LOCUS       sequence-216918-       12207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the drug-preservable Cas9-NS3-NLS/VPR, which can be used to 
            activate gene expression when combined with an NS3 inhibitor and 
            appropriately designed sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12207)
  AUTHORS   Tague EP, Dotson HL, Tunney SN, Sloas DC, Ngo JT
  TITLE     Chemogenetic control of gene expression and cell signaling with 
            antiviral drugs.
  JOURNAL   Nat Methods. 2018 Jul;15(7):519-522. doi: 10.1038/s41592-018-0042-y.
            Epub 2018 Jul 2.
  PUBMED    29967495
REFERENCE   2  (bases 1 to 12207)
  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"; doi: 
            "10.1038/s41592-018-0042-y"; journalName: "Nat Methods"; date: 
            "2018-07"; volume: "15"; issue: "7"; pages: "519-522"
COMMENT     Sequence Label: dCas9-NS3-NLS/VPR
FEATURES             Location/Qualifiers
     source          1..12207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(923..949,950..952,953..3436,3437..3442,3443..3508,
                     3509..3511,3512..5026)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5873..5899
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5915..5935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5972..5996
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6026..6175
                     /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             6200..6220
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6650..7006
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             7025..7594
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     primer_bind     complement(7629..7646)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7635..7859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7905..8333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7992..8011)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8202..8223
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8342..8362)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8347..8676
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8527..8662
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8589..8608
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             8725..9750
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             8725..9750
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    9880..10001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9917..9936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9971..9990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10050..10066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10050..10066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10063..10085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10074..10090
                     /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(10098..10104,10105..10122,10123..10128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10143..10164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10281..10298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10452..11040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10532..10551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11211..12002,12003..12071))
                     /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     11834..11853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12072..12176)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggacaa gaagtactcc attgggctcg ctatcggcac
      961 aaacagcgtc ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa
     1021 agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt
     1081 cgactccggg gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac
     1141 ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt
     1201 ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca
     1261 cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc
     1321 aaccatatat catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt
     1381 gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga
     1441 cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa
     1501 tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag
     1561 cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa
     1621 gaagaacggc ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa
     1681 atctaacttc gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga
     1741 tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc
     1801 aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac
     1861 caaagctccg ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac
     1921 tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga
     1981 tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta
     2041 caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct
     2101 taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca
     2161 gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt
     2221 gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg
     2281 ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat
     2341 cactccctgg aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga
     2401 aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct
     2461 gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg
     2521 gatgagaaag ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt
     2581 caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga
     2641 atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac
     2701 gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga
     2761 ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga
     2821 agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag
     2881 gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa
     2941 gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt
     3001 catgcagttg atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt
     3061 ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat
     3121 caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag
     3181 gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg
     3241 acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc
     3301 ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct
     3361 gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct
     3421 ctccgactac gacgtggctg ctatcgtgcc ccagtctttt ctcaaagatg attctattga
     3481 taataaagtg ttgacaagat ccgataaagc tagagggaag agtgataacg tcccctcaga
     3541 agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac
     3601 acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa
     3661 agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca
     3721 aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt
     3781 gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta
     3841 taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt
     3901 aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta
     3961 taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac
     4021 cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc
     4081 caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt
     4141 gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa
     4201 catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa
     4261 aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg
     4321 attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa
     4381 gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag
     4441 cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga
     4501 cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat
     4561 gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt
     4621 taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga
     4681 gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat
     4741 aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc
     4801 ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt
     4861 tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag
     4921 aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac
     4981 ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgacat
     5041 ggccagcatg actggtggac agcaaatggg gtcgacggag gacgtggtgt gctgccactc
     5101 aatctacggc aagaagaagg gtgatatcga cacctaccga tacataggct cttccgggac
     5161 aggctgcgtg gtcatagtgg gcaggatcgt cttgtccgga tccggcacta gtgcgcccat
     5221 cacggcgtac gcccagcaga cgagaggcct cctagggtgt ataatcacca gcctgactgg
     5281 ccgggacaaa aaccaagtgg agggtgaggt ccagatcgtg tcaactgcta cccaaacctt
     5341 cctggcaacg tgcatcaatg gggtatgctg ggcagtctac cacggggccg gaacgaggac
     5401 catcgcatca cccaagggtc ctgtcatcca gatgtatacc aatgtggacc aagaccttgt
     5461 gggctggccc gctcctcaag gttcccgctc attgacaccc tgtacctgcg gctcctcgga
     5521 cctttacctg gtcacgaggc acgccgatgt cattcccgtg cgccggcgag gtgatagcag
     5581 gggtagcctg ctttcgcccc ggcccatttc ctacttgaaa ggctcctcgg ggggtccgct
     5641 gttgtgcccc gcgggacacg ccgtgggcct attcagggcc gcggtgtgca cccgtggagt
     5701 ggctaaagcg gtggacttta tccctgtgga gaacctagag acaaccatga gatccccggt
     5761 gttcacggac aactcctctc caccagcagt caccctgacg cacccaatca ccaaaatcga
     5821 tagggaggtt ctctaccagg agttcgatga gatggaagag tgctctcagc actatcccta
     5881 cgatgtgccc gattacgctg gcgcgtctgc atgccccaag aagaagagga aggtgtcgcc
     5941 agggatccgt cgacttgacg cgttgatatc aacaagtttg tacaaaaaag caggctacaa
     6001 agaggccagc ggttccggac gggctgacgc attggacgat tttgatctgg atatgctggg
     6061 aagtgacgcc ctcgatgatt ttgaccttga catgcttggt tcggatgccc ttgatgactt
     6121 tgacctcgac atgctcggca gtgacgccct tgatgatttc gacctggaca tgctgattaa
     6181 ctctagaagt tccggatctc cgaaaaagaa acgcaaagtt ggtagccagt acctgcccga
     6241 caccgacgac cggcaccgga tcgaggaaaa gcggaagcgg acctacgaga cattcaagag
     6301 catcatgaag aagtccccct tcagcggccc caccgaccct agacctccac ctagaagaat
     6361 cgccgtgccc agcagatcca gcgccagcgt gccaaaacct gccccccagc cttacccctt
     6421 caccagcagc ctgagcacca tcaactacga cgagttccct accatggtgt tccccagcgg
     6481 ccagatctct caggcctctg ctctggctcc agcccctcct caggtgctgc ctcaggctcc
     6541 tgctcctgca ccagctccag ccatggtgtc tgcactggct caggcaccag cacccgtgcc
     6601 tgtgctggct cctggacctc cacaggctgt ggctccacca gcccctaaac ctacacaggc
     6661 cggcgagggc acactgtctg aagctctgct gcagctgcag ttcgacgacg aggatctggg
     6721 agccctgctg ggaaacagca ccgatcctgc cgtgttcacc gacctggcca gcgtggacaa
     6781 cagcgagttc cagcagctgc tgaaccaggg catccctgtg gcccctcaca ccaccgagcc
     6841 catgctgatg gaataccccg aggccatcac ccggctcgtg acaggcgctc agaggcctcc
     6901 tgatccagct cctgcccctc tgggagcacc aggcctgcct aatggactgc tgtctggcga
     6961 cgaggacttc agctctatcg ccgatatgga tttctcagcc ttgctgggct ctggcagcgg
     7021 cagccgggat tccagggaag ggatgttttt gccgaagcct gaggccggct ccgctattag
     7081 tgacgtgttt gagggccgcg aggtgtgcca gccaaaacga atccggccat ttcatcctcc
     7141 aggaagtcca tgggccaacc gcccactccc cgccagcctc gcaccaacac caaccggtcc
     7201 agtacatgag ccagtcgggt cactgacccc ggcaccagtc cctcagccac tggatccagc
     7261 gcccgcagtg actcccgagg ccagtcacct gttggaggat cccgatgaag agacgagcca
     7321 ggctgtcaaa gcccttcggg agatggccga tactgtgatt ccccagaagg aagaggctgc
     7381 aatctgtggc caaatggacc tttcccatcc gcccccaagg ggccatctgg atgagctgac
     7441 aaccacactt gagtccatga ccgaggatct gaacctggac tcacccctga ccccggaatt
     7501 gaacgagatt ctggatacct tcctgaacga cgagtgcctc ttgcatgcca tgcatatcag
     7561 cacaggactg tccatcttcg acacatctct gttttgatct agagggcccg tttaaacccg
     7621 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     7681 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     7741 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     7801 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     7861 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     7921 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     7981 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     8041 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     8101 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     8161 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     8221 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     8281 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     8341 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     8401 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     8461 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     8521 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     8581 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     8641 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     8701 attttcggat ctgatcagca cgtgatgaaa aagcctgaac tcaccgcgac gtctgtcgag
     8761 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     8821 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     8881 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     8941 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     9001 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     9061 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     9121 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     9181 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     9241 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     9301 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     9361 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     9421 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     9481 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     9541 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     9601 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     9661 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     9721 cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga ttccaccgcc
     9781 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     9841 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     9901 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     9961 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
    10021 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
    10081 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
    10141 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
    10201 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
    10261 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
    10321 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
    10381 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
    10441 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
    10501 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
    10561 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
    10621 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
    10681 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
    10741 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
    10801 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
    10861 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
    10921 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
    10981 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
    11041 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
    11101 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
    11161 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
    11221 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
    11281 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
    11341 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
    11401 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
    11461 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
    11521 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
    11581 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
    11641 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
    11701 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
    11761 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
    11821 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
    11881 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
    11941 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
    12001 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
    12061 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
    12121 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
    12181 tttccccgaa aagtgccacc tgacgtc
//
