LOCUS       sequence-308770-       11241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11241)
  TITLE     Tunable and temporal expression control of Cas9
REFERENCE   2  (bases 1 to 11241)
  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_KRAB_T2A_BFP
FEATURES             Location/Qualifiers
     source          1..11241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..368
                     /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    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    81..99
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    117..135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    189..207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    225..243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             390..437
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(438..461,462..464,465..3020,3021..3023,3024..4538)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4593..4613
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5028..5222
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             5238..5291
                     /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             5304..6002
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    6195..6329
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   6605..6639
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6650..6667)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6650..6666)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6659..6681)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6875..6894)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6994..7016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7054..7072)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7140..7244
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7245..7313,7314..8105)
                     /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(7463..7482)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8276..8864
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8765..8784
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9018..9035
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9152..9173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9188..9193,9194..9211,9212..9218)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9226..9242
                     /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     9231..9253
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9250..9266
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9250..9266
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   9292..9354
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    9530..9611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(9728..10126)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(10127..10183)
                     /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(10193..10936)
                     /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(10937..10960)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      10960..10969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(10989..11200)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag
       61 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc
      121 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta
      181 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag
      241 agaacgtata agctttgctt atgtaaacca gggcgcctat aaaagagtgc tgattttttg
      301 agtaaacttc aattccacaa cacttttgtc ttataccaac tttccgtacc acttcctacc
      361 ctcgtaaagg tacccgtacg gccaccatga aaaggccggc ggccacgaaa aaggccggcc
      421 aggcaaaaaa gaaaaaggac aagaagtaca gcatcggcct ggccatcggc accaactctg
      481 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
      541 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
      601 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
      661 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
      721 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
      781 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
      841 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
      901 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
      961 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     1021 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     1081 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     1141 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     1201 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     1261 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     1321 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     1381 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     1441 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     1501 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     1561 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     1621 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     1681 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     1741 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     1801 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     1861 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     1921 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     1981 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     2041 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     2101 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     2161 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     2221 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     2281 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     2341 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     2401 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     2461 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     2521 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     2581 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     2641 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     2701 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     2761 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     2821 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     2881 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     2941 acgatgtgga ccacatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     3001 tgctgaccag aagcgacaag gcccggggca agagcgacaa cgtgccctcc gaagaggtcg
     3061 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     3121 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     3181 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     3241 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     3301 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     3361 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     3421 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     3481 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     3541 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     3601 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     3661 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     3721 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     3781 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     3841 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     3901 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     3961 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     4021 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     4081 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     4141 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     4201 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     4261 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     4321 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     4381 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     4441 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     4501 acgagacacg gatcgacctg tctcagctgg gaggcgacag cgctggagga ggtggaagcg
     4561 gaggaggagg aagcggagga ggaggtagcg gacctaagaa aaagaggaag gtggcggccg
     4621 ctggatccat gaacatccag atgctgctgg aggccgctga ctacctggaa cggagagagc
     4681 gcgaagccga gcacggatat gcttcaatgc tgcccggaag cggcatgaat attcagatgc
     4741 tgctggaggc tgctgattac ctggaaaggc gcgaacggga ggccgaacat ggctatgctt
     4801 ccatgctgcc tgggtctgga atgaatatcc aaatgctgct ggaggcagcc gattacctgg
     4861 aacggagaga aagagaagcc gagcacggat acgccagcat gctgccaggc agcgggatga
     4921 acatacaaat gctgctggag gctgccgatt acctggagag gcgcgagaga gaagctgaac
     4981 atggctatgc ctctatgctg cccagtgggc agggaggagg tggaagcaga acactggtta
     5041 cgttcaagga cgtgtttgtg gactttacac gtgaggagtg gaaattgctg gatactgcgc
     5101 aacaaattgt gtatcgaaat gtcatgcttg agaattacaa gaacctcgtc agtctcggat
     5161 accagttgac gaaaccggat gtgatcctta ggctcgaaaa gggggaagaa ccttggctgg
     5221 taggtaccgg atccggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     5281 atcctggccc accggtcgcc accatgagcg agctgattaa ggagaacatg cacatgaagc
     5341 tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca
     5401 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     5461 tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca
     5521 cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag
     5581 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     5641 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga
     5701 tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg
     5761 gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa
     5821 acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct
     5881 actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc
     5941 agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta
     6001 atggatccgg ctaaactagt agaccacctc ccctgcgagc taagctggac agccaatgac
     6061 gggtaagaga gtgacatttt tcactaacct aagacaggag ggccgtcaga gctactgcct
     6121 aatccaaaga cgggtaaaag tgataaaaat gtatcactcc aacctaagac aggcgcagct
     6181 tccgagggat ttgagatcca gacatgataa gatacattga tgagtttgga caaaccaaaa
     6241 ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg
     6301 taaccattat aagctgcaat aaacaagttt gatatctata acaagaaaat atatatataa
     6361 taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt
     6421 aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa
     6481 atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag
     6541 atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag
     6601 aatgcatgcg tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg
     6661 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     6721 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     6781 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     6841 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6901 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6961 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7021 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     7081 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     7141 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     7201 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     7261 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     7321 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     7381 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     7441 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     7501 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7561 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7621 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     7681 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7741 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7801 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7861 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7921 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     7981 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     8041 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     8101 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     8161 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     8221 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     8281 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     8341 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     8401 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     8461 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     8521 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     8581 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     8641 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     8701 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8761 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8821 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8881 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     8941 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     9001 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     9061 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     9121 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     9181 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     9241 aatttcacac aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa
     9301 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
     9361 atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
     9421 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
     9481 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa
     9541 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     9601 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     9661 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc
     9721 tggagattta gccctcccac acataaccag agggcagcaa ttcacgaatc ccaactgccg
     9781 tcggctgtcc atcactgtcc ttcactatgg ctttgatccc aggatgcaga tcgagaagca
     9841 cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg atcgcgacga
     9901 tacaagtcag gttgccagct gccgcagcag cagcagtgcc cagcaccacg agttctgcac
     9961 aaggtccccc agtaaaatga tatacattga caccagtgaa gatgcggccg tcgctagaga
    10021 gagctgcgct ggcgacgctg tagtcttcag agatggggat gctgttgatt gtagccgttg
    10081 ctctttcaat gagggtggat tcttcttgag acaaaggctt ggccatgggg ccggggttct
    10141 cctccacgtc gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga
    10201 gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt
    10261 caagggcatc ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc
    10321 cgctttcgca ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga
    10381 aggcaggttc ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca
    10441 tagaatcggt ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca
    10501 atacgcagcc cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg
    10561 agaagccttg ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg
    10621 ttgggcgggt gccgagatgc actttagccc cgtcgcgatg tgagaggaga gcacagcggt
    10681 atgacttggc gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt
    10741 gggtatgatg cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct
    10801 tcacgtgcca gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg
    10861 tcaggccttc gataccgact ccattgagta attccagagc agagtttatg actttgctct
    10921 tgtccagtct agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt
    10981 cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact
    11041 gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca
    11101 ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc
    11161 cccaacttct cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcca
    11221 ctcgagtgga attatcacct c
//
