LOCUS       sequence-260738-       14477 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 14477)
  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 14477)
  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..14477
                     /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          3729..4745
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4669..4691
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4753..4772
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(4845..5462,5463..5468,5469..5504,5505..5507,5508..5543,
                     5544..5546,5547..5582,5583..5591)
                     /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    5701..5782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5789..6013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6046..6070
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(6096..6143)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6144..10244)
                     /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(6144..10244)
                     /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(10269..10289)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(10296..10361)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      10361..10370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(10395..10419)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(10436..10750)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(10439..10463)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    10510..10528
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10546..10564
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10581..10599
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10617..10635
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10653..10671
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10688..10706
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    10724..10742
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    10767..11603
                     /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(11637..11656)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(11638..11656)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11663..11680)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(11663..11679)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11672..11694)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             11817..12119
                     /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     11998..12017
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(12522..12541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     13136..13155
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(13516..14307,14308..14376))
                     /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     14139..14158
                     /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 rggsggggsg ggggggggsg
     3601 gggggggggg sggggsgrgg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     3661 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     3721 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     3781 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     3841 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc
     3901 tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg
     3961 gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct
     4021 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg
     4081 cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc
     4141 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc
     4201 ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta
     4261 cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg
     4321 gcggggcggg gccgcctcgg gccggggrgg gctcggggga ggggcgcggc ggcccccgga
     4381 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     4441 agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc
     4501 gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg
     4561 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg
     4621 gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct
     4681 ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc
     4741 tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattcg
     4801 gcttgatcga agctagtgga tcccccgggc tgcaggaatt caccatgtct agactggaca
     4861 agagcaaagt cataaacggc gctctggaat tactcaatgg agtcggtatc gaaggcctga
     4921 cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga
     4981 agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc
     5041 acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat
     5101 tccgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa
     5161 cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct
     5221 ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat
     5281 tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta
     5341 tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct
     5401 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg
     5461 gcgggccggc cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     5521 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     5581 tccccgggta aatcgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     5641 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     5701 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5761 aataaagcat ttttttcact gcctcgaccc atagagccca ccgcatcccc agcatgcctg
     5821 ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa
     5881 tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga
     5941 gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa
     6001 ctagaaggca cagacgcgtc agctgactag aggatccccg gatcaaccac tttgtacaag
     6061 aaagctgggt cggcgcgccc acccttgaat tcttactttt tcttttttgc ctggccggcc
     6121 tttttcgtgg ccgccggcct tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg
     6181 tacaggccgg tgatgctctg gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg
     6241 tacctcttcc ggtcgatggt ggtgtcaaag tacttgaagg cggcaggggc tcccagattg
     6301 gtcagggtaa acaggtggat gatattctcg gcctgctctc tgatgggctt atcccggtgc
     6361 ttgttgtagg cggacagcac tttgtccaga ttagcgtcgg ccaggatcac tctcttggag
     6421 aactcgctga tctgctcgat gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc
     6481 tgtttctgct cattatcctc gggggagccc ttcagcttct catagtggct ggccaggtac
     6541 aggaagttca catatttgga gggcagggcc agttcgtttc ccttctgcag ttcgccggca
     6601 gaggccagca ttctcttccg gccgttttcc agctcgaaca gggagtactt aggcagcttg
     6661 atgatcaggt cctttttcac ttctttgtag cccttggctt ccagaaagtc gatgggattc
     6721 ttctcgaagc tgcttctttc catgatggtg atccccagca gctctttcac actcttcagt
     6781 ttcttggact tgcccttttc cactttggcc accaccagca cagaataggc cacggtgggg
     6841 ctgtcgaagc cgccgtactt cttagggtcc cagtccttct ttctggcgat cagcttatcg
     6901 ctgttcctct tgggcaggat agactctttg ctgaagccgc ctgtctgcac ctcggtcttt
     6961 ttcacgatat tcacttgggg catgctcagc actttccgca cggtggcaaa atcccggccc
     7021 ttatcccaca cgatctcccc ggtttcgccg tttgtctcga tcagaggccg cttccggatc
     7081 tcgccgttgg ccagggtaat ctcggtcttg aaaaagttca tgatgttgct gtagaagaag
     7141 tacttggcgg tagccttgcc gatttcctgc tcgctcttgg cgatcatctt ccgcacgtcg
     7201 tacaccttgt agtcgccgta cacgaactcg ctttccagct tagggtactt tttgatcagg
     7261 gcggttccca cgacggcgtt caggtaggcg tcgtgggcgt ggtggtagtt gttgatctcg
     7321 cgcactttgt aaaactggaa atccttccgg aaatcggaca ccagcttgga cttcagggtg
     7381 atcactttca cttcccggat cagcttgtca ttctcgtcgt acttagtgtt catccgggag
     7441 tccaggatct gtgccacgtg ctttgtgatc tgccgggttt ccaccagctg tctcttgatg
     7501 aagccggcct tatccagttc gctcaggccg cctctctcgg ccttggtcag attgtcgaac
     7561 tttctctggg taatcagctt ggcgttcagc agctgccgcc agtagttctt catcttcttc
     7621 acgacctctt cggagggcac gttgtcgctc ttgccccggt tcttgtcgct tctggtcagc
     7681 accttgttgt cgatggagtc gtccttcaga aagctctgag gcacgatatg gtccacatcg
     7741 tagtcggaca gccggttgat gtccagttcc tggtccacgt acatatcccg cccattctgc
     7801 aggtagtaca ggtacagctt ctcgttctgc agctgggtgt tttccacggg gtgttctttc
     7861 aggatctggc tgcccagctc tttgatgccc tcttcgatcc gcttcattct ctcgcggctg
     7921 ttcttctgtc ccttctgggt ggtctggttc tctctggcca tttcgatcac gatgttctcg
     7981 ggcttgtgcc ggcccatcac tttcacgagc tcgtccacca ccttcactgt ctgcaggatg
     8041 cccttcttaa tggcggggct gccggccaga ttggcaatgt gctcgtgcag gctatcgccc
     8101 tggccggaca cctgggcttt ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc
     8161 agctgcatga agtttctgtt ggcgaagccg tcggacttca ggaaatccag gattgtcttg
     8221 ccggactgct tgtcccggat gccgttgatc agcttccggc tcagcctgcc ccagccggtg
     8281 tatctccgcc gcttcagctg cttcatcact ttgtcgtcga acaggtgggc ataggttttc
     8341 agccgttcct cgatcatctc tctgtcctca aacagtgtca gggtcagcac gatatcttcc
     8401 agaatgtcct cgttttcctc attgtccagg aagtccttgt ccttgataat tttcagcaga
     8461 tcgtggtatg tgcccaggga ggcgttgaac cgatcttcca cgccggagat ttccacggag
     8521 tcgaagcact cgattttctt gaagtagtcc tctttcagct gcttcacggt cactttccgg
     8581 ttggtcttga acagcaggtc cacgatggcc tttttctgct cgccgctcag gaaggcgggc
     8641 tttctcattc cctcggtcac gtatttcact ttggtcagct cgttatacac ggtgaagtac
     8701 tcgtacagca ggctgtgctt gggcagcacc ttctcgttgg gcaggttctt atcgaagttg
     8761 gtcatccgct cgatgaagct ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc
     8821 cagggggtga tggtttcctc gctctttctg gtcatccagg cgaatctgct gtttcccctg
     8881 gccagagggc ccacgtagta ggggatgcgg aaggtcagga tcttctcgat cttttcccgg
     8941 ttgtccttca ggaatgggta aaaatcttcc tgccgccgca gaatggcgtg cagctctccc
     9001 aggtggatct ggtgggggat gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc
     9061 tctctgttca gcttcacgag cagttcctcg gtgccgtcca tcttttccag gatgggcttg
     9121 atgaacttgt agaactcttc ctggctggct ccgccgtcaa tgtagccggc gtagccgttc
     9181 ttgctctggt cgaagaaaat ctctttgtac ttctcaggca gctgctgccg cacgagagct
     9241 ttcagcaggg tcaggtcctg gtggtgctcg tcgtatctct tgatcataga ggcgctcagg
     9301 ggggccttgg tgatctcggt gttcactctc aggatgtcgc tcagcaggat ggcgtcggac
     9361 aggttcttgg cggccagaaa caggtcggcg tactggtcgc cgatctgggc cagcaggttg
     9421 tccaggtcgt cgtcgtaggt gtccttgctc agctgcagtt tggcatcctc ggccaggtcg
     9481 aagttgctct tgaagttggg ggtcaggccc aggctcaggg caatcaggtt tccgaacagg
     9541 ccattcttct tctcgccggg cagctgggcg atcagatttt ccagccgtct gctcttgctc
     9601 agtctggcag acaggatggc cttggcgtcc acgccgctgg cgttgatggg gttttcctcg
     9661 aacagctggt tgtaggtctg caccagctgg atgaacagct tgtccacgtc gctgttgtcg
     9721 gggttcaggt cgccctcgat caggaagtgg ccccggaact tgatcatgtg ggccagggcc
     9781 agatagatca gccgcaggtc ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg
     9841 tagatggtgg ggtacttctc gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg
     9901 tgccgctcgt gcttcttatc ctcttccacc aggaaggact cttccagtct gtggaagaag
     9961 ctgtcgtcca ccttggccat ctcgttgctg aagatctctt gcagatagca gatccggttc
    10021 ttccgtctgg tgtatcttct tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg
    10081 ccgctgtcga acagcagggc tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg
    10141 cccagcacct tgaatttctt gctgggcacc ttgtactcgt cggtgatcac ggcccagccc
    10201 acagagttgg tgccgatgtc caggccgatg ctgtacttct tgtcggctgc tgggactccg
    10261 tggataccga ccttccgctt cttctttggg gccatcttat cgtcatcgtc tttgtaatca
    10321 atatcatgat ccttgtagtc tccgtcgtgg tccttatagt ccatggtggc accggtccaa
    10381 cctgaaaacc gcggagcctg cttttttgta caaacttgtt gatcggaatt ctccaggcga
    10441 tctgacggtt cactaaacga gctctgctta tataggcctc ccaccgtaca cgcctacctc
    10501 gacatacgtt ctctatcact gatagggagt aaactcgaca tacgttctct atcactgata
    10561 gggataaact cgacatacgt tctctatcac tgatagggag taaactcgac atacgttctc
    10621 tatcactgat agggagtaaa ctcgacatac gttctctatc actgataggg agtaaactcg
    10681 acatcgttct ctatcactga tagggagtaa actcgacata cgttctctat cactgatagg
    10741 gagtaaactc gacagtacta agctttacta gggacaggat tggtgacaga aaagccccat
    10801 ccttaggcct cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg
    10861 cagattcctt atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa
    10921 cctctaaggt ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg
    10981 ggtgggaggg aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac
    11041 cctctcccag aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt
    11101 gctgcagctt ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata
    11161 agttggtcct gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc
    11221 ctccgtgcgt cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc
    11281 tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag
    11341 gtgttcacca ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta
    11401 aagagtcccc agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct
    11461 aaggccctgc tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg
    11521 cttccctgtc ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct
    11581 acaggggttc ctggctctgc tctaagggcg aattcgcggc cgctaaattc aattcgccct
    11641 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
    11701 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
    11761 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctatac gtacggcagt
    11821 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
    11881 atattattga cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
    11941 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
    12001 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
    12061 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
    12121 tgtcaggcat gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca
    12181 gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg
    12241 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag
    12301 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta
    12361 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc
    12421 gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
    12481 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
    12541 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
    12601 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
    12661 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
    12721 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
    12781 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
    12841 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
    12901 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
    12961 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
    13021 tcgtgaccca tgcatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
    13081 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
    13141 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
    13201 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
    13261 ttctgaatta ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc
    13321 ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
    13381 tttatttttc taaatacatt caaatatgta tccgctcatg agattatcaa aaaggatctt
    13441 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
    13501 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
    13561 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
    13621 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
    13681 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
    13741 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
    13801 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
    13861 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
    13921 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
    13981 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
    14041 cgtaagatgc ttttctgtga ctggtgaata ctcaaccaag tcattctgag aatagtgtat
    14101 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
    14161 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
    14221 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
    14281 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
    14341 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
    14401 aagcatttat cagggttatt gtctcatgac caaaatccct taacgtgagt tttcgttcca
    14461 ctgagcgtca gaccccg
//
