LOCUS       sequence-354104-       13909 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 13909)
  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 13909)
  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_Hygro
FEATURES             Location/Qualifiers
     source          1..13909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          102..1118
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1042..1064
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1126..1145
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1215..1224
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1221..5324
                     /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             1221..5324
                     /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             1221..5324
                     /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             5337..5357
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5394..5418
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5448..5597
                     /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             5622..5642
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6072..6428
                     /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             6447..7016
                     /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(7041..7060)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7085..7133
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7365..7576
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7586..7595
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7607..8626
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             7607..8626
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     polyA_signal    8630..8751
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8667..8686)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8721..8740
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9514..9548
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10039..10055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10039..10055)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10052..10074)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10063..10079
                     /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(10087..10093,10094..10111,10112..10117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10132..10153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10270..10287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10441..11029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10521..10540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11200..11991,11992..12060))
                     /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     11823..11842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12061..12165)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(12191..12646)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12328..12349)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12540..12559
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12773..12795
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12787..12804
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12788..12804
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12934..12996
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13417..13796
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
       61 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      121 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      181 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      241 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      301 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      361 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      421 tttttgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      481 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
      541 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
      601 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
      661 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
      721 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
      781 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
      841 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
      901 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
      961 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1021 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1081 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1141 ttgtgctgtc tcatcatttt ggcaaagaat ctagataccc tcgtaaaggt ctagagctag
     1201 cggatcgaac ccttgccacc atggacaaga agtactccat tgggctcgat atcggcacaa
     1261 acagcgtcgg ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag
     1321 ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg
     1381 actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc
     1441 gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg
     1501 atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg
     1561 agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa
     1621 ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga
     1681 tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc
     1741 tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc
     1801 agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg
     1861 ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga
     1921 agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat
     1981 ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg
     2041 atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt ttagcggcaa
     2101 agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca
     2161 aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt
     2221 tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc
     2281 agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca
     2341 aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta
     2401 acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga
     2461 ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga
     2521 aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc
     2581 ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca
     2641 ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa
     2701 ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc
     2761 tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga
     2821 tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca
     2881 agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat
     2941 gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt
     3001 atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg
     3061 acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag
     3121 aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc
     3181 gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc
     3241 agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca
     3301 tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt
     3361 ctggccaggg ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca
     3421 aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc
     3481 ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac
     3541 agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc
     3601 aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt
     3661 actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct
     3721 ccgactacga cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata
     3781 ataaagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag
     3841 aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac
     3901 aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag
     3961 ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa
     4021 ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga
     4081 aagttattac tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata
     4141 aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag
     4201 gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata
     4261 aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg
     4321 ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt acactggcca
     4381 atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt
     4441 gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca
     4501 tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa
     4561 ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat
     4621 tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt
     4681 ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct
     4741 tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc
     4801 tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc
     4861 tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta
     4921 atttcttgta tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc
     4981 agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa
     5041 gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt
     5101 acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta
     5161 ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa
     5221 agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg
     5281 ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacccca
     5341 agaagaagag gaaggtgtcg ccagggatcc gtcgacttga cgcgttaata tcaacaagtt
     5401 tgtacaaaaa agcaggctac aaagaggcca gcggttccgg acgggctgac gcattggacg
     5461 attttgatct ggatatgctg ggaagtgacg ccctcgatga ttttgacctt gacatgcttg
     5521 gttcggatgc ccttgatgac tttgacctcg acatgctcgg cagtgacgcc cttgatgatt
     5581 tcgacctgga catgctgatt aactctagaa gttccggatc tccgaaaaag aaacgcaaag
     5641 ttggtagcca gtacctgccc gacaccgacg accggcaccg gatcgaggaa aagcggaagc
     5701 ggacctacga gacattcaag agcatcatga agaagtcccc cttcagcggc cccaccgacc
     5761 ctagacctcc acctagaaga atcgccgtgc ccagcagatc cagcgccagc gtgccaaaac
     5821 ctgcccccca gccttacccc ttcaccagca gcctgagcac catcaactac gacgagttcc
     5881 ctaccatggt gttccccagc ggccagatct ctcaggcctc tgctctggct ccagcccctc
     5941 ctcaggtgct gcctcaggct cctgctcctg caccagctcc agccatggtg tctgcactgg
     6001 ctcaggcacc agcacccgtg cctgtgctgg ctcctggacc tccacaggct gtggctccac
     6061 cagcccctaa acctacacag gccggcgagg gcacactgtc tgaagctctg ctgcagctgc
     6121 agttcgacga cgaggatctg ggagccctgc tgggaaacag caccgatcct gccgtgttca
     6181 ccgacctggc cagcgtggac aacagcgagt tccagcagct gctgaaccag ggcatccctg
     6241 tggcccctca caccaccgag cccatgctga tggaataccc cgaggccatc acccggctcg
     6301 tgacaggcgc tcagaggcct cctgatccag ctcctgcccc tctgggagca ccaggcctgc
     6361 ctaatggact gctgtctggc gacgaggact tcagctctat cgccgatatg gatttctcag
     6421 ccttgctggg ctctggcagc ggcagccggg attccaggga agggatgttt ttgccgaagc
     6481 ctgaggccgg ctccgctatt agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac
     6541 gaatccggcc atttcatcct ccaggaagtc catgggccaa ccgcccactc cccgccagcc
     6601 tcgcaccaac accaaccggt ccagtacatg agccagtcgg gtcactgacc ccggcaccag
     6661 tccctcagcc actggatcca gcgcccgcag tgactcccga ggccagtcac ctgttggagg
     6721 atcccgatga agagacgagc caggctgtca aagcccttcg ggagatggcc gatactgtga
     6781 ttccccagaa ggaagaggct gcaatctgtg gccaaatgga cctttcccat ccgcccccaa
     6841 ggggccatct ggatgagctg acaaccacac ttgagtccat gaccgaggat ctgaacctgg
     6901 actcacccct gaccccggaa ttgaacgaga ttctggatac cttcctgaac gacgagtgcc
     6961 tcttgcatgc catgcatatc agcacaggac tgtccatctt cgacacatct ctgttttgag
     7021 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     7081 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg
     7141 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     7201 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     7261 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgaa atcggatccg
     7321 ctgccgcact gattctagct gcagaggctc cggtgcccgt cagtgggcag agcgcacatc
     7381 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     7441 gtggcgcggg gtaaactggg aaagtgatgt cgcgtactgg ctccgccttt ttcccgaggg
     7501 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacaggtt
     7561 tgccgccaga acacaggatc ctcgagccac catggataga tccggaaagc ctgaactcac
     7621 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtttccg acctgatgca
     7681 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     7741 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     7801 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     7861 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     7921 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     7981 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     8041 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     8101 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     8161 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     8221 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     8281 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     8341 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     8401 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     8461 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     8521 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     8581 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataggtta acttgtttat
     8641 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     8701 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     8761 gaattgactc aaatgatgtc aattagtcta tcagaagctc atctggtctc ccttccgggg
     8821 gacaagacat ccctgtttaa tatttaaaca gcagtgttcc caaactgggt tcttatatcc
     8881 cttgctctgg tcaaccaggt tgcagggttt cctgtcctca caggaacgaa gtccctaaag
     8941 aaacagtggc agccaggttt agccccggaa ttgactggat tcctttttta gggcccattg
     9001 gtatggcttt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg
     9061 ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc
     9121 ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc
     9181 cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg gctgtccctg
     9241 atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     9301 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     9361 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     9421 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     9481 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     9541 ttctagggtt aatctagctg catcaggatc atatcgtcgg gtcttttttc cggctcagtc
     9601 atcgcccaag ctggcgctat ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg
     9661 aggttgaagc ggcatggaaa gagtttgccg aggatgactg ctgctgcatt gacgttgagc
     9721 gaaaacgcac gtttaccatg atgattcggg aaggtgtggc catgcacgcc tttaacggtg
     9781 aactgttcgt tcaggccacc tgggatacca gttcgtcgcg gcttttccgg acacagttcc
     9841 ggatggtcag cccgaagcgc atcagcaacc cgaacaatac cggcgacagc cggaactgcc
     9901 gtgccggtgt gcagattaat gacagcggtg cggcgctggg atattacgtc agcgaggacg
     9961 ggtatcctgg ctggatgccg cagaaatgga catggatacc ccgtgagtta cccggcgggc
    10021 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
    10081 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
    10141 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
    10201 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
    10261 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
    10321 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
    10381 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
    10441 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
    10501 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
    10561 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
    10621 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
    10681 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
    10741 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
    10801 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
    10861 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
    10921 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
    10981 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
    11041 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
    11101 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
    11161 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
    11221 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
    11281 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
    11341 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
    11401 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
    11461 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
    11521 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
    11581 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
    11641 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
    11701 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
    11761 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
    11821 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
    11881 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
    11941 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
    12001 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
    12061 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
    12121 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
    12181 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
    12241 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
    12301 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
    12361 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
    12421 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
    12481 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
    12541 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
    12601 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
    12661 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
    12721 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
    12781 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
    12841 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
    12901 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
    12961 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
    13021 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
    13081 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
    13141 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
    13201 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
    13261 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
    13321 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
    13381 cacctggggg gatacgggga aaaggcctcc acggccgaca ttgattattg actagttatt
    13441 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
    13501 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
    13561 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
    13621 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
    13681 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
    13741 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
    13801 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
    13861 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcgggggg
//
