pPB-R1R2_EF1aVP64dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA

LOCUS      pPB-R1R2_EF1aVP64dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA               12532 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses VP64-dCas9-VP64 and MS2-p65-HSF1 fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pPB-R1R2_EF1aVP64dCas9VP64_T2A_MS2p65HSF1-IRESbsdpA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12532)
  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 12532)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..12532
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..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        1474..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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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             2902..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             7102..7251
                     /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             7282..7335
                     /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      7342..7351
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             7351..7737
                     /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             7792..7812
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7828..8370
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             8395..8766
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     misc_feature    8791..9377
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8958..8975)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     9185..9204
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             9378..9776
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9780..10004
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(10025..10049)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   10231..10293
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10344..10364)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(10344..10362)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(10383..10399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10383..10399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10396..10418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10407..10423
                     /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(10431..10461)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10476..10497
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10614..10631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10785..11373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10865..10884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(11544..12404)
                     /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     12167..12186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12405..12509)
                     /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 gccgctggat ccggacgggc tgacgcattg gacgattttg atctggatat gctgggaagt
     7141 gacgccctcg atgattttga ccttgacatg cttggttcgg atgcccttga tgactttgac
     7201 ctcgacatgc tcggcagtga cgcccttgat gatttcgacc tggacatgct gattaactgt
     7261 acgggcggcg ggtccggagg agagggcaga ggaagtctcc taacatgcgg tgacgtggag
     7321 gagaatcctg gcccatgtac ggccaccatg gcttcaaact ttactcagtt cgtgctcgtg
     7381 gacaatggtg ggacagggga tgtgacagtg gctccttcta atttcgctaa tggggtggca
     7441 gagtggatca gctccaactc acggagccag gcctacaagg tgacatgcag cgtcaggcag
     7501 tctagtgccc agaagagaaa gtataccatc aaggtggagg tccccaaagt ggctacccag
     7561 acagtgggcg gagtcgaact gcctgtcgcc gcttggaggt cctacctgaa catggagctc
     7621 actatcccaa ttttcgctac caattctgac tgtgaactca tcgtgaaggc aatgcagggg
     7681 ctcctcaaag acggtaatcc tatcccttcc gccatcgccg ctaactcagg tatctacagc
     7741 gctggaggag gtggaagcgg aggaggagga agcggaggag gaggtagcgg acctaagaaa
     7801 aagaggaagg tggcggccgc tggatcccct tcagggcaga tcagcaacca ggccctggct
     7861 ctggccccta gctccgctcc agtgctggcc cagactatgg tgccctctag tgctatggtg
     7921 cctctggccc agccacctgc tccagcccct gtgctgaccc caggaccacc ccagtcactg
     7981 agcgctccag tgcccaagtc tacacaggcc ggcgagggga ctctgagtga agctctgctg
     8041 cacctgcagt tcgacgctga tgaggacctg ggagctctgc tggggaacag caccgatccc
     8101 ggagtgttca cagatctggc ctccgtggac aactctgagt ttcagcagct gctgaatcag
     8161 ggcgtgtcca tgtctcatag tacagccgaa ccaatgctga tggagtaccc cgaagccatt
     8221 acccggctgg tgaccggcag ccagcggccc cccgaccccg ctccaactcc cctgggaacc
     8281 agcggcctgc ctaatgggct gtccggagat gaagacttct caagcatcgc tgatatggac
     8341 tttagtgccc tgctgtcaca gatttcctct agtgggcagg gaggaggtgg aagcggcttc
     8401 agcgtggaca ccagtgccct gctggacctg ttcagcccct cggtgaccgt gcccgacatg
     8461 agcctgcctg accttgacag cagcctggcc agtatccaag agctcctgtc tccccaggag
     8521 ccccccaggc ctcccgaggc agagaacagc agcccggatt cagggaagca gctggtgcac
     8581 tacacagcgc agccgctgtt cctgctggac cccggctccg tggacaccgg gagcaacgac
     8641 ctgccggtgc tgtttgagct gggagagggc tcctacttct ccgaagggga cggcttcgcc
     8701 gaggacccca ccatctccct gctgacaggc tcggagcctc ccaaagccaa ggaccccact
     8761 gtctcctgta catgagaatt ccgccccccc cccctctccc tccccccccc ctaacgttac
     8821 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     8881 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     8941 tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga
     9001 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     9061 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     9121 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     9181 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     9241 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     9301 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     9361 ataatatggc cacaaccatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     9421 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     9481 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     9541 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     9601 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     9661 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     9721 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctgacgac
     9781 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     9841 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     9901 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     9961 ggaagtcaat agcaggcatg ctggggatgc ggtgggctct atggactagt ctcgagatat
    10021 ctagacccag ctttcttgta caaagtggta ctagttaaaa gttttgttac tttatagaag
    10081 aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga
    10141 atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa
    10201 ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacgcat gattatcttt
    10261 aacgtacgtc acaatatgat tatctttcta gggttaatct agtatacgcg tatgcggccg
    10321 cttaattaat ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
    10381 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
    10441 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
    10501 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
    10561 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
    10621 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
    10681 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    10741 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    10801 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    10861 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    10921 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    10981 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    11041 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    11101 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    11161 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    11221 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    11281 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    11341 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    11401 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    11461 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    11521 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    11581 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    11641 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    11701 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    11761 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    11821 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    11881 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    11941 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    12001 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    12061 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    12121 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    12181 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    12241 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    12301 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    12361 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    12421 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    12481 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//