LOCUS       sequence-354044-       11488 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PiggyBac compatible plasmid expressing spCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11488)
  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 11488)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_iRFP713
FEATURES             Location/Qualifiers
     source          1..11488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     318..340
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     402..421
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      490..499
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             496..4599
                     /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             496..4599
                     /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             496..4599
                     /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             4612..4632
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4687..4706)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4731..4779
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5011..5222
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5232..5241
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5238..6188
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    6192..6313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6229..6248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6283..6302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7076..7110
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7601..7617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7601..7617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7614..7636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7625..7641
                     /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(7649..7655,7656..7673,7674..7679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7694..7715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7832..7849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8003..8591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8083..8102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8762..9553,9554..9622))
                     /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     9385..9404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9623..9727)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(9753..10208)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9890..9911)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10102..10121
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10335..10357
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10349..10366
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10350..10366
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   10496..10558
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        10979..11358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg
       61 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
      121 ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa
      181 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
      241 ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg
      301 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc
      361 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt
      421 gctgtctcat cattttggca aagaatctag ataccctcgt aaaggtctag agctagcgaa
      481 ttcgaatttg ccaccatgga caagaagtac tccattgggc tcgatatcgg cacaaacagc
      541 gtcggctggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg
      601 ggcaataccg atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc
      661 ggggagacgg ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga
      721 aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac
      781 tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc
      841 cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata
      901 tatcatctga ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat
      961 ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac
     1021 ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta caatcagctt
     1081 ttcgaagaga acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg
     1141 ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac
     1201 ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac
     1261 ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc
     1321 gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac
     1381 ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct
     1441 ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg
     1501 aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct
     1561 aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt
     1621 attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga
     1681 gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac
     1741 ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat
     1801 aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc
     1861 gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc
     1921 tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg
     1981 actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac
     2041 gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga
     2101 aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg
     2161 aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc
     2221 gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac
     2281 gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt
     2341 cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc
     2401 ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga
     2461 tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga caagcagagt
     2521 ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag
     2581 ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc
     2641 cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag
     2701 ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag
     2761 cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag
     2821 aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc
     2881 cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac
     2941 ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac
     3001 tacgacgtgg atcatatcgt gccccagtct tttctcaaag atgattctat tgataataaa
     3061 gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt
     3121 gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg
     3181 aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc
     3241 ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc
     3301 gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt
     3361 attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg
     3421 agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact
     3481 gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg
     3541 tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag
     3601 tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga
     3661 gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac
     3721 aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt
     3781 aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac
     3841 agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat
     3901 tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa
     3961 aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa
     4021 aaaaacccca tcgactttct cgaggcgaaa ggatataaag aggtcaaaaa agacctcatc
     4081 attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct
     4141 agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc
     4201 ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag
     4261 cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgaa
     4321 ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat
     4381 aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg
     4441 accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg
     4501 tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc
     4561 tatgaaacaa gaatcgacct ctctcagctc ggtggagaca gcagggctga ccccaagaag
     4621 aagaggaagg tgtgaaaggg ttcgatccct accggttagt aatgagttta aacgggggag
     4681 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     4741 aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4801 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     4861 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     4921 tcggggcggc aggccctgcc atagcagatc tgcgaaatcg gatccgcggc cgcactgatt
     4981 ctagctgcag aggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     5041 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     5101 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     5161 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     5221 aggatcctcg agccaccatg gcggaaggat ccgtcgccag gcagcctgac ctcttgacct
     5281 gcgacgatga gccgatccat atccccggtg ccatccaacc gcatggactg ctgctcgccc
     5341 tcgccgccga catgacgatc gttgccggca gcgacaacct tcccgaactc accggactgg
     5401 cgatcggcgc cctgatcggc cgctctgcgg ccgatgtctt cgactcggaa acgcacaacc
     5461 gtctgacgat cgccttggcc gagcccgggg cggccgtcgg agcaccgatc actgtcggct
     5521 tcacgatgcg aaaggacgca ggcttcatcg gctcctggca tcgccatgat cagctcatct
     5581 tcctcgagct cgagcctccc cagcgggacg tcgccgagcc gcaggcgttc ttccgccgca
     5641 ccaacagcgc catccgccgc ctgcaggccg ccgaaacctt ggaaagcgcc tgcgccgccg
     5701 cggcgcaaga ggtgcggaag attaccggct tcgatcgggt gatgatctat cgcttcgcct
     5761 ccgacttcag cggcgaagtg atcgcagagg atcggtgcgc cgaggtcgag tcaaaactag
     5821 gcctgcacta tcctgcctca accgtgccgg cgcaggcccg tcggctctat accatcaacc
     5881 cggtacggat cattcccgat atcaattatc ggccggtgcc ggtcacccca gacctcaatc
     5941 cggtcaccgg gcggccgatt gatcttagct tcgccatcct gcgcagcgta tcgcccgtcc
     6001 atctggagtt catgcgcaac ataggcatgc acggcacgat gtcgatctcg attttgcgcg
     6061 gcgagcgact gtggggattg atcgtttgcc atcaccgaac gccgtactac gtcgatctcg
     6121 atggccgcca agcctgcgag ctagtcgccc aggttctggc ctggcagatc ggcgtgatgg
     6181 aagagtgagt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     6241 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     6301 tcaatgtatc ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc
     6361 tcatctggtc tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt
     6421 cccaaactgg gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct
     6481 cacaggaacg aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg
     6541 attccttttt tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag
     6601 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
     6661 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
     6721 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg
     6781 ctttgggggg gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc
     6841 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     6901 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     6961 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     7021 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     7081 gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc
     7141 gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa
     7201 gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac
     7261 tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg
     7321 gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg
     7381 cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat
     7441 accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg
     7501 ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata
     7561 ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     7621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     7681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     7741 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7801 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     7861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     7921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     7981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     8041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     8101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     8161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     8221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     8281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     8341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     8401 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     8461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     8521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     8581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     8641 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     8701 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     8761 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     8821 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     8881 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     8941 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     9001 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     9061 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     9121 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     9181 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     9241 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     9301 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     9361 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     9421 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     9481 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     9541 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     9601 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     9661 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     9721 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     9781 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     9841 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     9901 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     9961 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
    10021 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
    10081 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
    10141 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
    10201 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
    10261 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
    10321 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt
    10381 cattcacgtt tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag
    10441 cgtgatggag cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta
    10501 gaaagataat catattgtga cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg
    10561 ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta agttttatta
    10621 tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt
    10681 attaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt tatgagggac
    10741 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
    10801 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
    10861 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
    10921 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaggcct ccacggccga
    10981 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
    11041 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
    11101 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
    11161 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
    11221 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
    11281 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
    11341 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
    11401 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
    11461 ggggcggggg gggggggggg gggggccc
//
