LOCUS       sequence-135035-        9711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express hCas9 byT7 promoter with polyadenine tails.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9711)
  AUTHORS   Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T
  TITLE     ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in
            zygotes.
  JOURNAL   Nat Commun. 2016 Jan 20;7:10431. doi: 10.1038/ncomms10431.
  PUBMED    26786405
REFERENCE   2  (bases 1 to 9711)
  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: "Nat 
            Commun."; date: "2016-01-20"; volume: "7:10431. doi"; pages: " 
            10.1038/ncomms10431"
COMMENT     Sequence Label: pCas9-polyA
FEATURES             Location/Qualifiers
     source          1..9711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     728..747
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        728..746
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      754..763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             766..786
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             811..4911
                     /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             811..4911
                     /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             811..4911
                     /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             4924..4944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4999..5018)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     primer_bind     complement(5350..5369)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     rep_origin      5391..5819
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5478..5497)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5688..5709
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5828..5848)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5833..6162
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6013..6148
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6075..6094
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6229..7023
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6283..6302)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6893..6912
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7199..7320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7236..7255)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7290..7309
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7369..7385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7369..7385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7382..7404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7393..7409
                     /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(7417..7423,7424..7441,7442..7447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7462..7483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7600..7617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7771..8359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7851..7870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8530..9321,9322..9390))
                     /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     9153..9172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9391..9495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9570..9589)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagtaa tacgactcac tatagggaga gccgccacca tggccccaaa gaagaagcgg
      781 aaggtcggta tccacggagt cccagcagcc gacaagaagt actccattgg gctcgatatc
      841 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      901 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      961 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
     1021 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1081 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1141 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1201 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1261 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1321 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1381 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1441 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1501 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1561 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1621 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1681 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1741 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1801 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1861 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1921 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1981 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     2041 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2101 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2161 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2221 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2281 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2341 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2401 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2461 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2521 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2581 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2641 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2701 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2761 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2821 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2881 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2941 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     3001 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3061 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3121 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3181 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3241 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3301 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct
     3361 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
     3421 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3481 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3541 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3601 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3661 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3721 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3781 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3841 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3901 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3961 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     4021 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4081 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4141 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4201 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4261 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4321 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4381 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4441 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4501 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4561 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4621 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4681 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4741 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4801 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4861 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4921 gaccccaaga agaagaggaa ggtgtgaaag ggttcgatcc ctaccggtta gtaatgagtt
     4981 taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     5041 gacggcaata aaaagacaga ataaaacgca cggaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5101 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5161 aaagctagct gtacatcgat accccaccga gaccccattg aaggtgcaca aataccactg
     5221 agatcttttg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     5281 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcagatc
     5341 tgcgcagctc actcggaagg acatatgggg gggctctagg gggtatcccc acgcgccctg
     5401 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5461 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5521 ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta gtgctttacg
     5581 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5641 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5701 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5761 ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     5821 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     5881 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     5941 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     6001 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     6061 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     6121 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     6181 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     6241 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     6301 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     6361 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     6421 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     6481 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     6541 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     6601 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     6661 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     6721 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     6781 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     6841 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     6901 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     6961 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     7021 tgagcgggac tctggggttc gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     7081 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     7141 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     7201 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     7261 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     7321 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     7381 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     7441 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     7501 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     7561 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     7621 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     7681 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     7741 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     7801 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     7861 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     7921 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     7981 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     8041 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     8101 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     8161 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     8221 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     8281 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     8341 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     8401 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     8461 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     8521 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     8581 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     8641 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     8701 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     8761 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     8821 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     8881 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8941 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     9001 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     9061 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     9121 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     9181 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     9241 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     9301 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     9361 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     9421 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     9481 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     9541 tctcccgatc ccctatggtc gactctcagt acaatctgct ctgatgccgc atagttaagc
     9601 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     9661 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag g
//
