LOCUS       sequence-354105-       13844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac compatible plasmid expressing Cas9-VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13844)
  AUTHORS   Nghi Dang , Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J 
            Cecchi, Joanna Buchthal, Alejandro Chavez, Nan Cher Yeo
  TITLE     Generation and application of a versatile CRISPR toolkit for 
            mammalian cell engineering
  JOURNAL   Synthetic Biology, Volume 6, Issue 1, 2021
REFERENCE   2  (bases 1 to 13844)
  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: "Synthetic 
            Biology, Volume 6, Issue 1, 2021"
COMMENT     Sequence Label: NAX_CAGGS_CAS9_VPR_iRFP713
FEATURES             Location/Qualifiers
     source          1..13844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          122..1138
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1062..1084
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1146..1165
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1235..1244
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1241..5344
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1241..5344
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1241..5344
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5357..5377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5414..5438
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5468..5617
                     /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             5642..5662
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6092..6448
                     /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             6467..7036
                     /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(7061..7080)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7105..7153
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7385..7596
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7606..7615
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7612..8562
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDSSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    8566..8687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8603..8622)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8657..8676
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9450..9484
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9975..9991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9975..9991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9988..10010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9999..10015
                     /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(10023..10029,10030..10047,10048..10053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10068..10089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10206..10223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10377..10965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10457..10476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11136..11927,11928..11996))
                     /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     11759..11778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11997..12101)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12127..12582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12264..12285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12476..12495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12709..12731
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12723..12740
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12724..12740
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12870..12932
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13353..13732
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
       61 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      121 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      181 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      241 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      301 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      361 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      421 ctgcgggcgc ggcgcggggc tttttgcgct ccgcagtgtg cgcgagggga gcgcggccgg
      481 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
      541 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
      601 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
      661 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
      721 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
      781 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
      841 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
      901 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
      961 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1021 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1081 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1141 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat ctagataccc
     1201 tcgtaaaggt ctagagctag aggatcgaac ccttgccacc atggacaaga agtactccat
     1261 tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc attacggacg agtacaaggt
     1321 gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct
     1381 cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac
     1441 agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag
     1501 taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt
     1561 ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc
     1621 gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga
     1681 taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca
     1741 cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca
     1801 actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga
     1861 cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc
     1921 acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg
     1981 gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag
     2041 caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc
     2101 agaccttttt ttagcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg
     2161 agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga
     2221 gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta
     2281 caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc
     2341 aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga
     2401 ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa
     2461 tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga
     2521 ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg
     2581 gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg
     2641 caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc
     2701 tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt
     2761 gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt
     2821 caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc
     2881 tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga
     2941 ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt
     3001 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct
     3061 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga
     3121 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt
     3181 catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat
     3241 caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg
     3301 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga
     3361 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct
     3421 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
     3481 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
     3541 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
     3601 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
     3661 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
     3721 actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct
     3781 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag
     3841 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     3901 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     3961 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     4021 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     4081 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     4141 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     4201 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga
     4261 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     4321 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     4381 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     4441 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct
     4501 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     4561 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     4621 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     4681 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     4741 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata
     4801 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga
     4861 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     4921 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     4981 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     5041 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct
     5101 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     5161 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     5221 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact
     5281 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     5341 agacagcagg gctgacccca agaagaagag gaaggtgtcg ccagggatcc gtcgacttga
     5401 cgcgttaata tcaacaagtt tgtacaaaaa agcaggctac aaagaggcca gcggttccgg
     5461 acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg ccctcgatga
     5521 ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg acatgctcgg
     5581 cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa gttccggatc
     5641 tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg accggcaccg
     5701 gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga agaagtcccc
     5761 cttcagcggc cccaccgacc ctagacctcc acctagaaga atcgccgtgc ccagcagatc
     5821 cagcgccagc gtgccaaaac ctgcccccca gccttacccc ttcaccagca gcctgagcac
     5881 catcaactac gacgagttcc ctaccatggt gttccccagc ggccagatct ctcaggcctc
     5941 tgctctggct ccagcccctc ctcaggtgct gcctcaggct cctgctcctg caccagctcc
     6001 agccatggtg tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc
     6061 tccacaggct gtggctccac cagcccctaa acctacacag gccggcgagg gcacactgtc
     6121 tgaagctctg ctgcagctgc agttcgacga cgaggatctg ggagccctgc tgggaaacag
     6181 caccgatcct gccgtgttca ccgacctggc cagcgtggac aacagcgagt tccagcagct
     6241 gctgaaccag ggcatccctg tggcccctca caccaccgag cccatgctga tggaataccc
     6301 cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag ctcctgcccc
     6361 tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact tcagctctat
     6421 cgccgatatg gatttctcag ccttgctggg ctctggcagc ggcagccggg attccaggga
     6481 agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt ttgagggccg
     6541 cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc catgggccaa
     6601 ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg agccagtcgg
     6661 gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag tgactcccga
     6721 ggccagtcac ctgttggagg atcccgatga agagacgagc caggctgtca aagcccttcg
     6781 ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg gccaaatgga
     6841 cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac ttgagtccat
     6901 gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga ttctggatac
     6961 cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac tgtccatctt
     7021 cgacacatct ctgttttgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa
     7081 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt
     7141 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     7201 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     7261 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca
     7321 gatctgcgaa atcggatccg cggccgcact gattctagct gcagaggctc cggtgcccgt
     7381 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     7441 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     7501 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     7561 gttctttttc gcaacgggtt tgccgccaga acacaggatc ctcgagccac catggcggaa
     7621 ggatccgtcg ccaggcagcc tgacctcttg acctgcgacg atgagccgat ccatatcccc
     7681 ggtgccatcc aaccgcatgg actgctgctc gccctcgccg ccgacatgac gatcgttgcc
     7741 ggcagcgaca accttcccga actcaccgga ctggcgatcg gcgccctgat cggccgctct
     7801 gcggccgatg tcttcgactc ggaaacgcac aaccgtctga cgatcgcctt ggccgagccc
     7861 ggggcggccg tcggagcacc gatcactgtc ggcttcacga tgcgaaagga cgcaggcttc
     7921 atcggctcct ggcatcgcca tgatcagctc atcttcctcg agctcgagcc tccccagcgg
     7981 gacgtcgccg agccgcaggc gttcttccgc cgcaccaaca gcgccatccg ccgcctgcag
     8041 gccgccgaaa ccttggaaag cgcctgcgcc gccgcggcgc aagaggtgcg gaagattacc
     8101 ggcttcgatc gggtgatgat ctatcgcttc gcctccgact ccagcggcga agtgatcgca
     8161 gaggatcggt gcgccgaggt cgagtcaaaa ctaggcctgc actatcctgc ctcaaccgtg
     8221 ccggcgcagg cccgtcggct ctataccatc aacccggtac ggatcattcc cgatatcaat
     8281 tatcggccgg tgccggtcac cccagacctc aatccggtca ccgggcggcc gattgatctt
     8341 agcttcgcca tcctgcgcag cgtatcgccc gtccatctgg agttcatgcg caacataggc
     8401 atgcacggca cgatgtcgat ctcgattttg cgcggcgagc gactgtgggg attgatcgtt
     8461 tgccatcacc gaacgccgta ctacgtcgat ctcgatggcc gccaagcctg cgagctagtc
     8521 gcccaggttc tggcctggca gatcggcgtg atggaagagt gagttaactt gtttattgca
     8581 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     8641 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggaat
     8701 tgactcaaat gatgtcaatt agtctatcag aagctcatct ggtctccctt ccgggggaca
     8761 agacatccct gtttaatatt taaacagcag tgttcccaaa ctgggttctt atatcccttg
     8821 ctctggtcaa ccaggttgca gggtttcctg tcctcacagg aacgaagtcc ctaaagaaac
     8881 agtggcagcc aggtttagcc ccggaattga ctggattcct tttttagggc ccattggtat
     8941 ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca
     9001 gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct
     9061 cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc
     9121 tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccctgatat
     9181 ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat
     9241 ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg
     9301 actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc
     9361 acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt
     9421 tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct
     9481 agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc tcagtcatcg
     9541 cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt cccgcgaggt
     9601 tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg ttgagcgaaa
     9661 acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta acggtgaact
     9721 gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac agttccggat
     9781 ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga actgccgtgc
     9841 cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg aggacgggta
     9901 tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg gcgggcgcgc
     9961 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
    10021 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
    10081 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
    10141 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
    10201 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
    10261 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
    10321 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
    10381 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
    10441 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
    10501 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
    10561 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
    10621 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
    10681 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
    10741 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
    10801 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
    10861 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
    10921 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
    10981 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
    11041 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
    11101 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
    11161 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
    11221 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
    11281 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
    11341 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
    11401 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
    11461 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
    11521 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
    11581 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
    11641 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
    11701 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
    11761 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
    11821 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
    11881 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
    11941 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
    12001 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
    12061 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
    12121 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
    12181 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
    12241 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
    12301 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
    12361 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
    12421 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
    12481 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
    12541 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
    12601 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
    12661 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
    12721 gttgtaaaac gacggccagt gagcgcgcct cgttcattca cgtttttgaa cccgtggagg
    12781 acgggcagac tcgcggtgca aatgtgtttt acagcgtgat ggagcagatg aagatgctcg
    12841 acacgctgca gaacacgcag ctagattaac cctagaaaga taatcatatt gtgacgtacg
    12901 ttaaagataa tcatgtgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt
    12961 atttattaat ttgaatagat attaagtttt attatattta cacttacata ctaataataa
    13021 attcaacaaa caatttattt atgtttattt atttattaaa aaaaacaaaa actcaaaatt
    13081 tcttctataa agtaacaaaa cttttatgag ggacagcccc cccccaaagc ccccagggat
    13141 gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt
    13201 ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa
    13261 ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc
    13321 tggggggata cggggaaaag gcctccacgg ccgacattga ttattgacta gttattaata
    13381 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
    13441 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
    13501 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
    13561 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
    13621 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
    13681 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
    13741 gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta
    13801 tttatttatt ttttaattat tttgtgcagc gatgggggcg gggg
//
