LOCUS       sequence-354038-       11978 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 11978)
  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 11978)
  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_EBFP2
FEATURES             Location/Qualifiers
     source          1..11978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          112..1128
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1052..1074
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1136..1155
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1224..1233
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1230..5333
                     /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             1230..5333
                     /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             1230..5333
                     /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             5346..5366
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5421..5440)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5465..5513
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        5745..5956
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5966..5975
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5972..5974,5975..5977,5978..6691)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(6017..6038)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    6695..6816
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6732..6751)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6786..6805
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7579..7613
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8104..8120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8104..8120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8117..8139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8128..8144
                     /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(8152..8158,8159..8176,8177..8182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8197..8218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8335..8352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8506..9094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8586..8605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9265..10056,10057..10125))
                     /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     9888..9907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10126..10230)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(10256..10711)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10393..10414)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     10605..10624
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10838..10860
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     10852..10869
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     10853..10869
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   10999..11061
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        11482..11861
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
       61 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
      121 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
      181 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
      241 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag
      301 gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg
      361 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
      421 ggcgcggggc tttttgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc
      481 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg
      541 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc
      601 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
      661 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
      721 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
      781 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
      841 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
      901 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
      961 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     1021 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     1081 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     1141 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat ctagataccc tcgtaaaggt
     1201 ctagagctag cgaattcgaa tttgccacca tggacaagaa gtactccatt gggctcgata
     1261 tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg ccgagcaaaa
     1321 aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc attggcgccc
     1381 tcctgttcga ctccggggag acggccgaag ccacgcggct caaaagaaca gcacggcgca
     1441 gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt aatgagatgg
     1501 ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg gaggaggata
     1561 aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg taccatgaaa
     1621 agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat aaggctgact
     1681 tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac ttcctcatcg
     1741 agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa ctggttcaga
     1801 cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac gccaaagcaa
     1861 tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca cagctccctg
     1921 gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg ctgaccccca
     1981 actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc aaagacacct
     2041 acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca gacctttttt
     2101 tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga gtgaacacgg
     2161 agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag caccaccaag
     2221 acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac aaggaaattt
     2281 tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca agccaggagg
     2341 aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag gagctgctgg
     2401 taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat ggaagcatcc
     2461 cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag gatttctacc
     2521 cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg ataccctact
     2581 atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc aaatcagaag
     2641 agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct gcccagtcct
     2701 tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg cttcctaaac
     2761 actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc aaatacgtca
     2821 cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct atcgtggacc
     2881 tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac tatttcaaaa
     2941 agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc aacgcatccc
     3001 tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg gacaatgagg
     3061 agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa gatagggaga
     3121 tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc atgaaacagc
     3181 tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc aatgggatcc
     3241 gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga tttgccaacc
     3301 ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac atccagaaag
     3361 cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt gcaggtagcc
     3421 cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc gtcaaagtaa
     3481 tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac caaactaccc
     3541 agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt ataaaagaac
     3601 tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag aatgagaagc
     3661 tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa ctggacatca
     3721 atcggctctc cgactacgac gtggatcata tcgtgcccca gtcttttctc aaagatgatt
     3781 ctattgataa taaagtgttg acaagatccg ataaaaatag agggaagagt gataacgtcc
     3841 cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg aacgccaaac
     3901 tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc ctgtctgagt
     3961 tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc accaagcacg
     4021 tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac aaactgattc
     4081 gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga aaggactttc
     4141 agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc tacctgaatg
     4201 cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa tttgtttacg
     4261 gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag gaaataggca
     4321 aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag accgagatta
     4381 cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga gaaacaggag
     4441 aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg tccatgccgc
     4501 aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag gaaagtatcc
     4561 tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac cccaagaaat
     4621 acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc aaagtggaga
     4681 aagggaagtc taaaaaactc aaaagcgtca aggaactgct gggcatcaca atcatggagc
     4741 gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat aaagaggtca
     4801 aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa aacggccgga
     4861 aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca ctgccctcta
     4921 aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg tctcccgaag
     4981 ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat gagatcatcg
     5041 agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc gataaggtgc
     5101 tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa aacattatcc
     5161 acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc gacaccacca
     5221 tagacagaaa gcggtacacc tctacaaagg aggtcctgga cgccacactg attcatcagt
     5281 caattacggg gctctatgaa acaagaatcg acctctctca gctcggtgga gacagcaggg
     5341 ctgaccccaa gaagaagagg aaggtgtgaa agggttcgat ccctaccggt tagtaatgag
     5401 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     5461 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg
     5521 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     5581 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     5641 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgaa atcggatccg
     5701 cggccgcact gattctagct gcagaggctc cggtgcccgt cagtgggcag agcgcacatc
     5761 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     5821 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     5881 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     5941 tgccgccaga acacaggatc ctcgagccac catggtgagc aagggcgagg agctgttcac
     6001 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     6061 gaggggcgag ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac
     6121 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgagcc acggcgtgca
     6181 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     6241 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcacct acaagacccg
     6301 cgccgaggtg aagttcgagg gcgacaccct agtgaaccgc atcgagctga agggcgtcga
     6361 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa
     6421 catctatatc atggccgtca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     6481 caacgtggag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     6541 cgacggcccc gtgctgctgc ccgacagcca ctacctgagc acccagtccg tgctgagcaa
     6601 agaccccaac gagaagcgcg atcacatggt cctgctggag ttccgcaccg ccgccgggat
     6661 cactctcggc atggacgagc tgtacaagta agttaacttg tttattgcag cttataatgg
     6721 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6781 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg
     6841 atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg
     6901 tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac
     6961 caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca
     7021 ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc
     7081 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
     7141 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
     7201 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
     7261 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga
     7321 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
     7381 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
     7441 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
     7501 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
     7561 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct
     7621 agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc
     7681 gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat
     7741 ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta
     7801 ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg
     7861 ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga
     7921 agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga
     7981 ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga
     8041 tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc
     8101 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     8161 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     8221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     8281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     8341 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     8401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     8461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     8521 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     8581 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     8641 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     8701 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     8761 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     8821 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     8881 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     8941 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     9001 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     9061 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     9121 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     9181 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     9241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     9301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     9361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     9421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     9481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     9541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     9601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     9661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     9721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     9781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     9841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     9901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     9961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
    10021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
    10081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
    10141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
    10201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
    10261 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
    10321 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
    10381 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
    10441 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
    10501 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
    10561 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
    10621 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
    10681 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
    10741 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
    10801 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
    10861 acggccagtg agcgcgcctc gttcattcac gtttttgaac ccgtggagga cgggcagact
    10921 cgcggtgcaa atgtgtttta cagcgtgatg gagcagatga agatgctcga cacgctgcag
    10981 aacacgcagc tagattaacc ctagaaagat aatcatattg tgacgtacgt taaagataat
    11041 catgtgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt
    11101 tgaatagata ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac
    11161 aatttattta tgtttattta tttattaaaa aaaacaaaaa ctcaaaattt cttctataaa
    11221 gtaacaaaac ttttatgagg gacagccccc ccccaaagcc cccagggatg taattacgtc
    11281 cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc
    11341 cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg
    11401 atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac
    11461 ggggaaaagg cctccacggc cgacattgat tattgactag ttattaatag taatcaatta
    11521 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
    11581 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
    11641 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
    11701 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
    11761 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
    11821 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
    11881 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
    11941 tttaattatt ttgtgcagcg atgggggcgg gggggggg
//
