LOCUS       sequence-202363-       14479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 upon induction with doxicycline..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14479)
  AUTHORS   Castano J, Bueno C, Jimenez-Delgado S, Roca-Ho H, Fraga MF, 
            Fernandez AF, Nakanishi M, Torres-Ruiz R, Rodriguez-Perales S, 
            Menendez P
  TITLE     Generation and characterization of a human iPSC cell line expressing
            inducible Cas9 in the ""safe harbor"" AAVS1 locus.
  JOURNAL   Stem Cell Res. 2017 May;21:137-140. doi: 10.1016/j.scr.2017.04.011. 
            Epub 2017 Apr 22.
  PUBMED    28677529
REFERENCE   2  (bases 1 to 14479)
  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: "Stem Cell 
            Res."; date: "2017-05"; volume: "21:137-140. doi"; pages: " 
            10.1016/j.scr.2017.04.011. Epub 2017 Apr 22"
COMMENT     Sequence Label: pAAVS1-TRE-Cas9- puro-polyA-CAG-rtTA
FEATURES             Location/Qualifiers
     source          1..14479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     765..782
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    899..920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(935..940,941..958,959..965)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    973..989
                     /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     978..1000
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     997..1013
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     997..1013
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1034..1052
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    1087..1890
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1897..1922
                     /label=SA
                     /note="splice acceptor site"
     CDS             1946..1999
                     /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             2009..2608
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2009..2028)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2505..2525
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2640..2657)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2646..2870
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    2877..2958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        3146..3449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3451..3728
                     /label=chicken beta-actin promoter
     intron          3730..4747
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4671..4693
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4755..4774
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(4847..5464,5465..5470,5471..5506,5507..5509,5510..5545,
                     5546..5548,5549..5584,5585..5593)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    5703..5784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5791..6015
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6048..6072
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(6098..6145)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6146..10246)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(6146..10246)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(10271..10291)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(10298..10363)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      10363..10372
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(10397..10421)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(10438..10752)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(10441..10465)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    10512..10530
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10548..10566
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10583..10601
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10619..10637
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10655..10673
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10690..10708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10726..10744
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    10769..11605
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(11639..11658)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11640..11658)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11665..11682)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11665..11681)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11674..11696)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             11819..12121
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     12000..12019
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(12524..12543)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     13138..13157
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(13518..14309,14310..14378))
                     /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     14141..14160
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      721 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      781 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      841 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      901 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      961 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     1021 acgccaagct cagaattaac cctcactaaa gggactagtc ctgcaggttt aaacgaattc
     1081 gccctttgct ttctctgacc agcattctct cccctgggcc tgtgccgctt tctgtctgca
     1141 gcttgtggcc tgggtcacct ctacggctgg cccagatcct tccctgccgc ctccttcagg
     1201 ttccgtcttc ctccactccc tcttcccctt gctctctgct gtgttgctgc ccaaggatgc
     1261 tctttccgga gcacttcctt ctcggcgctg caccacgtga tgtcctctga gcggatcctc
     1321 cccgtgtctg ggtcctctcc gggcatctct cctccctcac ccaaccccat gccgtcttca
     1381 ctcgctgggt tcccttttcc ttctccttct ggggcctgtg ccatctctcg tttcttagga
     1441 tggccttctc cgacggatgt ctcccttgcg tcccgcctcc ccttcttgta ggcctgcatc
     1501 atcaccgttt ttctggacaa ccccaaagta ccccgtctcc ctggctttag ccacctctcc
     1561 atcctcttgc tttctttgcc tggacacccc gttctcctgt ggattcgggt cacctctcac
     1621 tcctttcatt tgggcagctc ccctaccccc cttacctctc tagtctgtgc tagctcttcc
     1681 agccccctgt catggcatct tccaggggtc cgagagctca gctagtcttc ttcctccaac
     1741 ccgggcccct atgtccactt caggacagca tgtttgctgc ctccagggat cctgtgtccc
     1801 cgagctggga ccaccttata ttcccagggc cggttaatgt ggctctggtt ctgggtactt
     1861 ttatctgtcc cctccacccc acagtggggc aagcttctga cctcttctct tcctcccaca
     1921 gggcctcgag agatctggca gcggagaggg cagaggaagt cttctaacat gcggtgacgt
     1981 ggaggagaat cccggcccta ggctcgagat gaccgagtac aagcccacgg tgcgcctcgc
     2041 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2101 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     2161 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     2221 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     2281 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     2341 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     2401 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     2461 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     2521 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     2581 gtgcatgacc cgcaagcccg gtgcctgatc tagagggccc gtttaaaccc gctgatcagc
     2641 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2701 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2761 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2821 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtcgaggcag
     2881 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     2941 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     3001 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
     3061 gatcctctag agatatcgtc gacactagtt attaatagta atcaattacg gggtcattag
     3121 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     3181 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     3241 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     3301 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     3361 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     3421 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     3481 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     3541 gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg
     3601 aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     3661 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     3721 ggcgggcggg gagtcgctgc gacgctgcct tcgccccgtg ccccgctccg ccgccgcctc
     3781 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
     3841 cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg
     3901 gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg
     3961 gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg
     4021 ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag
     4081 cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt
     4141 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc
     4201 ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg
     4261 tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc
     4321 gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg
     4381 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg
     4441 cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg
     4501 ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat
     4561 gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg
     4621 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
     4681 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
     4741 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
     4801 cggcttgatc gaagctagtg gatcccccgg gctgcaggaa ttcaccatgt ctagactgga
     4861 caagagcaaa gtcataaacg gcgctctgga attactcaat ggagtcggta tcgaaggcct
     4921 gacgacaagg aaactcgctc aaaagctggg agttgagcag cctaccctgt actggcacgt
     4981 gaagaacaag cgggccctgc tcgatgccct gccaatcgag atgctggaca ggcatcatac
     5041 ccacttctgc cccctggaag gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc
     5101 attccgctgt gctctcctct cacatcgcga cggggctaaa gtgcatctcg gcacccgccc
     5161 aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt
     5221 ctccctggag aacgcactgt acgctctgtc cgccgtgggc cactttacac tgggctgcgt
     5281 attggaggaa caggagcatc aagtagcaaa agaggaaaga gagacaccta ccaccgattc
     5341 tatgccccca cttctgagac aagcaattga gctgttcgac cggcagggag ccgaacctgc
     5401 cttccttttc ggcctggaac taatcatatg tggcctggag aaacagctaa agtgcgaaag
     5461 cggcgggccg gccgacgccc ttgacgattt tgacttagac atgctcccag ccgatgccct
     5521 tgacgacttt gaccttgata tgctgcctgc tgacgctctt gacgattttg accttgacat
     5581 gctccccggg taaatcgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     5641 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     5701 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5761 caaataaagc atttttttca ctgcctcgac ccatagagcc caccgcatcc ccagcatgcc
     5821 tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag
     5881 aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg
     5941 gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc
     6001 aactagaagg cacagacgcg tcagctgact agaggatccc cggatcaacc actttgtaca
     6061 agaaagctgg gtcggcgcgc ccacccttga attcttactt tttctttttt gcctggccgg
     6121 cctttttcgt ggccgccggc cttttgtcgc ctcccagctg agacaggtcg atccgtgtct
     6181 cgtacaggcc ggtgatgctc tggtggatca gggtggcgtc cagcacctct ttggtgctgg
     6241 tgtacctctt ccggtcgatg gtggtgtcaa agtacttgaa ggcggcaggg gctcccagat
     6301 tggtcagggt aaacaggtgg atgatattct cggcctgctc tctgatgggc ttatcccggt
     6361 gcttgttgta ggcggacagc actttgtcca gattagcgtc ggccaggatc actctcttgg
     6421 agaactcgct gatctgctcg atgatctcgt ccaggtagtg cttgtgctgt tccacaaaca
     6481 gctgtttctg ctcattatcc tcgggggagc ccttcagctt ctcatagtgg ctggccaggt
     6541 acaggaagtt cacatatttg gagggcaggg ccagttcgtt tcccttctgc agttcgccgg
     6601 cagaggccag cattctcttc cggccgtttt ccagctcgaa cagggagtac ttaggcagct
     6661 tgatgatcag gtcctttttc acttctttgt agcccttggc ttccagaaag tcgatgggat
     6721 tcttctcgaa gctgcttctt tccatgatgg tgatccccag cagctctttc acactcttca
     6781 gtttcttgga cttgcccttt tccactttgg ccaccaccag cacagaatag gccacggtgg
     6841 ggctgtcgaa gccgccgtac ttcttagggt cccagtcctt ctttctggcg atcagcttat
     6901 cgctgttcct cttgggcagg atagactctt tgctgaagcc gcctgtctgc acctcggtct
     6961 ttttcacgat attcacttgg ggcatgctca gcactttccg cacggtggca aaatcccggc
     7021 ccttatccca cacgatctcc ccggtttcgc cgtttgtctc gatcagaggc cgcttccgga
     7081 tctcgccgtt ggccagggta atctcggtct tgaaaaagtt catgatgttg ctgtagaaga
     7141 agtacttggc ggtagccttg ccgatttcct gctcgctctt ggcgatcatc ttccgcacgt
     7201 cgtacacctt gtagtcgccg tacacgaact cgctttccag cttagggtac tttttgatca
     7261 gggcggttcc cacgacggcg ttcaggtagg cgtcgtgggc gtggtggtag ttgttgatct
     7321 cgcgcacttt gtaaaactgg aaatccttcc ggaaatcgga caccagcttg gacttcaggg
     7381 tgatcacttt cacttcccgg atcagcttgt cattctcgtc gtacttagtg ttcatccggg
     7441 agtccaggat ctgtgccacg tgctttgtga tctgccgggt ttccaccagc tgtctcttga
     7501 tgaagccggc cttatccagt tcgctcaggc cgcctctctc ggccttggtc agattgtcga
     7561 actttctctg ggtaatcagc ttggcgttca gcagctgccg ccagtagttc ttcatcttct
     7621 tcacgacctc ttcggagggc acgttgtcgc tcttgccccg gttcttgtcg cttctggtca
     7681 gcaccttgtt gtcgatggag tcgtccttca gaaagctctg aggcacgata tggtccacat
     7741 cgtagtcgga cagccggttg atgtccagtt cctggtccac gtacatatcc cgcccattct
     7801 gcaggtagta caggtacagc ttctcgttct gcagctgggt gttttccacg gggtgttctt
     7861 tcaggatctg gctgcccagc tctttgatgc cctcttcgat ccgcttcatt ctctcgcggc
     7921 tgttcttctg tcccttctgg gtggtctggt tctctctggc catttcgatc acgatgttct
     7981 cgggcttgtg ccggcccatc actttcacga gctcgtccac caccttcact gtctgcagga
     8041 tgcccttctt aatggcgggg ctgccggcca gattggcaat gtgctcgtgc aggctatcgc
     8101 cctggccgga cacctgggct ttctggatgt cctctttaaa ggtcaggctg tcgtcgtgga
     8161 tcagctgcat gaagtttctg ttggcgaagc cgtcggactt caggaaatcc aggattgtct
     8221 tgccggactg cttgtcccgg atgccgttga tcagcttccg gctcagcctg ccccagccgg
     8281 tgtatctccg ccgcttcagc tgcttcatca ctttgtcgtc gaacaggtgg gcataggttt
     8341 tcagccgttc ctcgatcatc tctctgtcct caaacagtgt cagggtcagc acgatatctt
     8401 ccagaatgtc ctcgttttcc tcattgtcca ggaagtcctt gtccttgata attttcagca
     8461 gatcgtggta tgtgcccagg gaggcgttga accgatcttc cacgccggag atttccacgg
     8521 agtcgaagca ctcgattttc ttgaagtagt cctctttcag ctgcttcacg gtcactttcc
     8581 ggttggtctt gaacagcagg tccacgatgg cctttttctg ctcgccgctc aggaaggcgg
     8641 gctttctcat tccctcggtc acgtatttca ctttggtcag ctcgttatac acggtgaagt
     8701 actcgtacag caggctgtgc ttgggcagca ccttctcgtt gggcaggttc ttatcgaagt
     8761 tggtcatccg ctcgatgaag ctctgggcgg aagcgccctt gtccaccact tcctcgaagt
     8821 tccagggggt gatggtttcc tcgctctttc tggtcatcca ggcgaatctg ctgtttcccc
     8881 tggccagagg gcccacgtag taggggatgc ggaaggtcag gatcttctcg atcttttccc
     8941 ggttgtcctt caggaatggg taaaaatctt cctgccgccg cagaatggcg tgcagctctc
     9001 ccaggtggat ctggtggggg atgctgccgt tgtcgaaggt ccgctgcttc cgcagcaggt
     9061 cctctctgtt cagcttcacg agcagttcct cggtgccgtc catcttttcc aggatgggct
     9121 tgatgaactt gtagaactct tcctggctgg ctccgccgtc aatgtagccg gcgtagccgt
     9181 tcttgctctg gtcgaagaaa atctctttgt acttctcagg cagctgctgc cgcacgagag
     9241 ctttcagcag ggtcaggtcc tggtggtgct cgtcgtatct cttgatcata gaggcgctca
     9301 ggggggcctt ggtgatctcg gtgttcactc tcaggatgtc gctcagcagg atggcgtcgg
     9361 acaggttctt ggcggccaga aacaggtcgg cgtactggtc gccgatctgg gccagcaggt
     9421 tgtccaggtc gtcgtcgtag gtgtccttgc tcagctgcag tttggcatcc tcggccaggt
     9481 cgaagttgct cttgaagttg ggggtcaggc ccaggctcag ggcaatcagg tttccgaaca
     9541 ggccattctt cttctcgccg ggcagctggg cgatcagatt ttccagccgt ctgctcttgc
     9601 tcagtctggc agacaggatg gccttggcgt ccacgccgct ggcgttgatg gggttttcct
     9661 cgaacagctg gttgtaggtc tgcaccagct ggatgaacag cttgtccacg tcgctgttgt
     9721 cggggttcag gtcgccctcg atcaggaagt ggccccggaa cttgatcatg tgggccaggg
     9781 ccagatagat cagccgcagg tcggccttgt cggtgctgtc caccagtttc tttctcaggt
     9841 ggtagatggt ggggtacttc tcgtggtagg ccacctcgtc cacgatgttg ccgaagatgg
     9901 ggtgccgctc gtgcttctta tcctcttcca ccaggaagga ctcttccagt ctgtggaaga
     9961 agctgtcgtc caccttggcc atctcgttgc tgaagatctc ttgcagatag cagatccggt
    10021 tcttccgtct ggtgtatctt cttctggcgg ttctcttcag ccgggtggcc tcggctgttt
    10081 cgccgctgtc gaacagcagg gctccgatca ggttcttctt gatgctgtgc cggtcggtgt
    10141 tgcccagcac cttgaatttc ttgctgggca ccttgtactc gtcggtgatc acggcccagc
    10201 ccacagagtt ggtgccgatg tccaggccga tgctgtactt cttgtcggct gctgggactc
    10261 cgtggatacc gaccttccgc ttcttctttg gggccatctt atcgtcatcg tctttgtaat
    10321 caatatcatg atccttgtag tctccgtcgt ggtccttata gtccatggtg gcaccggtcc
    10381 aacctgaaaa ccgcggagcc tgcttttttg tacaaacttg ttgatcggaa ttctccaggc
    10441 gatctgacgg ttcactaaac gagctctgct tatataggcc tcccaccgta cacgcctacc
    10501 tcgacatacg ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga
    10561 tagggataaa ctcgacatac gttctctatc actgataggg agtaaactcg acatacgttc
    10621 tctatcactg atagggagta aactcgacat acgttctcta tcactgatag ggagtaaact
    10681 cgacatcgtt ctctatcact gatagggagt aaactcgaca tacgttctct atcactgata
    10741 gggagtaaac tcgacagtac taagctttac tagggacagg attggtgaca gaaaagcccc
    10801 atccttaggc ctcctccttc ctagtctcct gatattgggt ctaaccccca cctcctgtta
    10861 ggcagattcc ttatctggtg acacaccccc atttcctgga gccatctctc tccttgccag
    10921 aacctctaag gtttgcttac gatggagcca gagaggatcc tgggagggag agcttggcag
    10981 ggggtgggag ggaagggggg gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct
    11041 accctctccc agaacctgag ctgctctgac gcggctgtct ggtgcgtttc actgatcctg
    11101 gtgctgcagc ttccttacac ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa
    11161 taagttggtc ctgagttcta actttggctc ttcacctttc tagtccccaa tttatattgt
    11221 tcctccgtgc gtcagtttta cctgtgagat aaggccagta gccagccccg tcctggcagg
    11281 gctgtggtga ggaggggggt gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct
    11341 aggtgttcac caggtcgtgg ccgcctctac tccctttctc tttctccatc cttctttcct
    11401 taaagagtcc ccagtgctat ctgggacata ttcctccgcc cagagcaggg tcccgcttcc
    11461 ctaaggccct gctctgggct tctgggtttg agtccttggc aagcccagga gaggcgctca
    11521 ggcttccctg tcccccttcc tcgtccacca tctcatgccc ctggctctcc tgccccttcc
    11581 ctacaggggt tcctggctct gctctaaggg cgaattcgcg gccgctaaat tcaattcgcc
    11641 ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa
    11701 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
    11761 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca
    11821 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
    11881 tgatattatt gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
    11941 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
    12001 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
    12061 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
    12121 aatgtcaggc atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc
    12181 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
    12241 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
    12301 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
    12361 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg
    12421 gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
    12481 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
    12541 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
    12601 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
    12661 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
    12721 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
    12781 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
    12841 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
    12901 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
    12961 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
    13021 cgtcgtgacc catgcatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
    13081 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
    13141 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
    13201 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
    13261 tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt
    13321 gcggtatttc acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
    13381 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagattatc aaaaaggatc
    13441 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
    13501 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
    13561 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
    13621 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
    13681 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
    13741 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
    13801 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
    13861 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
    13921 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
    13981 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
    14041 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
    14101 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
    14161 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
    14221 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
    14281 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
    14341 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
    14401 tgaagcattt atcagggtta ttgtctcatg accaaaatcc cttaacgtga gttttcgttc
    14461 cactgagcgt cagaccccg
//
