LOCUS       sequence-354041-       12021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac compatible plasmid expressing spCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12021)
  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 12021)
  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_EYFP
FEATURES             Location/Qualifiers
     source          1..12021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          149..1165
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1089..1111
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1173..1192
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1261..1270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1267..5370
                     /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             1267..5370
                     /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             1267..5370
                     /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             5383..5403
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5458..5477)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5502..5550
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5782..5993
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6003..6012
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6009..6011,6012..6014,6015..6728)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6054..6075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6315..6334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6662..6683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6732..6853
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6769..6788)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6823..6842
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7616..7650
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8141..8157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8141..8157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8154..8176)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8165..8181
                     /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(8189..8195,8196..8213,8214..8219))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8234..8255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8372..8389)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8543..9131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8623..8642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9302..10093,10094..10162))
                     /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     9925..9944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10163..10267)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10293..10748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10430..10451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10642..10661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10875..10897
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10889..10906
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10890..10906
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   11036..11098
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        11519..11898
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
       61 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      121 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
      181 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      241 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg
      301 gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg
      361 cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg
      421 ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt ttgcgctccg
      481 cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga
      541 ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc
      601 gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
      661 ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
      721 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
      781 ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
      841 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
      901 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
      961 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
     1021 tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg
     1081 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt
     1141 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
     1201 tcattttggc aaagaatcta gataccctcg taaaggtcta gagctagcga attcgaattt
     1261 gccaccatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag cgtcggctgg
     1321 gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc
     1381 gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg
     1441 gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg
     1501 atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc
     1561 cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc
     1621 tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg
     1681 aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg
     1741 gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac
     1801 agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag
     1861 aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa
     1921 tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt
     1981 ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg
     2041 gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg
     2101 ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac
     2161 gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc
     2221 gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt
     2281 gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc
     2341 tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc
     2401 atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg
     2461 ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa
     2521 ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa
     2581 aagattgaga aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga
     2641 aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc
     2701 gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt
     2761 gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc
     2821 acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca
     2881 ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa
     2941 gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt
     3001 gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg
     3061 aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac
     3121 attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact
     3181 tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga
     3241 tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca
     3301 atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat
     3361 gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac
     3421 agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg
     3481 cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat
     3541 atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg
     3601 gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa
     3661 cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac
     3721 ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg
     3781 gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca
     3841 agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa
     3901 atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat
     3961 aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa
     4021 aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc
     4081 atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg
     4141 aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc
     4201 aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc
     4261 aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt
     4321 aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt
     4381 tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg
     4441 aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg
     4501 gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc
     4561 gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag
     4621 ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca
     4681 gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa
     4741 agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc
     4801 atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt
     4861 cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc
     4921 gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg
     4981 gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc
     5041 gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa
     5101 agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg
     5161 gataagccca tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg
     5221 ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
     5281 acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca
     5341 agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag
     5401 gtgtgaaagg gttcgatccc taccggttag taatgagttt aaacggggga ggctaactga
     5461 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     5521 taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     5581 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     5641 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     5701 caggccctgc catagcagat ctgcgaaatc ggatccgcgg ccgcactgat tctagctgca
     5761 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     5821 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     5881 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     5941 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggatcctc
     6001 gagccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     6061 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     6121 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     6181 ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg cttcgcccgc taccccgacc
     6241 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     6301 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     6361 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     6421 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     6481 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     6541 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     6601 acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     6661 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     6721 acaagtaagt taacttgttt attgcagctt ataatggtta caagtaaagc aatagcatca
     6781 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     6841 tcaatgtatc ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc
     6901 tcatctggtc tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt
     6961 cccaaactgg gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct
     7021 cacaggaacg aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg
     7081 attccttttt tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag
     7141 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
     7201 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
     7261 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg
     7321 ctttgggggg gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc
     7381 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     7441 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     7501 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     7561 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     7621 gtcaatttta cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc
     7681 gggtcttttt tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa
     7741 gaagcccgtg ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac
     7801 tgctgctgca ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg
     7861 gccatgcacg cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg
     7921 cggcttttcc ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat
     7981 accggcgaca gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg
     8041 ggatattacg tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata
     8101 ccccgtgagt tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     8161 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     8221 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     8281 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     8341 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     8401 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     8461 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     8521 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     8581 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     8641 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     8701 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     8761 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     8821 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     8881 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     8941 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     9001 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     9061 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     9121 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     9181 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     9241 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     9301 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     9361 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     9421 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     9481 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     9541 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     9601 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     9661 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     9721 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     9781 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     9841 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     9901 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     9961 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    10021 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    10081 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    10141 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    10201 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    10261 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
    10321 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
    10381 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
    10441 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
    10501 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
    10561 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
    10621 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
    10681 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
    10741 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
    10801 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
    10861 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt
    10921 cattcacgtt tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag
    10981 cgtgatggag cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta
    11041 gaaagataat catattgtga cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg
    11101 ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta agttttatta
    11161 tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt
    11221 attaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt tatgagggac
    11281 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
    11341 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
    11401 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
    11461 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaggcct ccacggccga
    11521 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
    11581 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
    11641 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
    11701 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
    11761 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
    11821 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
    11881 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
    11941 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
    12001 ggggcggggg gggggggggg g
//
