LOCUS       sequence-354108-       13544 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 13544)
  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 13544)
  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_Puro
FEATURES             Location/Qualifiers
     source          1..13544
                     /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"
     CDS             7635..8234
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7635..7654)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8131..8151
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    8253..8374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8290..8309)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8344..8363
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   9137..9171
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9662..9678)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9662..9678)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9675..9697)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9686..9702
                     /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(9710..9716,9717..9734,9735..9740))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9755..9776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9893..9910)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10064..10652)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10144..10163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10823..11614,11615..11683))
                     /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     11446..11465
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11684..11788)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11814..12269)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11951..11972)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12163..12182
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12396..12418
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12410..12427
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12411..12427
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   12557..12619
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        13040..13419
                     /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 tagcggatcg 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 caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     7681 tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     7741 ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     7801 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     7861 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     7921 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     7981 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     8041 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     8101 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     8161 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     8221 agcccggtgc ctgaatctag tgtcgacaag ttaacttgtt tattgcagct tataatggtt
     8281 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     8341 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggaattga ctcaaatgat
     8401 gtcaattagt ctatcagaag ctcatctggt ctcccttccg ggggacaaga catccctgtt
     8461 taatatttaa acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca
     8521 ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg
     8581 tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg
     8641 tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc
     8701 ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg
     8761 ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg
     8821 acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa
     8881 atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat
     8941 gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg
     9001 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
     9061 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
     9121 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag
     9181 ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc
     9241 tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg
     9301 aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc
     9361 atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc
     9421 acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag
     9481 cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt
     9541 aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg
     9601 ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat
     9661 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     9721 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     9781 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     9841 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     9901 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     9961 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
    10021 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
    10081 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
    10141 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
    10201 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
    10261 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
    10321 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
    10381 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
    10441 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
    10501 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
    10561 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
    10621 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
    10681 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
    10741 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
    10801 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
    10861 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
    10921 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
    10981 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
    11041 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
    11101 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
    11161 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
    11221 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
    11281 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
    11341 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
    11401 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
    11461 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
    11521 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
    11581 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
    11641 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
    11701 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
    11761 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
    11821 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
    11881 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
    11941 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
    12001 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
    12061 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
    12121 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
    12181 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
    12241 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
    12301 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
    12361 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
    12421 ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg ggcagactcg
    12481 cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag atgctcgaca cgctgcagaa
    12541 cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta aagataatca
    12601 tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt tattaatttg
    12661 aatagatatt aagttttatt atatttacac ttacatacta ataataaatt caacaaacaa
    12721 tttatttatg tttatttatt tattaaaaaa aacaaaaact caaaatttct tctataaagt
    12781 aacaaaactt ttatgaggga cagccccccc ccaaagcccc cagggatgta attacgtccc
    12841 tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc
    12901 cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat
    12961 cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg
    13021 ggaaaaggcc tccacggccg acattgatta ttgactagtt attaatagta atcaattacg
    13081 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
    13141 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
    13201 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
    13261 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
    13321 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
    13381 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
    13441 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
    13501 taattatttt gtgcagcgat gggggcgggg gggggggggg gggg
//
