LOCUS       sequence-354102-       13716 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 13716)
  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 13716)
  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_EYFP
FEATURES             Location/Qualifiers
     source          1..13716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          145..1161
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1085..1107
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1169..1188
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1258..1267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1264..5367
                     /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             1264..5367
                     /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             1264..5367
                     /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             5380..5400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5437..5461
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5491..5640
                     /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             5665..5685
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6115..6471
                     /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             6490..7059
                     /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(7084..7103)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7128..7176
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7408..7619
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7629..7638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7635..7637,7638..7640,7641..8354)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7680..7701)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7941..7960)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8288..8309
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    8358..8479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8395..8414)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8449..8468
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9242..9276
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9767..9783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9767..9783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9780..9802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9791..9807
                     /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(9815..9821,9822..9839,9840..9845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9860..9881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9998..10015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10169..10757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10249..10268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10928..11719,11720..11788))
                     /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     11551..11570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11789..11893)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11919..12374)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12056..12077)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12268..12287
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12501..12523
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12515..12532
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12516..12532
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12662..12724
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13145..13524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
       61 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      121 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
      181 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
      241 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
      301 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
      361 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
      421 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttttgc gctccgcagt
      481 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
      541 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
      601 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
      661 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
      721 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
      781 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
      841 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
      901 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
      961 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1021 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     1081 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     1141 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1201 tttggcaaag aatctagata ccctcgtaaa ggtctagagc tagaggatcg aacccttgcc
     1261 accatggaca agaagtactc cattgggctc gatatcggca caaacagcgt cggctgggcc
     1321 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
     1381 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc
     1441 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
     1501 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     1561 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     1621 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     1681 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     1741 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     1801 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     1861 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     1921 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     1981 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc
     2041 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     2101 gcccagatcg gcgaccagta cgcagacctt tttttagcgg caaagaacct gtcagacgcc
     2161 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     2221 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     2281 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     2341 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     2401 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     2461 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg
     2521 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag
     2581 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat
     2641 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag
     2701 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat
     2761 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca
     2821 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc
     2881 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt
     2941 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa
     3001 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa
     3061 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt
     3121 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac
     3181 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg
     3241 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc
     3301 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat
     3361 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt
     3421 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag
     3481 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc
     3541 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa
     3601 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac
     3661 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc
     3721 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat
     3781 catatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga
     3841 tccgataaaa atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg
     3901 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat
     3961 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg
     4021 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg
     4081 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag
     4141 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac
     4201 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa
     4261 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg
     4321 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac
     4381 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag
     4441 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat
     4501 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa
     4561 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg
     4621 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc
     4681 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc
     4741 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc
     4801 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc
     4861 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag
     4921 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc
     4981 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg
     5041 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga
     5101 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat
     5161 aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc
     5221 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca
     5281 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga
     5341 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg
     5401 tcgccaggga tccgtcgact tgacgcgtta atatcaacaa gtttgtacaa aaaagcaggc
     5461 tacaaagagg ccagcggttc cggacgggct gacgcattgg acgattttga tctggatatg
     5521 ctgggaagtg acgccctcga tgattttgac cttgacatgc ttggttcgga tgcccttgat
     5581 gactttgacc tcgacatgct cggcagtgac gcccttgatg atttcgacct ggacatgctg
     5641 attaactcta gaagttccgg atctccgaaa aagaaacgca aagttggtag ccagtacctg
     5701 cccgacaccg acgaccggca ccggatcgag gaaaagcgga agcggaccta cgagacattc
     5761 aagagcatca tgaagaagtc ccccttcagc ggccccaccg accctagacc tccacctaga
     5821 agaatcgccg tgcccagcag atccagcgcc agcgtgccaa aacctgcccc ccagccttac
     5881 cccttcacca gcagcctgag caccatcaac tacgacgagt tccctaccat ggtgttcccc
     5941 agcggccaga tctctcaggc ctctgctctg gctccagccc ctcctcaggt gctgcctcag
     6001 gctcctgctc ctgcaccagc tccagccatg gtgtctgcac tggctcaggc accagcaccc
     6061 gtgcctgtgc tggctcctgg acctccacag gctgtggctc caccagcccc taaacctaca
     6121 caggccggcg agggcacact gtctgaagct ctgctgcagc tgcagttcga cgacgaggat
     6181 ctgggagccc tgctgggaaa cagcaccgat cctgccgtgt tcaccgacct ggccagcgtg
     6241 gacaacagcg agttccagca gctgctgaac cagggcatcc ctgtggcccc tcacaccacc
     6301 gagcccatgc tgatggaata ccccgaggcc atcacccggc tcgtgacagg cgctcagagg
     6361 cctcctgatc cagctcctgc ccctctggga gcaccaggcc tgcctaatgg actgctgtct
     6421 ggcgacgagg acttcagctc tatcgccgat atggatttct cagccttgct gggctctggc
     6481 agcggcagcc gggattccag ggaagggatg tttttgccga agcctgaggc cggctccgct
     6541 attagtgacg tgtttgaggg ccgcgaggtg tgccagccaa aacgaatccg gccatttcat
     6601 cctccaggaa gtccatgggc caaccgccca ctccccgcca gcctcgcacc aacaccaacc
     6661 ggtccagtac atgagccagt cgggtcactg accccggcac cagtccctca gccactggat
     6721 ccagcgcccg cagtgactcc cgaggccagt cacctgttgg aggatcccga tgaagagacg
     6781 agccaggctg tcaaagccct tcgggagatg gccgatactg tgattcccca gaaggaagag
     6841 gctgcaatct gtggccaaat ggacctttcc catccgcccc caaggggcca tctggatgag
     6901 ctgacaacca cacttgagtc catgaccgag gatctgaacc tggactcacc cctgaccccg
     6961 gaattgaacg agattctgga taccttcctg aacgacgagt gcctcttgca tgccatgcat
     7021 atcagcacag gactgtccat cttcgacaca tctctgtttt gagtttaaac gggggaggct
     7081 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     7141 acagaataaa acgcacgggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     7201 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     7261 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     7321 gggcggcagg ccctgccata gcagatctgc gaaatcggat ccgcggccgc actgattcta
     7381 gctgcagagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     7441 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     7501 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     7561 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     7621 atcctcgagc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     7681 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     7741 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     7801 tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc gcccgctacc
     7861 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     7921 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     7981 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     8041 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     8101 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     8161 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     8221 tgcccgacaa ccactacctg agctaccagt ccaagctgag caaagacccc aacgagaagc
     8281 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     8341 agctgtacaa gtaagttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     8401 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     8461 aactcatcaa tgtatcttat catgtctgga attgactcaa atgatgtcaa ttagtctatc
     8521 agaagctcat ctggtctccc ttccggggga caagacatcc ctgtttaata tttaaacagc
     8581 agtgttccca aactgggttc ttatatccct tgctctggtc aaccaggttg cagggtttcc
     8641 tgtcctcaca ggaacgaagt ccctaaagaa acagtggcag ccaggtttag ccccggaatt
     8701 gactggattc cttttttagg gcccattggt atggcttttt ccccgtatcc ccccaggtgt
     8761 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
     8821 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     8881 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     8941 tgggggcttt gggggggggc tgtccctgat atctataaca agaaaatata tatataataa
     9001 gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa
     9061 agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc
     9121 agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg
     9181 tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat
     9241 gcatgcgtca attttacgca gactatcttt ctagggttaa tctagctgca tcaggatcat
     9301 atcgtcgggt cttttttccg gctcagtcat cgcccaagct ggcgctatct gggcatcggg
     9361 gaggaagaag cccgtgcctt ttcccgcgag gttgaagcgg catggaaaga gtttgccgag
     9421 gatgactgct gctgcattga cgttgagcga aaacgcacgt ttaccatgat gattcgggaa
     9481 ggtgtggcca tgcacgcctt taacggtgaa ctgttcgttc aggccacctg ggataccagt
     9541 tcgtcgcggc ttttccggac acagttccgg atggtcagcc cgaagcgcat cagcaacccg
     9601 aacaataccg gcgacagccg gaactgccgt gccggtgtgc agattaatga cagcggtgcg
     9661 gcgctgggat attacgtcag cgaggacggg tatcctggct ggatgccgca gaaatggaca
     9721 tggatacccc gtgagttacc cggcgggcgc gcttggcgta atcatggtca tagctgtttc
     9781 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     9841 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     9901 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     9961 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
    10021 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
    10081 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
    10141 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
    10201 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
    10261 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
    10321 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
    10381 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
    10441 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
    10501 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
    10561 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
    10621 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
    10681 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
    10741 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
    10801 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
    10861 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
    10921 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
    10981 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
    11041 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
    11101 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
    11161 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
    11221 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
    11281 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
    11341 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
    11401 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
    11461 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
    11521 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
    11581 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
    11641 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
    11701 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
    11761 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
    11821 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
    11881 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
    11941 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
    12001 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
    12061 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
    12121 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
    12181 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
    12241 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
    12301 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
    12361 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
    12421 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
    12481 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc
    12541 ctcgttcatt cacgtttttg aacccgtgga ggacgggcag actcgcggtg caaatgtgtt
    12601 ttacagcgtg atggagcaga tgaagatgct cgacacgctg cagaacacgc agctagatta
    12661 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgtgt aaaattgacg
    12721 catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt
    12781 ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat
    12841 ttatttatta aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttatg
    12901 agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg
    12961 cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg
    13021 gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac
    13081 gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa aggcctccac
    13141 ggccgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
    13201 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
    13261 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
    13321 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
    13381 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
    13441 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
    13501 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
    13561 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
    13621 gcgatggggg cggggggggg ggggggggcg cgcccgcggg gggggggggg gggggggggg
    13681 gggggggggg gggggggggg gggggggggg gggggg
//
