LOCUS       sequence-179978-        8507 bp    DNA     linear   UNA 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      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8507)
  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 8507)
  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..8507
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        118..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     669..688
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        767..871
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(872..940,941..1732)
                     /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(1090..1109)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1903..2491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2392..2411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2645..2662
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2779..2800
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2815..2820,2821..2838,2839..2845)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2853..2869
                     /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     2858..2880
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2877..2893
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2877..2893
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2942..3063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2953..2972)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3007..3026
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(3106..3819,3820..3822,3823..3825))
                     /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             complement(join(3109..3819,3820..3822,3823..3825))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3151..3172)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3500..3519
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3759..3780
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3826..3879)
                     /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             complement(3895..3915)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(3928..8001,8002..8004,8005..8031))
                     /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             complement(3928..8031)
                     /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"
     regulatory      8028..8037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(8071..8090)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(8152..8174)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cccccccccc gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg
       61 gggtggggag gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat
      121 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
      181 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
      241 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
      301 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
      361 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
      421 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
      481 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
      541 cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta
      601 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
      661 taactatgcg gcatcagagc agattgtact gagagtcgac cctaggggat cgggagatcg
      721 atctcccgat ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      781 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      841 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      901 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      961 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     1021 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     1081 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1141 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1201 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1261 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1321 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1381 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1441 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1501 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1561 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1621 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1681 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1741 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1801 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1861 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1921 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1981 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2041 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2101 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2161 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2221 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2281 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2341 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2401 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2461 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2521 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2581 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2641 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2701 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2761 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcaccct aggctttaca
     2821 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     2881 aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg
     2941 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3001 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3061 gttggggtgg gcgaagaact ccagcatgag atccccgcgc tgcagttact tgtacagctc
     3121 gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg
     3181 cttctcgttg gggtctttgc tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag
     3241 cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct
     3301 gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc
     3361 ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt
     3421 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     3481 gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc
     3541 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     3601 gtagcggctg aagcactgca cgccgtaggt cagggtggtc acgagggtgg gccagggcac
     3661 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     3721 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     3781 gatgggcacc accccggtga acagctcctc gcccttgctc accattgggc cgggattctc
     3841 ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccgccgccgg acctcacctt
     3901 cctcttcttc ttggggtcag ccctgctgtc tccaccgagc tgagagaggt cgattcttgt
     3961 ttcatagagc cccgtaattg actgatgaat cagtgtggcg tccaggacct cctttgtaga
     4021 ggtgtaccgc tttctgtcta tggtggtgtc gaagtacttg aaggctgcag gcgcgcccaa
     4081 gttggtcaga gtaaacaagt ggataatgtt ttctgcctgc tccctgatgg gcttatccct
     4141 gtgcttattg taagcagaaa gcaccttatc gaggttagcg tcggcgagga tcactctttt
     4201 ggagaattcg cttatttgct cgatgatctc atcaaggtag tgtttgtgtt gttccacgaa
     4261 cagctgcttc tgctcattat cttcgggaga ccctttgagc ttttcatagt ggctggccag
     4321 atacaagaaa ttaacgtatt tagagggcag tgccagctcg ttacctttct gcagctcgcc
     4381 cgcactagcg agcattcgtt tccggccgtt ttcaagctca aagagagagt acttgggaag
     4441 cttaatgatg aggtcttttt tgacctcttt atatcctttc gcctcgagaa agtcgatggg
     4501 gtttttttcg aagcttgatc gctccatgat tgtgatgccc agcagttcct tgacgctttt
     4561 gagtttttta gacttccctt tctccacttt ggccacaacc agtacactgt aagcgactgt
     4621 aggagaatcg aatccgccgt atttcttggg gtcccaatct tttttgcgtg cgatcagctt
     4681 gtcgctgttc cttttcggga ggatactttc cttggagaag cctccggtct gtacttcggt
     4741 ctttttaacg atgttcacct gcggcatgga caggaccttc cggactgtcg cgaaatccct
     4801 acccttgtcc cacacgattt ctcctgtttc tccgtttgtt tcgataagtg gtcgcttccg
     4861 aatctctcca ttggccagtg taatctcggt cttgaaaaaa ttcataatat tgctgtaaaa
     4921 gaagtactta gcggtggcct tgcctatttc ctgctcagac tttgcgatca ttttcctaac
     4981 atcgtacact ttatagtctc cgtaaacaaa ttcagattca agcttgggat attttttgat
     5041 aagtgcagtg cctaccactg cattcaggta ggcatcatgc gcatggtggt aattgttgat
     5101 ctctctcacc ttataaaact gaaagtcctt tctgaaatct gagaccagct tagacttcag
     5161 agtaataact ttcacctctc gaatcagttt gtcattttca tcgtacttgg tgttcatgcg
     5221 tgaatcgaga atttgggcca cgtgcttggt gatctggcgt gtctcaacaa gctgcctttt
     5281 gatgaagccg gctttatcca actcagacag gccacctcgt tcagccttag tcagattatc
     5341 gaacttccgt tgtgtgatca gtttggcgtt cagcagctgc cgccaataat ttttcatttt
     5401 cttgacaact tcttctgagg ggacgttatc actcttccct ctatttttat cggatcttgt
     5461 caacacttta ttatcaatag aatcatcttt gagaaaagac tggggcacga tatgatccac
     5521 gtcgtagtcg gagagccgat tgatgtccag ttcctgatcc acgtacatgt ccctgccgtt
     5581 ctgcaggtag tacaggtaga gcttctcatt ctgaagctgg gtgttttcaa ctgggtgttc
     5641 cttaaggatt tgggacccca gttcttttat accctcttca atcctcttca tcctttccct
     5701 actgttcttc tgtcccttct gggtagtttg gttctctcgg gccatctcga taacgatatt
     5761 ctcgggctta tgccttccca ttactttgac gagttcatcc acgaccttaa cggtctgcag
     5821 tattcccttt ttgatagctg ggctacctgc aagattagcg atgtgctcgt gaagactgtc
     5881 cccctggcca gaaacttgtg ctttctggat gtcctcctta aaggtgagag agtcatcatg
     5941 gatcaactgc atgaagttcc ggttggcaaa tccatcggac ttaagaaaat ccaggattgt
     6001 ctttccactc tgcttgtctc ggatcccatt gatcagtttt cttgacagcc gcccccatcc
     6061 tgtatatcgg cgcctcttga gctgtttcat gactttgtcg tcgaagagat gagcgtaagt
     6121 tttcaagcgt tcttcaatca tctccctatc ttcaaacaac gtaagggtga ggacaatgtc
     6181 ctcaagaatg tcctcgttct cctcattgtc caggaagtcc ttgtctttaa tgattttcag
     6241 gagatcgtga tacgttccca gggatgcgtt gaagcgatcc tccactccgc tgatttcaac
     6301 agagtcgaaa cattcaatct ttttgaaata gtcttctttg agctgtttca cggtaacttt
     6361 ccggttcgtc ttgaagagga ggtccacgat agctttcttc tgctctccag acaggaatgc
     6421 tggctttctc atcccttctg tgacgtattt gaccttggtg agctcgttat aaactgtgaa
     6481 gtactcgtac agcagagagt gtttaggaag caccttttcg ttaggcagat ttttatcaaa
     6541 gttagtcatc ctttcgatga aggactgggc agaggccccc ttatccacga cttcctcgaa
     6601 gttccaggga gtgatggtct cttctgattt gcgagtcatc cacgcgaatc tggaatttcc
     6661 ccgggcgagg gggcctacat agtagggtat ccgaaatgtg aggattttct caatcttttc
     6721 cctgttatct ttcaaaaagg ggtagaaatc ctcttgccgc ctgaggatag cgtgcagttc
     6781 gcccaggtga atctggtggg ggatgcttcc attgtcgaaa gtgcgctgtt tgcgcaacag
     6841 atcttctctg ttaagcttta ccagcagctc ctcggtgccg tccatttttt ccaagatggg
     6901 cttaataaat ttgtaaaatt cctcctggct tgctccgccg tcaatgtatc cggcgtagcc
     6961 atttttagac tgatcgaaga aaatttcctt gtacttctca ggcagttgct gtctgacaag
     7021 ggccttcagc aaagtcaagt cttggtggtg ctcatcatag cgcttgatca tactagcgct
     7081 cagcggagct ttggtgatct ccgtgttcac tcgcagaata tcactcagca gaatggcgtc
     7141 tgacaggttc tttgccgcca aaaaaaggtc tgcgtactgg tcgccgatct gggccagcag
     7201 attgtcgaga tcatcatcgt aggtgtcttt gctcagttga agcttggcat cttcggccag
     7261 gtcgaagtta gatttaaagt tgggggtcag cccgagtgac agggcgataa gattaccaaa
     7321 caggccgttc ttcttctccc cagggagctg tgcgatgagg ttttcgagcc gccgggattt
     7381 ggacagccta gcgctcagga ttgctttggc gtcaactccg gatgcgttga tcgggttctc
     7441 ttcgaaaagc tgattgtaag tctgaaccag ttggataaag agtttgtcga catcgctgtt
     7501 gtctgggttc aggtccccct cgatgaggaa gtgtccccga aatttgatca tatgcgccag
     7561 cgcgagatag atcaaccgca agtcagcctt atcagtactg tctacaagct tcttcctcag
     7621 atgatatatg gttgggtact tttcatggta cgccacctcg tccacgatat tgccaaagat
     7681 tgggtggcgc tcgtgctttt tatcctcctc caccaaaaag gactcctcca gcctatggaa
     7741 gaaagagtca tccaccttag ccatctcatt actaaagatc tcctgcaggt agcagatccg
     7801 attctttctg cgggtatatc tgcgccgtgc tgttcttttg agccgcgtgg cttcggccgt
     7861 ctccccggag tcgaacagga gggcgccaat gaggttcttc tttatgctgt ggcgatcggt
     7921 attgcccaga actttgaatt ttttgctcgg caccttgtac tcgtccgtaa tgacggccca
     7981 gccgacgctg tttgtgccga tagcgagccc aatggagtac ttcttgtcca tggtggcggc
     8041 cgcgaattct ttgccaaaat gatgagacag cacaataacc agcacgttgc ccaggagctg
     8101 taggaaaaag aagaaggcat gaacatggtt agcagaggcg ctagagccgc cggtcacacg
     8161 ccagaagccg aaccccgccc tgccccgtcc cccccgaagg cagccgtccc cccgcggaca
     8221 gccccgaggc tggagaggga gaaggggacg gcggcgcggc gacgcacgaa ggccctcccc
     8281 gcccatttcc ttcctgccgg cgccgcaccg cttcgccccg cgcccgctag agggggtgcg
     8341 gcggcgcctc ccagatttcg gctccgcaca gatttgggac aaaggaagtc cctgcgccct
     8401 ctcgcacgat taccataaaa ggcaatggct gcggctcgcc gcgcctcgac agccgccggc
     8461 gctccggggg ccgccgcgcc cctcccccga gccctccccg gcccgag
//
