LOCUS       sequence-436575-       12657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible CRISPR nuclease (CRISPRn) knock in construct into the 
            AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12657)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen 
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, 
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
  PUBMED    26971820
REFERENCE   2  (bases 1 to 12657)
  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"; doi: 
            "10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date: 
            "2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
COMMENT     Sequence Label: pAAVS1-PDi-CRISPRn
FEATURES             Location/Qualifiers
     source          1..12657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(54..122,123..914)
                     /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(272..291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1085..1673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1574..1593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1721..2557
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    2590..2608
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2626..2644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2662..2680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2698..2716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2734..2752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2770..2788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2806..2824
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2869..2893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2977..2986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2986..3051
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3058..3078
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3103..7203
                     /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             3103..7203
                     /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             7204..7251
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    7286..7407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7323..7342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7377..7396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7456..7472)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7456..7472)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7469..7491)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7480..7496
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7504..7510,7511..7528,7529..7534))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7549..7570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7687..7704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7760..7793
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8124..8143
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    8142..8197
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     8243..8262
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(8353..9099)
                     /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"
     primer_bind     complement(9131..9150)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          9158..10174
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(9212..9234)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(10175..10452)
                     /label=chicken beta-actin promoter
     enhancer        10454..10833
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    10839..11063
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     11052..11069
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(11101..11700)
                     /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(11184..11204)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     11681..11700
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(11710..11763)
                     /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"
     misc_feature    11787..11812
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    11819..11852
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    11854..12657
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
       61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
      421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
     1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
     1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
     1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
     1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
     1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
     2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
     2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
     2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
     2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
     2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
     2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
     2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
     2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
     2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
     2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     2941 ccacttccta ccctcgtaaa cttaaggtta attaacgcca ccatggacta taaggaccac
     3001 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
     3061 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
     3121 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     3181 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     3241 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     3301 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     3361 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     3421 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     3481 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     3541 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     3601 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     3661 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     3721 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     3781 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     3841 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     3901 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     3961 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     4021 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     4081 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     4141 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     4201 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     4261 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     4321 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     4381 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     4441 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     4501 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     4561 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     4621 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     4681 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     4741 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     4801 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     4861 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     4921 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     4981 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     5041 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     5101 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     5161 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     5221 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     5281 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     5341 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     5401 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     5461 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     5521 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     5581 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     5641 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     5701 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     5761 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     5821 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     5881 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     5941 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     6001 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     6061 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     6121 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     6181 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     6241 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     6301 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     6361 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     6421 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     6481 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     6541 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     6601 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     6661 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     6721 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     6781 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     6841 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     6901 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     6961 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     7021 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     7081 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     7141 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     7201 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaaaccggt
     7261 gcggccgcaa tcgatcgccc accccaactt gtttattgca gcttataatg gttacaaata
     7321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     7381 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     7441 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     7501 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     7561 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     7621 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     7681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     7741 tcactcaaag gacggccgca taacttcgta taatgtatgc tatacgaagt tataagcttg
     7801 ggctgcaggt cgagggatct ccataagaga agagggacag ctatgactgg gagtagtcag
     7861 gagaggagga aaaatctggc tagtaaaaca tgtaaggaaa attttaggga tgttaaagaa
     7921 aaaaataaca caaaacaaaa tataaaaaaa atctaacctc aagtcaaggc ttttctatgg
     7981 aataaggaat ggacagcagg gggctgtttc atatactgat gacctcttta tagccaacct
     8041 ttgttcatgg cagccagcat atgggcatat gttgccaaac tctaaaccaa atactcattc
     8101 tgatgtttta aatgatttgc cctcccatat gtccttccga gtgagagaca caaaaaattc
     8161 caacacacta ttgcaatgaa aataaatttc ctttattagc cagaagtcag atgctcaagg
     8221 ggcttcatga tgtccccata atttttggca gagggaaaaa gatctcagtg gtatttgtga
     8281 gccagggcat tggccacacc agccaccacc ttctgatagg cagcctgcac ctgaggagtg
     8341 aattcacgcg ttttagcctg gcagcatatc gagatcgaag tcatcgagag cgtcagcagg
     8401 gagcatatcc agatcaaaat catccagggc atcggcgggc agcatgtcca ggtcgaagtc
     8461 gtccagggcg tcggtgggtc cgccgctctc gcacttcagc tgcttctcga ggccgcagat
     8521 gatcagttcc aggccgaaca ggaaggcggg ctcggcgccc tgtctgtcga acagctcgat
     8581 ggcctgcttc agcagggggg gcatgctgtc ggtggtgggt gtctctcttt cctctttggc
     8641 gacctggtgt tcctgttctt ccagcacgca gcccagggtg aagtggccca cggcgctcag
     8701 ggcgtacagg gcgttttcca ggctgaagcc ctgctggcac aggaaggcca gctggttttc
     8761 cagtgtctcg tactgcttct cggtgggtct ggtgcccagg tgcactttgg cgccgtcccg
     8821 gtggctcagc agggcgcatc tgtagctctt ggcgttgttc cgcaggaagt cctgccagct
     8881 ctcgccttcc agagggcagc tgtgggtgtg gtgccggtcc agcatctcga tgggcagggc
     8941 gtccagcagg gcccgcttgt tcttcacgtg ccagtacagg gtgggctgtt ccacgcccag
     9001 tttctgggcc agcttccggg tggtcaggcc ctcgatgccc acgccgttca gcagttccag
     9061 ggcgctgttg atcactttgc tcttgtccag ccggctcatg tcgacattct ttgccaaaat
     9121 gatgagacag cacaataacc agcacgttgc ccaggagctg taggaaaaag aagaaggcat
     9181 gaacatggtt agcagaggct ctagagccgc cggtcacacg ccagaagccg aaccccgccc
     9241 tgccccgtcc cccccgaagg cagccgtccc cccgcggaca gccccgaggc tggagaggga
     9301 gaaggggacg gcggcgcggc gacgcacgaa ggccctcccc gcccatttcc ttcctgccgg
     9361 cgccgcaccg cttcgccccg cgcccgctag agggggtgcg gcggcgcctc ccagatttcg
     9421 gctccgcaca gatttgggac aaaggaagtc cctgcgccct ctcgcacgat taccataaaa
     9481 ggcaatggct gcggctcgcc gcgcctcgac agccgccggc gctccggggg ccgccgcgcc
     9541 ccycccccga gcccyccccg gcccgmggcg gccccgcccc gcccggcacc cccmcctgcc
     9601 gccacccccc gcccggcacg gcgagccccg cgccacgccc cgtacggagc cccgcacccg
     9661 aagccgggcc gtgctcagca actcggggag gggggtgcag ggggggttgc agcccgaccg
     9721 acgcgcccac accccctgct caccccccca cgcacacacc ccgcacgcag cctttgttcc
     9781 cctcgcagcc ccccccgcac cgcggggcac cgcccccggc cgcgctcccc tcgcgcacac
     9841 tgcggagcgc acaaagcccc gcgccgcgcc cgcagcgctc acagccgccg ggcagcgcgg
     9901 agccgcacgc ggcgctcccc acgcacacac acacgcacgc accccccgag ccgctccccc
     9961 cgcacaaagg gccctcccgg agcccctcaa ggctttcacg cagccacaga aaagaaacaa
    10021 gccgtcatta aaccaagcgc taattacagc ccggaggaga agggccgtcc cgcccgctca
    10081 cctgtgggag taacgcggtc agtcagagcc ggggcgggcg gcgcgaggcg gcggcggagc
    10141 ggggcacggg gcgaaggcag cgcgcagcga ctcccgcccg ccgcgcgctt cgctttttat
    10201 agggccgccg ccgccgccgc ctcgccataa aaggaaactt tcggagcgcg ccgctctgat
    10261 tggctgccgc cgcacctctc cgccycsccc csccccsccc cycsccccsc cccsccccsc
    10321 ccggcgcgcg cccccccccc ccccccgccc ccatcgctgc acaaaataat taaaaaataa
    10381 ataaatacaa aattgggggt ggggaggggg gggagatggg gagagtgaag cagaacgtgg
    10441 ggctcacctc gaccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
    10501 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
    10561 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
    10621 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
    10681 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
    10741 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
    10801 attactatta ataactagtc aataatcaat gtcgagaccc atagagccca ccgcatcccc
    10861 agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc
    10921 cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag
    10981 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga
    11041 tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga
    11101 tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc
    11161 ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc
    11221 caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct
    11281 gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg
    11341 ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga
    11401 accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct
    11461 ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat
    11521 gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg
    11581 gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg
    11641 tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat
    11701 ctcgagccta gggccgggat tctcctccac gtcaccgcat gttagaagac ttcctctgcc
    11761 ctctccgctg ccagatctct cgaggccctg tgggaggaag agaagaggtc agaagcttat
    11821 aacttcgtat aatgtatgct atacgaagtt attgccccac tgtggggtgg aggggacaga
    11881 taaaagtacc cagaaccaga gccacattaa ccggccctgg gaatataagg tggtcccagc
    11941 tcggggacac aggatccctg gaggcagcaa acatgctgtc ctgaagtgga cataggggcc
    12001 cgggttggag gaagaagact agctgagctc tcggacccct ggaagatgcc atgacagggg
    12061 gctggaagag ctagcacaga ctagagaggt aaggggggta ggggagctgc ccaaatgaaa
    12121 ggagtgagag gtgacccgaa tccacaggag aacggggtgt ccaggcaaag aaagcaagag
    12181 gatggagagg tggctaaagc cagggagacg gggtactttg gggttgtcca gaaaaacggt
    12241 gatgatgcag gcctacaaga aggggaggcg ggacgcaagg gagacatccg tcggagaagg
    12301 ccatcctaag aaacgagaga tggcacaggc cccagaagga gaaggaaaag ggaacccagc
    12361 gagtgaagac ggcatggggt tgggtgaggg aggagagatg cccggagagg acccagacac
    12421 ggggaggatc cgctcagagg acatcacgtg gtgcagcgcc gagaaggaag tgctccggaa
    12481 agagcatcct tgggcagcaa cacagcagag agcaagggga agagggagtg gaggaagacg
    12541 gaacctgaag gaggcggcag ggaaggatct gggccagccg tagaggtgac ccaggccaca
    12601 agctgcagac agaaagcggc acaggcccag gggagagaat gctggtcaga gaaagca
//
