LOCUS       sequence-354101-       13715 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 13715)
  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 13715)
  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_EBFP2
FEATURES             Location/Qualifiers
     source          1..13715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          214..1230
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1154..1176
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1238..1257
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1327..1336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1333..5436
                     /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             1333..5436
                     /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             1333..5436
                     /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             5449..5469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5506..5530
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5560..5709
                     /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             5734..5754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6184..6540
                     /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             6559..7128
                     /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(7153..7172)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7197..7245
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7477..7688
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7698..7707
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7704..7706,7707..7709,7710..8423)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(7749..7770)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    8427..8548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8464..8483)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8518..8537
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9311..9345
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9836..9852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9836..9852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9849..9871)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9860..9876
                     /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(9884..9890,9891..9908,9909..9914))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9929..9950
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10067..10084)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10238..10826)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10318..10337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10997..11788,11789..11857))
                     /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     11620..11639
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11858..11962)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11988..12443)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12125..12146)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12337..12356
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12570..12592
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12584..12601
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12585..12601
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12731..12793
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13214..13593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tttttttttt ttttttaatt gtttgggggg gggggggggg gggggggggg gggggggcgc
       61 gcgcgggggg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
      121 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
      181 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
      241 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      301 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
      361 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
      421 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
      481 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttttgcg
      541 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
      601 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
      661 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
      721 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
      781 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg
      841 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
      901 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
      961 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     1021 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     1081 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1141 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     1201 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     1261 tctcatcatt ttggcaaaga atctagatac cctcgtaaag gtctagagct agcggatcga
     1321 acccttgcca ccatggacaa gaagtactcc attgggctcg atatcggcac aaacagcgtc
     1381 ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc
     1441 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg
     1501 gagacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag
     1561 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct
     1621 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac
     1681 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat
     1741 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc
     1801 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca
     1861 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc
     1921 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg
     1981 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc
     2041 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc
     2101 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac
     2161 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttagcggc aaagaacctg
     2221 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg
     2281 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag
     2341 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa
     2401 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt
     2461 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa
     2521 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg
     2581 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac
     2641 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc
     2701 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg
     2761 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact
     2821 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag
     2881 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag
     2941 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac
     3001 cggaaagtta ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac
     3061 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat
     3121 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt
     3181 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg
     3241 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat
     3301 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga
     3361 aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt catgcagttg
     3421 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag
     3481 ggggacagtc ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga
     3541 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc
     3601 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac
     3661 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt
     3721 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg
     3781 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac
     3841 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg
     3901 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc
     3961 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag
     4021 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc
     4081 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat
     4141 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt
     4201 actctgaagt ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga
     4261 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca
     4321 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac
     4381 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac
     4441 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag
     4501 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag
     4561 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa
     4621 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc
     4681 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct
     4741 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa
     4801 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa
     4861 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt
     4921 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt
     4981 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg
     5041 tatctggcca gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag
     5101 ctgttcgtgg aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc
     5161 tccaaaagag tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag
     5221 cacagggata agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc
     5281 aacttgggcg cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac
     5341 acctctacaa aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat
     5401 gaaacaagaa tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag
     5461 aggaaggtgt cgccagggat ccgtcgactt gacgcgttaa tatcaacaag tttgtacaaa
     5521 aaagcaggct acaaagaggc cagcggttcc ggacgggctg acgcattgga cgattttgat
     5581 ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat
     5641 gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg
     5701 gacatgctga ttaactctag aagttccgga tctccgaaaa agaaacgcaa agttggtagc
     5761 cagtacctgc ccgacaccga cgaccggcac cggatcgagg aaaagcggaa gcggacctac
     5821 gagacattca agagcatcat gaagaagtcc cccttcagcg gccccaccga ccctagacct
     5881 ccacctagaa gaatcgccgt gcccagcaga tccagcgcca gcgtgccaaa acctgccccc
     5941 cagccttacc ccttcaccag cagcctgagc accatcaact acgacgagtt ccctaccatg
     6001 gtgttcccca gcggccagat ctctcaggcc tctgctctgg ctccagcccc tcctcaggtg
     6061 ctgcctcagg ctcctgctcc tgcaccagct ccagccatgg tgtctgcact ggctcaggca
     6121 ccagcacccg tgcctgtgct ggctcctgga cctccacagg ctgtggctcc accagcccct
     6181 aaacctacac aggccggcga gggcacactg tctgaagctc tgctgcagct gcagttcgac
     6241 gacgaggatc tgggagccct gctgggaaac agcaccgatc ctgccgtgtt caccgacctg
     6301 gccagcgtgg acaacagcga gttccagcag ctgctgaacc agggcatccc tgtggcccct
     6361 cacaccaccg agcccatgct gatggaatac cccgaggcca tcacccggct cgtgacaggc
     6421 gctcagaggc ctcctgatcc agctcctgcc cctctgggag caccaggcct gcctaatgga
     6481 ctgctgtctg gcgacgagga cttcagctct atcgccgata tggatttctc agccttgctg
     6541 ggctctggca gcggcagccg ggattccagg gaagggatgt ttttgccgaa gcctgaggcc
     6601 ggctccgcta ttagtgacgt gtttgagggc cgcgaggtgt gccagccaaa acgaatccgg
     6661 ccatttcatc ctccaggaag tccatgggcc aaccgcccac tccccgccag cctcgcacca
     6721 acaccaaccg gtccagtaca tgagccagtc gggtcactga ccccggcacc agtccctcag
     6781 ccactggatc cagcgcccgc agtgactccc gaggccagtc acctgttgga ggatcccgat
     6841 gaagagacga gccaggctgt caaagccctt cgggagatgg ccgatactgt gattccccag
     6901 aaggaagagg ctgcaatctg tggccaaatg gacctttccc atccgccccc aaggggccat
     6961 ctggatgagc tgacaaccac acttgagtcc atgaccgagg atctgaacct ggactcaccc
     7021 ctgaccccgg aattgaacga gattctggat accttcctga acgacgagtg cctcttgcat
     7081 gccatgcata tcagcacagg actgtccatc ttcgacacat ctctgttttg agtttaaacg
     7141 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     7201 aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc
     7261 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     7321 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     7381 ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg aaatcggatc cgcggccgca
     7441 ctgattctag ctgcagaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     7501 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     7561 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     7621 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     7681 gaacacagga tcctcgagcc accatggtga gcaagggcga ggagctgttc accggggtgg
     7741 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgaggggcg
     7801 agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca
     7861 agctgcccgt gccctggccc accctcgtga ccaccctgag ccacggcgtg cagtgcttcg
     7921 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     7981 acgtccagga gcgcaccatc ttcttcaagg acgacggcac ctacaagacc cgcgccgagg
     8041 tgaagttcga gggcgacacc ctagtgaacc gcatcgagct gaagggcgtc gacttcaagg
     8101 aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacatctata
     8161 tcatggccgt caagcagaag aacggcatca aggtgaactt caagatccgc cacaacgtgg
     8221 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     8281 ccgtgctgct gcccgacagc cactacctga gcacccagtc cgtgctgagc aaagacccca
     8341 acgagaagcg cgatcacatg gtcctgctgg agttccgcac cgccgccggg atcactctcg
     8401 gcatggacga gctgtacaag taagttaact tgtttattgc agcttataat ggttacaaat
     8461 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     8521 gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat
     8581 tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat
     8641 ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc
     8701 agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc
     8761 cccggaattg actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc
     8821 cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc
     8881 caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg
     8941 ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa
     9001 ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat
     9061 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     9121 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     9181 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     9241 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     9301 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat
     9361 caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg
     9421 ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag
     9481 tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg
     9541 attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg
     9601 gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc
     9661 agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac
     9721 agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag
     9781 aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat
     9841 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     9901 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     9961 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    10021 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    10081 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    10141 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    10201 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    10261 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    10321 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    10381 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    10441 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    10501 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    10561 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    10621 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    10681 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    10741 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    10801 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    10861 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    10921 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    10981 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    11041 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    11101 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    11161 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    11221 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    11281 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    11341 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    11401 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    11461 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    11521 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    11581 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    11641 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    11701 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    11761 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    11821 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    11881 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    11941 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
    12001 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
    12061 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
    12121 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
    12181 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
    12241 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
    12301 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
    12361 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
    12421 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
    12481 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
    12541 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
    12601 tgagcgcgcc tcgttcattc acgtttttga acccgtggag gacgggcaga ctcgcggtgc
    12661 aaatgtgttt tacagcgtga tggagcagat gaagatgctc gacacgctgc agaacacgca
    12721 gctagattaa ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgtgta
    12781 aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga
    12841 tattaagttt tattatattt acacttacat actaataata aattcaacaa acaatttatt
    12901 tatgtttatt tatttattaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
    12961 acttttatga gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
    13021 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
    13081 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
    13141 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
    13201 ggcctccacg gccgacattg attattgact agttattaat agtaatcaat tacggggtca
    13261 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
    13321 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
    13381 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
    13441 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
    13501 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
    13561 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
    13621 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
    13681 ttttgtgcag cgatgggggc gggggggggg ggggg
//
