LOCUS       sequence-151441-       10149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for in vitro transcription of SpCas9-Gem mRNA. Cas9 is fused to
            Geminin peptide and is degraded during G0/G1 phases of the 
            cell-cycle to minimize indels caused by non-homologous end joining.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10149)
  AUTHORS   Howden SE, McColl B, Glaser A, Vadolas J, Petrou S, Little MH, 
            Elefanty AG, Stanley EG
  TITLE     A Cas9 Variant for Efficient Generation of Indel-Free Knockin or 
            Gene-Corrected Human Pluripotent Stem Cells.
  JOURNAL   Stem Cell Reports. 2016 Sep 13;7(3):508-17. doi: 
            10.1016/j.stemcr.2016.07.001. Epub 2016 Aug 4.
  PUBMED    27499201
REFERENCE   2  (bases 1 to 10149)
  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.stemcr.2016.07.001"; journalName: "Stem Cell Reports"; 
            date: "2016-09-13- 13"; volume: "7"; issue: "3"; pages: "508-17"
COMMENT     Sequence Label: pDNR-SpCas9-Gem
FEATURES             Location/Qualifiers
     source          1..10149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             60..95
                     /codon_start=1
                     /product="6x(His-Asn) affinity tag"
                     /label=6xHN
                     /translation="HNHNHNHNHNHN"
     terminator      complement(131..225)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(246..905)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     820..839
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    complement(934..967)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        982..1427
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1428..1514,1515..2849)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        2917..3021
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3022..3090,3091..3882)
                     /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(3240..3259)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4053..4641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4542..4561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4757..4774
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4758..4774
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4782..4801
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4782..4800
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4822..4831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4828..8931
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4828..8931
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4828..8931
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             8977..9306
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     primer_bind     complement(9349..9368)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    9393..9441
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      9643..10071
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9730..9749)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9940..9961
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10080..10100)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 ggatccgctc gagaagcttt ctagaccatt cgtttggcgc gcgggcccag gtaagtggtc
       61 ataatcataa tcataatcat aatcataatc acaactagcc taggagatcc tggtcatgac
      121 tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
      181 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata
      241 tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
      301 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
      361 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
      421 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
      481 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
      541 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
      601 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
      661 agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga
      721 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
      781 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
      841 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
      901 tccattttag cttccttagc tcctgaaaga tccataactt cgtatagcat acattatacg
      961 aagttatgcg gccgcgacgt ccacatatac ctgccgttca ctattattta gtgaaatgag
     1021 atattatgat attttctgaa ttgtgattaa aaaggcaact ttatgcccat gcaacagaaa
     1081 ctataaaaaa tacagagaat gaaaagaaac agatagattt tttagttctt taggcccgta
     1141 gtctgcaaat ccttttatga ttttctatca aacaaaagag gaaaatagac cagttgcaat
     1201 ccaaacgaga gtctaataga atgaggtcga aaagtaaatc gcgcgggttt gttactgata
     1261 aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat actttggcgt caccccttac
     1321 atattttagg tcttttttta ttgtgcgtaa ctaacttgcc atcttcaaac aggagggctg
     1381 gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg aacatcaaaa
     1441 agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca ggaggcgcaa
     1501 ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac ggcatttccc
     1561 atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa aaatatcaag
     1621 ttcctgagtt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc ctggacgttt
     1681 gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac ggctaccaca
     1741 tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt tacatgttct
     1801 atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc gtctttaaag
     1861 acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa gaatggtcag
     1921 gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat ttctccggta
     1981 aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca tcagacagct
     2041 ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac ggaaaaacgt
     2101 atcaaaatgt acagcagttc atcgatgaag gcaactacag ctcaggcgac aaccatacgc
     2161 tgagagatcc tcactacgta gaagataaag gccacaaata cttagtattt gaagcaaaca
     2221 ctggaactga agatggctac caaggcgaag aatctttatt taacaaagca tactatggca
     2281 aaagcacatc attcttccgt caagaaagtc aaaaacttct gcaaagcgat aaaaaacgca
     2341 cggctgagtt agcaaacggc gctctcggta tgattgagct aaacgatgat tacacactga
     2401 aaaaagtgat gaaaccgctg attgcatcta acacagtaac agatgaaatt gaacgcgcga
     2461 acgtctttaa aatgaacggc aaatggtacc tgttcactga ctcccgcgga tcaaaaatga
     2521 cgattgacgg cattacgtct aacgatattt acatgcttgg ttatgtttct aattctttaa
     2581 ctggcccata caagccgctg aacaaaactg gccttgtgtt aaaaatggat cttgatccta
     2641 acgatgtaac ctttacttac tcacacttcg ctgtacctca agcgaaagga aacaatgtcg
     2701 tgattacaag ctatatgaca aacagaggat tctacgcaga caaacaatca acgtttgcgc
     2761 ctagcttcct gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg
     2821 aacaaggaca attaacagtt aacaaataaa aacgcaaaag aaaatgccga tatcctattg
     2881 gcattgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2941 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3001 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3061 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3121 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3181 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3241 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3301 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3361 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3421 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3481 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3541 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3601 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3661 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3721 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3781 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3841 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3901 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3961 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4021 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4081 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4141 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4201 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4261 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4321 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4381 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4441 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4501 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4561 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4621 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4681 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4741 attaccgcct tacgcgtgta aaacgacggc cagtagatct gtaatacgac tcactatagg
     4801 gcgctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgatatc
     4861 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
     4921 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
     4981 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
     5041 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     5101 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     5161 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     5221 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     5281 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     5341 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     5401 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     5461 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     5521 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     5581 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     5641 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     5701 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     5761 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     5821 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     5881 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     5941 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     6001 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     6061 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     6121 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     6181 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     6241 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     6301 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     6361 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     6421 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     6481 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     6541 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     6601 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     6661 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     6721 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     6781 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     6841 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     6901 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     6961 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     7021 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     7081 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     7141 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     7201 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     7261 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     7321 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct
     7381 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
     7441 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     7501 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     7561 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     7621 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     7681 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     7741 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     7801 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     7861 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     7921 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     7981 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     8041 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     8101 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     8161 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     8221 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     8281 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     8341 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     8401 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     8461 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     8521 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     8581 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     8641 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     8701 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     8761 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     8821 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     8881 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     8941 gacaagggat atccatcaca ctggcggccg ctcgagatga atcccagtat gaagcagaaa
     9001 caagaagaaa tcaaagagaa tataaagaat agttctgtcc caagaagaac tctgaagatg
     9061 attcagcctt ctgcatctgg atctcttgtt ggaagagaaa atgagctgtc cgcaggcttg
     9121 tccaaaagga aacatcggaa tgaccactta acatctacaa cttccagccc tggggttatt
     9181 gtcccagaat ctagtgaaaa taaaaatctt ggaggagtca cccaggagtc atttgatctt
     9241 atgattaaag aaaatccatc ctctcagtat tggaaggaag tggcagaaaa acggagaaag
     9301 gcgctgtaat ctaccggtta gtaatgagtt taaacggggg aggctaactg aaacacggaa
     9361 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     9421 cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg
     9481 ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc
     9541 accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg
     9601 ccatagcaga tctgcgcagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     9661 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     9721 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     9781 gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg
     9841 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     9901 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     9961 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt
    10021 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
    10081 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
    10141 aagcatgca
//
