LOCUS       sequence-304743-       11310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11310)
  TITLE     Tunable and temporal expression control of Cas9
REFERENCE   2  (bases 1 to 11310)
  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"
COMMENT     Sequence Label: XClone-Cas9-Cronus-T2A-BFP
FEATURES             Location/Qualifiers
     source          1..11310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             383..4486
                     /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             383..4486
                     /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"
     RBS             4399..4407
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5315..5368
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5381..6079
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6272..6406
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6682..6716
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6727..6744)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6727..6743)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6736..6758)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6952..6971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7071..7093
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7131..7149)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7217..7321
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7322..7390,7391..8182)
                     /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     complement(7540..7559)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8353..8941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8842..8861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9095..9112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9229..9250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9265..9270,9271..9288,9289..9295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9303..9319
                     /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)."
     primer_bind     9308..9330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9327..9343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9327..9343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   9369..9431
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    9607..9688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(9805..10203)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(10204..10260)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(10270..11013)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(11014..11037)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      11037..11046
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(11066..11277)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccatggataa gaaatacagc attggactgg acattgggac
      421 aaactccgtg ggatgggccg tgattacaga cgaatacaaa gtgccttcaa agaagttcaa
      481 ggtgctgggc aacaccgata gacacagcat caagaaaaat ctgattggag ccctgctgtt
      541 cgactccggc gagacagctg aagcaactcg gctgaaaaga actgctcgga gaaggtatac
      601 ccgccgaaag aataggatct gctacctgca ggagattttc agcaacgaaa tggccaaggt
      661 ggacgatagt ttctttcacc gcctggagga atcattcctg gtcgaggaag ataagaaaca
      721 cgagcggcat cccatctttg gcaacattgt ggacgaggtc gcttatcacg aaaagtaccc
      781 taccatctat catctgagga agaaactggt ggactccaca gataaagcag acctgcgcct
      841 gatctatctg gccctggctc acatgattaa gttccggggc cattttctga tcgaggggga
      901 tctgaaccca gacaattctg atgtggacaa gctgttcatc cagctggtcc agacatacaa
      961 tcagctgttt gaggaaaacc ccattaatgc atctggcgtg gacgcaaaag ccatcctgag
     1021 tgccagactg tctaagagtc ggagactgga gaacctgatc gctcagctgc caggggaaaa
     1081 gaaaaacggc ctgtttggga atctgattgc actgtcactg ggactgactc ccaacttcaa
     1141 gagcaatttt gatctggccg aggacgctaa actgcagctg tccaaggaca cctatgacga
     1201 tgacctggat aacctgctgg ctcagatcgg ggatcagtac gcagacctgt tcctggccgc
     1261 taagaatctg tctgacgcca tcctgctgag tgatattctg cgcgtgaaca ccgagattac
     1321 aaaagccccc ctgtcagcta gcatgatcaa gagatatgac gagcaccatc aggatctgac
     1381 cctgctgaag gctctggtga ggcagcagct gcctgagaag tacaaggaaa tcttctttga
     1441 tcagtctaag aacggatacg ccggctatat tgacggcggg gctagtcagg aggagttcta
     1501 caagtttatc aaacccattc tggagaagat ggatggcaca gaggaactgc tggtgaaact
     1561 gaatcgggaa gacctgctga ggaagcagcg cacttttgat aacggaagca tccctcacca
     1621 gattcatctg ggagagctgc acgcaatcct gaggcgccag gaagacttct acccatttct
     1681 gaaggataac agggagaaga tcgaaaaaat tctgacattc cgcatcccct actatgtggg
     1741 ccctctggca agaggcaaca gccggtttgc ctggatgact cgcaaatctg aggaaacaat
     1801 cactccctgg aacttcgagg aagtggtcga taagggcgct tccgcacagt ctttcattga
     1861 gcggatgaca aacttcgaca agaacctgcc aaacgaaaaa gtgctgccca agcactctct
     1921 gctgtacgag tatttcacag tctataacga actgactaag gtgaaatacg tcaccgaggg
     1981 gatgagaaag cctgccttcc tgagtggaga acagaagaaa gctatcgtgg acctgctgtt
     2041 taaaaccaat aggaaggtga cagtcaagca gctgaaagag gactatttca agaaaattga
     2101 atgtttcgat tctgtggaga tcagtggcgt cgaagacagg tttaacgcct ccctggggac
     2161 ctaccacgat ctgctgaaga tcattaagga taaagacttc ctggacaacg aggaaaatga
     2221 ggatatcctg gaagacattg tgctgaccct gacactgttt gaggataggg aaatgatcga
     2281 ggaacgcctg aagacctatg cccatctgtt cgatgacaaa gtgatgaaac agctgaagcg
     2341 acggagatac acaggatggg gccgactgtc tcggaagctg atcaatggga ttcgcgacaa
     2401 acagagtgga aagaccatcc tggactttct gaaatcagat ggcttcgcca accggaactt
     2461 catgcagctg attcacgatg acagcctgac attcaaagag gatatccaga aggcacaggt
     2521 gtccgggcag ggagactctc tgcacgagca tatcgcaaac ctggccggca gccctgccat
     2581 caagaaaggg attctgcaga ccgtgaaggt ggtggacgag ctggtgaaag tcatgggaag
     2641 acataagcca gaaaacatcg tgattgagat ggccagggaa aatcagacca cacagaaagg
     2701 ccagaagaac tcaagggagc gcatgaaaag aatcgaggaa ggaattaagg aactgggcag
     2761 ccagatcctg aaagagcacc ccgtggaaaa cacacagctg cagaatgaga agctgtatct
     2821 gtactatctg cagaatggac gcgatatgta cgtggaccag gagctggata ttaaccgact
     2881 gtccgattac gacgtggatc atatcgtccc acagtcattc ctgaaagatg acagcattga
     2941 caataaggtg ctgacccgct ctgacaaaaa ccgaggcaag agtgataatg tcccctcaga
     3001 ggaagtggtc aagaaaatga agaactactg gaggcagctg ctgaatgcca aactgatcac
     3061 acagcgaaag tttgataacc tgactaaagc tgagcgggga ggcctgagtg aactggacaa
     3121 agcaggcttc attaagcgac agctggtgga gacacggcag atcacaaagc acgtcgccca
     3181 gattctggat tcaagaatga acactaagta cgatgagaat gacaaactga tcagagaagt
     3241 gaaggtcatt accctgaagt caaaactggt gagcgacttt cggaaagatt tccagtttta
     3301 taaggtcaga gagatcaaca actaccacca tgctcatgac gcatacctga acgcagtggt
     3361 cggcacagcc ctgattaaga aataccctaa actggagtcc gagttcgtgt acggggacta
     3421 taaggtgtac gatgtcagaa aaatgatcgc caagtctgag caggaaattg gcaaagccac
     3481 tgctaagtat ttcttttaca gtaacatcat gaatttcttt aagactgaga tcaccctggc
     3541 aaatggggaa atccgaaagc ggccactgat tgagactaac ggcgagacag gagaaatcgt
     3601 gtgggacaaa ggaagagatt ttgctaccgt gaggaaggtc ctgagcatgc cccaagtgaa
     3661 tattgtcaag aaaacagagg tgcagactgg gggattcagt aaggaatcaa ttctgcctaa
     3721 acgcaactcc gataagctga tcgcccgaaa gaaagactgg gaccccaaga agtatggcgg
     3781 gttcgactcc ccaactgtgg cttactctgt cctggtggtc gcaaaggtgg agaagggaaa
     3841 aagcaagaaa ctgaaatccg tcaaggaact gctgggcatc accattatgg agcgcagctc
     3901 cttcgaaaag aatcctatcg attttctgga ggccaaaggc tataaggaag tgaagaaaga
     3961 cctgatcatc aagctgccaa agtactcact gtttgagctg gaaaacggga gaaagaggat
     4021 gctggcaagc gccggggagc tgcagaaagg aaatgaactg gccctgccct ccaagtacgt
     4081 gaacttcctg tatctggcta gccactacga gaagctgaaa gggtcccctg aggataacga
     4141 acagaaacag ctgtttgtgg agcagcacaa gcattatctg gacgagatca ttgaacagat
     4201 tagcgagttc tccaaaagag tgatcctggc tgacgcaaat ctggataagg tcctgagcgc
     4261 atacaacaaa caccgggata agccaatcag agagcaggcc gaaaatatca ttcatctgtt
     4321 cactctgacc aacctgggag cccccgcagc cttcaagtat tttgacacta ccatcgatcg
     4381 caaacgatac acaagcacta aggaggtgct ggacgctacc ctgattcatc agagcattac
     4441 tggcctgtat gaaacaagga ttgacctgtc tcagctgggc ggcgactccg gagctgacgg
     4501 aagcgaaaat cctggtaaca aaacaatagt tcctgcaacg ttaccacaac tcacccctac
     4561 cctggtgtca ctgttggagg ttattgaacc tgaagtgtta tatgcaggat atgatagctc
     4621 tgttccagac tcaacttgga ggatcatgac tacgctcaac atgttaggag ggcggcaagt
     4681 gattgcagca gtgaaatggg caaaggcaat accaggtttc aggaacttac acctggatga
     4741 ccaaatgacc ctactgcagt actcctggat gtttcttatg gcatttgctc tggggtggag
     4801 atcatataga caatcaagtg caaacctgct gtgttttgct cctgatctga ttattaatga
     4861 gcagagaatg actctaccct gcatgtacga ccaatgtaaa cacatgctgt atgtttcctc
     4921 tgagttacac aggcttcagg tatcttatga agagtatctc tgtatgaaaa ccttactgct
     4981 tctctcttca gttcctaagg acggtctgaa gagccaagag ctatttgatg aaattagaat
     5041 gacctacatc aaagagctag gaaaagccat tgtcaagagg gaaggaaact ccagccagaa
     5101 ctggcagcgg ttttatcaac tgacaaaact cttggattct atgcatgaag tggttgaaaa
     5161 tctccttaac tattgcttcc aaacattttt ggataagacc atgtccatcg agttccccga
     5221 gatgttagct gaaatcatca ccaatcagat accaaaatat tcaaatggaa atatcaaaaa
     5281 acttctgttt catcaaaagg gtaccggatc cggagagggc agaggaagtc tgctaacatg
     5341 cggtgacgtc gaggagaatc ctggcccacc ggtcgccacc atgagcgagc tgattaagga
     5401 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac
     5461 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
     5521 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa
     5581 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg
     5641 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga
     5701 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac
     5761 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct
     5821 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg
     5881 gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct
     5941 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa
     6001 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa
     6061 actggggcac aagcttaatg gatccggcta aactagtaga ccacctcccc tgcgagctaa
     6121 gctggacagc caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc
     6181 cgtcagagct actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac
     6241 ctaagacagg cgcagcttcc gagggatttg agatccagac atgataagat acattgatga
     6301 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga
     6361 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca
     6421 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat
     6481 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc
     6541 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc
     6601 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga
     6661 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa
     6721 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     6781 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     6841 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     6901 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     6961 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     7021 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     7081 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     7141 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     7201 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     7261 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     7321 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     7381 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     7441 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     7501 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     7561 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     7621 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7681 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7741 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7801 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7861 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7921 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7981 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     8041 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     8101 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     8161 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     8221 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     8281 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     8341 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     8401 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     8461 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     8521 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     8581 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     8641 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     8701 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8761 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8821 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8881 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8941 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     9001 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     9061 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     9121 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     9181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     9241 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     9301 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt
     9361 cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa
     9421 attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata
     9481 ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac aatttattta
     9541 tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa
     9601 cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     9661 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     9721 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     9781 ggctgattat gatcctctgg agatttagcc ctcccacaca taaccagagg gcagcaattc
     9841 acgaatccca actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg
     9901 atgcagatcg agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc
     9961 atttccgatc gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag
    10021 caccacgagt tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat
    10081 gcggccgtcg ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct
    10141 gttgattgta gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc
    10201 catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc
    10261 gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat
    10321 atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag
    10381 ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag
    10441 ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg
    10501 tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg
    10561 atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta
    10621 cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt
    10681 ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga
    10741 gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc
    10801 ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag
    10861 cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg
    10921 agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga
    10981 gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat
    11041 ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa
    11101 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga
    11161 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc
    11221 aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact
    11281 gacgggcacc ggagccactc gagtggaatt
//
