LOCUS       sequence-168995-       13554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 expression vector for targeting to the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13554)
  AUTHORS   Bertero A, Pawlowski M, Ortmann D, Snijders K, Yiangou L, Cardoso de
            Brito M, Brown S, Bernard WG, Cooper JD, Giacomelli E, Gambardella 
            L, Hannan NR, Iyer D, Sampaziotis F, Serrano F, Zonneveld MC, Sinha 
            S, Kotter M, Vallier L
  TITLE     Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro 
            studies of human development using hPSCs.
  JOURNAL   Development. 2016 Dec 1;143(23):4405-4418.
  PUBMED    27899508
REFERENCE   2  (bases 1 to 13554)
  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: 
            "Development."; date: "2016-12-1"; volume: "143(23)"; pages: 
            "4405-4418"
COMMENT     Sequence Label: pAAV-Neo_CAG-Cas9
FEATURES             Location/Qualifiers
     source          1..13554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    33..836
                     /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    843..868
                     /label=SA
                     /note="splice acceptor site"
     CDS             892..945
                     /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             955..1758
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1790..1807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1796..2020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        2026..2405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2407..2684
                     /label=chicken beta-actin promoter
     intron          2686..3703
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3627..3649
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3711..3730
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3759..3768
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3768..3833
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3840..3860
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3885..7985
                     /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             3885..7985
                     /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             7986..8033
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8121..8140)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    8186..8241
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(8240..8259)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(8602..8618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8626..8642
                     /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(8650..8656,8657..8674,8675..8680))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8695..8716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    8790..9626
                     /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(9660..9679)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9661..9679)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9686..9703)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9686..9702)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9695..9717)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             9840..10142
                     /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     10021..10040
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(join(11539..12330,12331..12399))
                     /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     12162..12181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12523..13111
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13012..13031
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13265..13282
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13399..13420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13435..13440,13441..13458,13459..13465)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13473..13489
                     /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     13497..13513
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        13534..13552
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ctagtcctgc aggtttaaac gaattcgccc tttgctttct ctgaccagca ttctctcccc
       61 tgggcctgtg ccgctttctg tctgcagctt gtggcctggg tcacctctac ggctggccca
      121 gatccttccc tgccgcctcc ttcaggttcc gtcttcctcc actccctctt ccccttgctc
      181 tctgctgtgt tgctgcccaa ggatgctctt tccggagcac ttccttctcg gcgctgcacc
      241 acgtgatgtc ctctgagcgg atcctccccg tgtctgggtc ctctccgggc atctctcctc
      301 cctcacccaa ccccatgccg tcttcactcg ctgggttccc ttttccttct ccttctgggg
      361 cctgtgccat ctctcgtttc ttaggatggc cttctccgac ggatgtctcc cttgcgtccc
      421 gcctcccctt cttgtaggcc tgcatcatca ccgtttttct ggacaacccc aaagtacccc
      481 gtctccctgg ctttagccac ctctccatcc tcttgctttc tttgcctgga caccccgttc
      541 tcctgtggat tcgggtcacc tctcactcct ttcatttggg cagctcccct acccccctta
      601 cctctctagt ctgtgctagc tcttccagcc ccctgtcatg gcatcttcca ggggtccgag
      661 agctcagcta gtcttcttcc tccaacccgg gcccctatgt ccacttcagg acagcatgtt
      721 tgctgcctcc agggatcctg tgtccccgag ctgggaccac cttatattcc cagggccggt
      781 taatgtggct ctggttctgg gtacttttat ctgtcccctc caccccacag tggggcaagc
      841 ttctgacctc ttctcttcct cccacagggc ctcgagagat ctggcagcgg agagggcaga
      901 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctaggct cgagatggga
      961 tcggccattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     1021 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     1081 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     1141 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     1201 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     1261 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     1321 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     1381 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     1441 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     1501 gacggcgatg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     1561 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     1621 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     1681 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     1741 cttgacgagt tcttctgatc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1801 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1861 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1921 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1981 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg gtctcgacat tgattattga
     2041 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2101 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2161 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2221 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2281 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2341 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2401 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
     2461 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
     2521 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     2581 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     2641 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcggggagt cgctgcgacg
     2701 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     2761 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     2821 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     2881 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     2941 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
     3001 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
     3061 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     3121 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
     3181 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
     3241 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
     3301 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
     3361 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     3421 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     3481 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     3541 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     3601 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     3661 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     3721 ggttattgtg ctgtctcatc attttggcaa agaattccgc caccatggac tataaggacc
     3781 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     3841 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     3901 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     3961 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     4021 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     4081 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     4141 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     4201 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     4261 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     4321 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     4381 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     4441 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     4501 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     4561 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     4621 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     4681 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     4741 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     4801 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     4861 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     4921 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     4981 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     5041 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     5101 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     5161 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     5221 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     5281 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     5341 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     5401 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     5461 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     5521 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     5581 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     5641 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     5701 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     5761 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     5821 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     5881 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     5941 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     6001 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     6061 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     6121 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     6181 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     6241 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     6301 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     6361 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     6421 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     6481 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     6541 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     6601 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     6661 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     6721 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     6781 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     6841 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     6901 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     6961 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     7021 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     7081 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     7141 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     7201 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     7261 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     7321 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     7381 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     7441 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     7501 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     7561 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     7621 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     7681 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     7741 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     7801 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     7861 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     7921 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     7981 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaatgaa
     8041 ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     8101 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     8161 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     8221 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     8281 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     8341 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     8401 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     8461 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     8521 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     8581 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     8641 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     8701 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     8761 gtgccagcgg atcgacagta ctaagcttta ctagggacag gattggtgac agaaaagccc
     8821 catccttagg cctcctcctt cctagtctcc tgatattggg tctaaccccc acctcctgtt
     8881 aggcagattc cttatctggt gacacacccc catttcctgg agccatctct ctccttgcca
     8941 gaacctctaa ggtttgctta cgatggagcc agagaggatc ctgggaggga gagcttggca
     9001 gggggtggga gggaaggggg ggatgcgtga cctgcccggt tctcagtggc caccctgcgc
     9061 taccctctcc cagaacctga gctgctctga cgcggctgtc tggtgcgttt cactgatcct
     9121 ggtgctgcag cttccttaca cttcccaaga ggagaagcag tttggaaaaa caaaatcaga
     9181 ataagttggt cctgagttct aactttggct cttcaccttt ctagtcccca atttatattg
     9241 ttcctccgtg cgtcagtttt acctgtgaga taaggccagt agccagcccc gtcctggcag
     9301 ggctgtggtg aggagggggg tgtccgtgtg gaaaactccc tttgtgagaa tggtgcgtcc
     9361 taggtgttca ccaggtcgtg gccgcctcta ctccctttct ctttctccat ccttctttcc
     9421 ttaaagagtc cccagtgcta tctgggacat attcctccgc ccagagcagg gtcccgcttc
     9481 cctaaggccc tgctctgggc ttctgggttt gagtccttgg caagcccagg agaggcgctc
     9541 aggcttccct gtcccccttc ctcgtccacc atctcatgcc cctggctctc ctgccccttc
     9601 cctacagggg ttcctggctc tgctctaagg gcgaattcgc ggccgctaaa ttcaattcgc
     9661 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     9721 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     9781 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta tacgtacggc
     9841 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     9901 gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     9961 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
    10021 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
    10081 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
    10141 aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc acgtagaaag
    10201 ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa
    10261 gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc
    10321 tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg
    10381 gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat
    10441 ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc atgattgaac
    10501 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
    10561 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
    10621 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
    10681 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
    10741 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
    10801 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
    10861 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
    10921 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
    10981 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
    11041 tcgtcgtgac ccatgcatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
    11101 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
    11161 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
    11221 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
    11281 ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg
    11341 tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat
    11401 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagattat caaaaaggat
    11461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
    11521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
    11581 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
    11641 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
    11701 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
    11761 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
    11821 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
    11881 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
    11941 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
    12001 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
    12061 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
    12121 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
    12181 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
    12241 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
    12301 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
    12361 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
    12421 ttgaagcatt tatcagggtt attgtctcat gaccaaaatc ccttaacgtg agttttcgtt
    12481 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
    12541 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
    12601 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
    12661 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
    12721 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
    12781 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
    12841 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
    12901 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
    12961 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
    13021 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
    13081 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
    13141 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
    13201 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
    13261 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
    13321 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
    13381 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
    13441 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
    13501 aaacagctat gaccatgatt acgccaagct cagaattaac cctcactaaa ggga
//
