LOCUS       sequence-222619-       16301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9-VP160 expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16301)
  AUTHORS   Vo BT, Kwon JA, Li C, Finkelstein D, Xu B, Orr BA, Sherr CJ, Roussel
            MF
  TITLE     Mouse medulloblastoma driven by CRISPR activation of cellular Myc.
  JOURNAL   Sci Rep. 2018 Jun 7;8(1):8733. doi: 10.1038/s41598-018-24956-1.
  PUBMED    29880921
REFERENCE   2  (bases 1 to 16301)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2018-06-7"; volume: "8(1):8733. doi"; pages: " 
            10.1038/s41598-018-24956-1"
COMMENT     Sequence Label: pT2K-CAGGS-U6-stuffer-dCAs9-VP160-T2A-IRES-mCh...
FEATURES             Location/Qualifiers
     source          1..16301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(34..462)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     356..375
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(2499..2510,2511..2673)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     misc_RNA        complement(3092..3167)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5049..5297)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5107..5126)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5277..5297)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        5300..5679
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          5958..6978
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     6898..6920
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     6986..7005
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             7048..7074
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7078..7098
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(7108..7131,7132..7134,7135..9690,9691..9693,
                     9694..11208)
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(7108..7131,7132..7134,7135..9621,9622..9624,
                     9625..11208)
                     /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 H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             11212..11232
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11266..11649
                     /codon_start=1
                     /product="10 tandem repeats of the minimal activation 
                     domain of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP160
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS
                     DALDDFDLDMLGSDALDDFDLDML"
     CDS             11674..11727
                     /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"
     misc_feature    11762..12311
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(11906..11923)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     12133..12152
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             12314..13024
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(12459..12477)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     12727..12746
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(13043..13062)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    13108..13163
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(13162..13181)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    join(13831..14018,14019..14030)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(14064..14080)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14064..14080)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(14117..14137)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(14117..14135)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(14156..14172)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14156..14172)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14169..14191)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14180..14196
                     /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(14204..14210,14211..14228,14229..14234))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14249..14270
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14387..14404)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14558..15146)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14638..14657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15317..16108,16109..16177))
                     /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     15940..15959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16178..16282)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
      361 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
      421 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccaaatagta ggaattaccc acctgtacaa gtgctgaaaa cttggatgaa taagcccgtt
      721 tgccattttc tactgctatt ttaaatcttt tctgtatttg tctgcatttg tctttaccct
      781 tgaataaatg ttttagttgt tttttttcaa ttatgactgt gtttaacaga cattattaca
      841 ataatatgta atagtagtac acactattat tatgtaatag tacttgttga ctgtatttga
      901 gaactggacc gagtgagtgt tacgtcaccc attcaaaatg acttacttct ggctccaaca
      961 aaatgaagtt aattcagttg ccatttttca ctgtatggac atcgccgtgt tggagctaga
     1021 cgttgccaat aagcaagaaa gagccgagat gcgtcgagtc tgagtcaccg ttcctatggc
     1081 aacccctcta accaatcaga agtaagcttg ttggaagtcc acagcctacc acttgaaagc
     1141 gggctgcaca aaatctgtca aacgttttga acgttggatg tgagagcaca tacttttatt
     1201 aaggcatctg gttggtcagt ttataatatc aacaacttgg gctacagaaa agaaaagtta
     1261 ttacagaaat tatgattaac aagtacatgt taaataaaga ttttaatatg aatgccacca
     1321 ctggagcatt catgccattt ggagcttctt cctgtttgga tcactagaag gaggaggtca
     1381 ctcattacag ttctcatata cagtcgttgg ttggttggtt ggttggtaga ttgattgatt
     1441 gattgattga ttgattgatt gattgattga ttgattgatt gattgattga ttgattgatt
     1501 gattggtagt caaaataaga aataatttcc acagattcat tacagaaatg attaaatgca
     1561 tacataaaaa actggggggg ggggggatac aacaacacac ttaagtcaca tttgcctacg
     1621 taaagaaaag taaagaaaat caatagctat attttacatc tccttttttt gctgtctttt
     1681 aattagcctt gttttgctgt tatcttgatt gaaactgtaa cttcttcacc tgcttctttt
     1741 ctttgtaggt tttgccagcc cagaaacagg catggctgtg caaggccaga gcaccatgca
     1801 caatgcgctg catgtcttca tgaacggctc aatgtcctca gtccagggct cagccaacga
     1861 ccccatcttc ctccttcacc atgctttcat tgacaggtaa caaacacgtc atgacattag
     1921 actgcacagt ttttgacaaa gttcatacaa tctgttgttt atagctgcta caattagtga
     1981 agtttgtgaa tgtacttgga tgagcagcga aagatcaatt gagatcaatt gttagagttt
     2041 ggttgccctg cagagcaaag aacaaaaaat aatctggtgg ctttactgcg tgaggttatt
     2101 attggtggaa tagaaacaca aaacataatt gcatttattt gtttaatttt ttatcttatc
     2161 ttaactttca tcttgcatat ttgtttctta catcatttct agcatctttg agcgctggct
     2221 aagaactcat cagcctcccc ggtccatcta cccacgtacc aatgcaccaa ttggccacaa
     2281 tgacggctac tacatggtgc cattccttcc tctttatagg aatggagact acctcctgtc
     2341 caacaatgct cttggatacg agtacgccta cctgttggac ccaggtcatt gcacaacacc
     2401 agaaatgccc tctgatctgc aaaagacgtg aatatctgtt cagacaccca tatccactct
     2461 gttccacaca ggtcagaggt ttgtccagga gttcttgaca gaggtgtaaa aagtactcaa
     2521 aaattttact caagtgaaag tacaagtact tagggaaaat tttactcaat taaaagtaaa
     2581 agtatctggc tagaatctta cttgagtaaa agtaaaaaag tactccatta aaattgtact
     2641 tgagtattaa ggaagtaaaa gtaaaagcaa gaaagaaaac tagagattct tgtttaagct
     2701 tttaatctca aaaaacatta aatgaaatgc atacaaggtt ttatcctgct ttagaactgt
     2761 ttgtatttaa ttatcaaact ataagacaga caatctaatg ccagtacacg ctactcaaag
     2821 ttgtaaaacc tcagatttaa cttcagtaga agctgattct caaaattgtt agtgtcaagc
     2881 ctagctcttt tggggctgaa aagcaatcct gcagtgctga aaagcctctc acaggcagcc
     2941 gatgcgggaa gaggtgtatt agtcttgata gagaggctgc aaatagcagg aaacgtgagc
     3001 agagactccc tggtgtctga aacacaggcc agatgggccc tcgtcgacca attcccactc
     3061 ctttcaagac ctagctagcg aattcaaaaa agcaccgact cggtgccact ttttcaagtt
     3121 gataacggac tagccttatt ttaacttgct atttctagct ctaaaacaga gacgtacaaa
     3181 aaagagcaag aagctaaaaa agatttaaaa attattttta gcgcagttaa tggaacagga
     3241 actaaattta ccccaaaaat attacgtgaa tcaggatata acgttattga ggttgaagag
     3301 catgcatttg aagatgaaac atttaaaaat gttgtaaatc caaatccaga atttgatcct
     3361 gcatgaaaaa taccgcttga atatggtatt aaacatgatg cagatattat tattatgaat
     3421 gacccagatg ctgacagatt tggaatggca ataaaacatg atggtcattt tgtaagatta
     3481 gatggaaatc aaacaggacc aattttaatt gattgaaaat tatcaaatct aaaacgctta
     3541 aatagcattc caaaaaatcc ggctctatat tcaagttttg taacaagtga tttgggtgat
     3601 agaatcgctc atgaaaaata tggagttaat attgtaaaaa ctttaactgg atttaaatga
     3661 atgggtagag aaattgctaa agaagaagat aacggattaa attttgtttt tgcttatgaa
     3721 gaaagttatg gatatgtaat tgatgactca gctagagata aagatggaat acaagcttct
     3781 atattaatag cagaggctgc ttgattttat aaaaaacaaa ataaaacatt agtagactat
     3841 ttagaagatt tatttaaaga aatgggtgca tattacactt tcactttaaa cttgaatttt
     3901 aaaccagaag aaaagaaatt aaaaattgaa ccattaatga aatcattgag agcaacaccc
     3961 ttaactcaaa ttgctggact taaagttgtt aatgttgaag actacatcga tggaatgtat
     4021 aatatgccag gacaagactt actaaaattt tatttagaag ataagtcatg atttgctgtt
     4081 cgcccaagtg gaactgaacc taaactaaaa atttatttta taggtgttgg tgaatctgtt
     4141 caaaacgcta aagttaaagt agacgaaatt attaaagaat taaaattaaa aatgaatata
     4201 taggagaaaa aatgaaacta aacaaatata tagatcacac attattaaaa caagatgcta
     4261 cgaaagctga aattaaacaa ttatgtgatg aagcaattga atttgatttt gcaacagttt
     4321 gtgttaattc atattgaaca agctattgta aagaattatt aaaaggcaca aatgtaggaa
     4381 taacaaatgt tgtaggtttt cctctaggtg catgcacaac agctacaaaa gcattcgaag
     4441 tttctgaagc aattaaagat ggtgcaacag aaattgatat ggtattaaat attggtgcat
     4501 taaaagacaa aaattatgaa ttagttttag aagacatgaa agctgtaaaa aaagcagctg
     4561 gatcacatgt tgttaaatgt attatggaaa attgtttatt aacaaaagaa gaaatcatga
     4621 aagcttgtga aatagctgtt gaagctggat tagaatttgt taaaacatca acaggatttt
     4681 caaaatcagg tgcaacattt gaagatgtta aactaatgaa gtcagttgtt aaagacaatg
     4741 ctttagttaa agcagctggt ggagttagaa catttgaaga tgctcaaaaa atgattgaag
     4801 caggagctga ccgcttagga acaagtggtg gagtagctat tattaaaggt gaagaaaaca
     4861 acgcgagtta ctaaaactag cgttttttta ttttgctcat ttttattaaa agtttgcaaa
     4921 aaggaacata aaaattctaa ttattgatac taaagttatt aaaaagaaga ttttggttga
     4981 ttttataaag gtcatagaat ataatatttt agcatgtgta ttttgtgtgc tcatttacaa
     5041 ccgtctccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     5101 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     5161 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     5221 aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     5281 atgggaaata ggccctctcg acattgatta ttgactagtt attaatagta atcaattacg
     5341 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     5401 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     5461 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     5521 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     5581 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     5641 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     5701 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     5761 taattatttt gtgcagcgat gggggcgggg gggggggggg nsgcgcgccr ggsggggsgg
     5821 ggsggggsgr ggggsggggs ggggcgrggc ggagaggtgc ggcggcagcc aatcagagcg
     5881 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     5941 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     6001 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     6061 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     6121 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     6181 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     6241 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     6301 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     6361 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     6421 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     6481 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     6541 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     6601 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     6661 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     6721 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     6781 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     6841 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     6901 tcggcttctg gcgtgtgacc ggcggctcta gctagagcct ctgctaacca tgttcatgcc
     6961 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     7021 ggcaaagaat tcaccggtgc caccatgtac ccatacgatg ttccagatta cgcttcgccg
     7081 aagaaaaagc gcaaggtcga agcgtccgac aagaagtaca gcatcggcct ggccatcggc
     7141 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     7201 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     7261 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     7321 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     7381 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     7441 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     7501 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     7561 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     7621 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     7681 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     7741 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     7801 aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc
     7861 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     7921 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     7981 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     8041 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     8101 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     8161 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     8221 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     8281 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     8341 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     8401 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     8461 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     8521 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     8581 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     8641 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     8701 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     8761 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     8821 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     8881 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     8941 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     9001 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     9061 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     9121 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     9181 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     9241 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     9301 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     9361 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     9421 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     9481 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     9541 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     9601 ctgtccgact acgatgtgga cgccatcgtg cctcagagct ttctgaagga cgactccatc
     9661 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     9721 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     9781 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     9841 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     9901 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     9961 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
    10021 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
    10081 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
    10141 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
    10201 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
    10261 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
    10321 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
    10381 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
    10441 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
    10501 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
    10561 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
    10621 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
    10681 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
    10741 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
    10801 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
    10861 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
    10921 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
    10981 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
    11041 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
    11101 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
    11161 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacag ccccaagaag
    11221 aagagaaagg tggaggccag cgggccggcc ggatccgggc gcgccgacgc gctggacgat
    11281 ttcgatctcg acatgctggg ttctgatgcc ctcgatgact ttgacctgga tatgttggga
    11341 agcgacgcat tggatgactt tgatctggac atgctcggct ccgatgctct ggacgatttc
    11401 gatctcgata tgttagggtc agacgcactg gatgatttcg accttgatat gttgggaagc
    11461 gatgcccttg atgatttcga cctggacatg ctcggcagcg acgccctgga cgatttcgat
    11521 ctggacatgc tggggtccga tgccttggat gattttgact tggatatgct ggggagtgat
    11581 gccctggacg actttgacct ggacatgctg ggctccgatg cgctcgatga cttcgatttg
    11641 gatatgttgt atatcgatag atctggcagc ggagagggca gaggaagcct tctaacatgc
    11701 ggtgacgtgg aggagaatcc cggccctagg ctcgagcggg atcaattccg ccccccccct
    11761 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
    11821 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
    11881 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
    11941 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
    12001 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
    12061 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
    12121 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
    12181 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
    12241 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
    12301 acacgataat accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
    12361 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
    12421 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
    12481 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
    12541 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
    12601 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
    12661 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
    12721 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
    12781 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
    12841 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
    12901 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
    12961 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
    13021 gtagtcgagt ctagatatca gatctttttc cctctgccaa aaattatggg gacatcatga
    13081 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt
    13141 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac
    13201 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga
    13261 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
    13321 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
    13381 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
    13441 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc
    13501 agcccaagct tctgcgaaga tacggccacg ggtgctcttg atcctgtggc tgattttgga
    13561 ctgtgctgct cgcagctgct gatgaatcac atacttcctc cattttcttc cactgattga
    13621 ctgttataat ttccctaatt tccaggtcaa ggtgctgtgc attgtggtaa tagatgtgac
    13681 atgacgtcac ttccaaagga ccaatgaaca tgtctgacca atttcatata atgtgaaaac
    13741 gattttcata ggcagaataa ataacattta aattaaactg ggcatcagcg caattcaatt
    13801 ggtttggtaa tagcaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
    13861 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
    13921 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
    13981 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg gttcttgacc
    14041 ccctaccttc agcaagccca gcagatccac tagttctaga gcggccgcca ccgcggtgga
    14101 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
    14161 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
    14221 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
    14281 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    14341 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    14401 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    14461 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    14521 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    14581 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    14641 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    14701 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    14761 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    14821 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    14881 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    14941 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    15001 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    15061 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    15121 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    15181 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    15241 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    15301 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    15361 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    15421 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    15481 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    15541 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    15601 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    15661 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    15721 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    15781 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    15841 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    15901 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    15961 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    16021 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    16081 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    16141 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    16201 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    16261 ataaacaaat aggggttccg cgcacatttc cccgaaaagt g
//
