861 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     4921 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     4981 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     5041 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     5101 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     5161 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     5221 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     5281 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     5341 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     5401 gactacgatg tggaccacat cgtgcctcag agctttctga aggacgactc catcgacaac
     5461 aaggtgctga ccagaagcga caaggcccgg ggcaagagcg acaacgtgcc ctccgaagag
     5521 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     5581 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     5641 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     5701 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     5761 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     5821 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     5881 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     5941 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     6001 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     6061 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     6121 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     6181 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     6241 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     6301 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     6361 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     6421 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     6481 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     6541 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     6601 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     6661 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     6721 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     6781 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     6841 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     6901 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     6961 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acagcgctgg aggaggtgga
     7021 agcggaggag gaggaagcgg aggaggaggt agcggaccta agaaaaagag gaaggtggcg
     7081 ggaggtggaa gcggaggaat tttcaaacca gaagaactac gacaggcact gatgccaact
     7141 ttggaggcac tttaccgtca ggatccagaa tcccttccct ttcgtcaacc tgtggaccct
     7201 cagcttttag gaatccctga ttactttgat attgtgaaga gccccatgga tctttctacc
     7261 attaagagga agttagacac tggacagtat caggagccct ggcagtatgt cgatgatatt
     7321 tggcttatgt tcaataatgc ctggttatat aaccggaaaa catcacgggt atacaaatac
     7381 tgctccaagc tctctgaggt ctttgaacaa gaaattgacc cagtgatgca aagccttgga
     7441 tactgttgtg gcagaaagtt ggagttctct ccacagacac tgtgttgcta cggcaaacag
     7501 ttgtgcacaa tacctcgtga tgccacttat tacagttacc agaacaggta tcatttctgt
     7561 gagaagtgtt tcaatgagat ccaaggggag agcgtttctt tgggggatga cccttcccag
     7621 cctcaaacta caataaataa agaacaattt tccaagagaa aaaatgacac actggatcct
     7681 gaactgtttg ttgaatgtac agagtgcgga agaaagatgc atcagatctg tgtccttcac
     7741 catgagatca tctggcctgc tggattcgtc tgtgatggct gtttaaagaa aagtgcacga
     7801 actaggaaag aaaataagtt ttctgctaaa aggttgccat ctaccagact tggcaccttt
     7861 cttgagaatc gtgtgaatga ctttctgagg cgacagaatc accctgagtc aggagaggtc
     7921 actgttagag tagttcatgc ttctgacaaa accgtggaag taaaaccagg catgaaagca
     7981 aggtttgtgg acagtggaga gatggcagaa tcctttccat accgaaccaa agccctcttt
     8041 gcctttgaag aaattgatgg tgttgacctg tgcttctttg gcatgcatgt tcaagagtat
     8101 ggctctgact gccctccacc caaccagagg agagtataca tatcttacct cgatagtgtt
     8161 catttcttcc gtcctaaatg cttgaggact gcagtctatc atgaaatcct aattggatat
     8221 ttagaatatg tcaagaaatt aggttacaca acagggcata tttgggcatg tccaccaagt
     8281 gagggagatg attatatctt ccattgccat cctcctgacc agaagatacc caagcccaag
     8341 cgactgcagg aatggtacaa aaaaatgctt gacaaggctg tatcagagcg tattgtccat
     8401 gactacaagg atatttttaa acaagctact gaagatagat taacaagtgc aaaggaattg
     8461 ccttatttcg agggtgattt ctggcccaat gttctggaag aaagcattaa ggaactggaa
     8521 caggaggaag aagagagaaa acgagaggaa aacaccagca atgaaagcac agatgtgacc
     8581 aagggagaca gcaaaaatgc taaaaagaag aataataaga aaaccagcaa aaataagagc
     8641 agcctgagta ggggcaacaa gaagaaaccc gggatgccca atgtatctaa cgacctctca
     8701 cagaaactat atgccaccat ggagaagcat aaagaggtct tctttgtgat ccgcctcatt
     8761 gctggccctg ctgccaactc cctgcctccc attgttgatc ctgatcctct catcccctgc
     8821 gatctgatgg atggtcggga tgcgtttctc acgctggcaa gggacaagca cctggagttc
     8881 tcttcactcc gaagagccca gtggtccacc atgtgcatgc tggtggagct gcacacgcag
     8941 agccaggacg agggcagagg aagtctccta acatgcggtg acgtggagga gaatcctggc
     9001 ccaggaggta tggcttcaaa ctttactcag ttcgtgctcg tggacaatgg tgggacaggg
     9061 gatgtgacag tggctccttc taatttcgct aatggggtgg cagagtggat cagctccaac
     9121 tcacggagcc aggcctacaa ggtgacatgc agcgtcaggc agtctagtgc ccagaagaga
     9181 aagtatacca tcaaggtgga ggtccccaaa gtggctaccc agacagtggg cggagtcgaa
     9241 ctgcctgtcg ccgcttggag gtcctacctg aacatggagc tcactatccc aattttcgct
     9301 accaattctg actgtgaact catcgtgaag gcaatgcagg ggctcctcaa agacggtaat
     9361 cctatccctt ccgccatcgc cgctaactca ggtatctaca gcgctggagg aggtggaagc
     9421 ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa ggtggcggcc
     9481 gctggatccc cttcagggca gatcagcaac caggccctgg ctctggcccc tagctccgct
     9541 ccagtgctgg cccagactat ggtgccctct agtgctatgg tgcctctggc ccagccacct
     9601 gctccagccc ctgtgctgac cccaggacca ccccagtcac tgagcgctcc agtgcccaag
     9661 tctacacagg ccggcgaggg gactctgagt gaagctctgc tgcacctgca gttcgacgct
     9721 gatgaggacc tgggagctct gctggggaac agcaccgatc ccggagtgtt cacagatctg
     9781 gcctccgtgg acaactctga gtttcagcag ctgctgaatc agggcgtgtc catgtctcat
     9841 agtacagccg aaccaatgct gatggagtac cccgaagcca ttacccggct ggtgaccggc
     9901 agccagcggc cccccgaccc cgctccaact cccctgggaa ccagcggcct gcctaatggg
     9961 ctgtccggag atgaagactt ctcaagcatc gctgatatgg actttagtgc cctgctgtca
    10021 cagatttcct ctagtgggca gggaggaggt ggaagcggct tcagcgtgga caccagtgcc
    10081 ctgctggacc tgttcagccc ctcggtgacc gtgcccgaca tgagcctgcc tgaccttgac
    10141 agcagcctgg ccagtatcca agagctcctg tctccccagg agccccccag gcctcccgag
    10201 gcagagaaca gcagcccgga ttcagggaag cagctggtgc actacacagc gcagccgctg
    10261 ttcctgctgg accccggctc cgtggacacc gggagcaacg acctgccggt gctgtttgag
    10321 ctgggagagg gctcctactt ctccgaaggg gacggcttcg ccgaggaccc caccatctcc
    10381 ctgctgacag gctcggagcc tcccaaagcc aaggacccca ctgtctcctg tacatgagga
    10441 attccgcccc ccccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg
    10501 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
    10561 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
    10621 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
    10681 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
    10741 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
    10801 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
    10861 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
    10921 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
    10981 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
    11041 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
    11101 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
    11161 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
    11221 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
    11281 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
    11341 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
    11401 cgtgaattgc tgccctctgg ttatgtgtgg gagggctgac gactgtgcct tctagttgcc
    11461 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
    11521 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
    11581 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagtc aatagcaggc
    11641 atgctgggga tgcggtgggc tctatggact agtctcgaga tatctagacc cagctttctt
    11701 gtacaaagtg gtactagtta aaagttttgt tactttatag aagaaatttt gagtttttgt
    11761 ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt
    11821 aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca
    11881 gaccgataaa acacatgcgt caattttacg catgattatc tttaacgtac gtcacaatat
    11941 gattatcttt ctagggttaa tctagtatac gcgtatgcgg ccgcttaatt aatccagctt
    12001 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
    12061 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
    12121 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
    12181 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
    12241 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
    12301 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
    12361 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    12421 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    12481 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    12541 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    12601 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    12661 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    12721 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    12781 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    12841 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    12901 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    12961 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    13021 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    13081 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    13141 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    13201 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    13261 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    13321 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    13381 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    13441 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    13501 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    13561 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    13621 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    13681 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    13741 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    13801 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    13861 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    13921 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    13981 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    14041 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    14101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
    14161 aggggttccg cgcacatttc cccgaaaagt gccac
//
