LOCUS       sequence-179979-        9270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of human codon-optimized Cas9 (D10A) mutant nickase 
            and GFP, plasmid optimized for expression in human pluripotent stem 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9270)
  AUTHORS   Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K
  TITLE     Enhanced efficiency of human pluripotent stem cell genome editing 
            through replacing TALENs with CRISPRs.
  JOURNAL   Cell Stem Cell. 2013 Apr 4;12(4):393-4. doi: 
            10.1016/j.stem.2013.03.006.
  PUBMED    23561441
REFERENCE   2  (bases 1 to 9270)
  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.2013.03"; journalName: "Cell Stem Cell"; date: 
            "2013-04-4- 4"; volume: "12"; issue: "4"; pages: "393-4"
COMMENT     Sequence Label: pCas9D10A_GFP
FEATURES             Location/Qualifiers
     source          1..9270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..664
                     /label=chicken beta-actin promoter
     intron          665..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1738..1747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1744..1770,1771..1773,1774..5847)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1744..5847
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5860..5880
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5896..5949
                     /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             join(5950..5952,5953..5955,5956..6669)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5950..5952,5953..5955,5956..6666)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5995..6016)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6256..6275)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6603..6624
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6712..6833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6749..6768)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6803..6822
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6882..6898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6882..6898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6895..6917)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6906..6922
                     /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(6930..6936,6937..6954,6955..6960))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6975..6996
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7113..7130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7284..7872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7364..7383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8043..8834,8835..8903))
                     /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     8666..8685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8904..9008)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9087..9106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga
      841 aagccttaaa gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagcgcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt cgcggccgcc
     1741 accatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc
     1801 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
     1861 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc
     1921 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
     1981 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     2041 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     2101 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     2161 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     2221 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     2281 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     2341 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     2401 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     2461 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc
     2521 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     2581 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc
     2641 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     2701 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     2761 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     2821 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     2881 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     2941 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg
     3001 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag
     3061 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat
     3121 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag
     3181 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat
     3241 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca
     3301 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc
     3361 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt
     3421 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa
     3481 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa
     3541 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt
     3601 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac
     3661 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg
     3721 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc
     3781 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat
     3841 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt
     3901 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag
     3961 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc
     4021 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa
     4081 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac
     4141 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc
     4201 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat
     4261 catatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga
     4321 tccgataaaa atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg
     4381 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat
     4441 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg
     4501 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg
     4561 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag
     4621 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac
     4681 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa
     4741 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg
     4801 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac
     4861 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag
     4921 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat
     4981 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa
     5041 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg
     5101 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc
     5161 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc
     5221 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc
     5281 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc
     5341 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag
     5401 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc
     5461 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg
     5521 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga
     5581 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat
     5641 aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc
     5701 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca
     5761 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga
     5821 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg
     5881 aggtccggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
     5941 cccggcccaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     6001 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     6061 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     6121 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     6181 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     6241 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     6301 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     6361 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     6421 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     6481 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     6541 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     6601 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     6661 tacaagtaac tgcagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     6721 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     6781 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     6841 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     6901 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     6961 gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     7021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7081 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     7141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     7201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     7261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     7321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     7381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     7441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     7501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     7561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7681 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     7921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     7981 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     8041 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     8101 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     8161 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     8221 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     8281 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     8341 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     8401 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     8461 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     8521 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     8581 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     8641 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     8701 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     8761 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     8821 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     8881 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     8941 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     9001 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcgatct
     9061 cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata gttaagccag
     9121 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     9181 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     9241 gcgctgcttc gcgatgtacg ggccagatat
//
