LOCUS       sequence-323775-        9955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9 and mRFP in mammalian cells (an insertion vector to
            generate Cas9-knockin cell lines).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9955)
  AUTHORS   Park J, Lim JM, Jung I, Heo SJ, Park J, Chang Y, Kim HK, Jung D, Yu 
            JH, Min S, Yoon S, Cho SR, Park T, Kim HH
  TITLE     Recording of elapsed time and temporal information about biological 
            events using Cas9.
  JOURNAL   Cell. 2021 Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014.
  PUBMED    33539780
REFERENCE   2  (bases 1 to 9955)
  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: "Cell. 2021 
            Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014."
COMMENT     Sequence Label: pcDNA5/FRT/CMV_promoter-Cas9-E2A-mRFP
FEATURES             Location/Qualifiers
     source          1..9955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      281..290
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             287..4390
                     /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             287..4390
                     /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             4406..4426
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4427..4453
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4427..4453
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4469..4528
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             4535..5248
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(5293..5310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5299..5523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5656..5675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5807..5854
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             5862..6884
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5862..6884
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    7014..7135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7051..7070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7105..7124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7184..7200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7184..7200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7197..7219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7208..7224
                     /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(7232..7238,7239..7256,7257..7262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7277..7298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7415..7432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7586..8174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7666..7685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8345..9136,9137..9205))
                     /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     8968..8987
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9206..9310)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9385..9404)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        9576..9955
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctt gccaccatgg acaagaagta
      301 cagcatcggc ctggacatcg gtaccaacag cgtgggctgg gccgtgatca ccgacgagta
      361 caaggtgccc agcaagaagt tcaaggtgct gggcaacacc gaccgccaca gcatcaagaa
      421 gaacctgatc ggcgccctgc tgttcgacag cggcgagacc gccgaggcca cccgcctgaa
      481 gcgcaccgcc cgccgccgct acacccgccg caagaaccgc atctgctacc tgcaggagat
      541 cttcagcaac gagatggcca aggtggacga cagcttcttc caccgcctgg aggagagctt
      601 cctggtggag gaggacaaga agcacgagcg ccaccccatc ttcggcaaca tcgtggacga
      661 ggtggcctac cacgagaagt accccaccat ctaccacctg cgcaagaagc tggtggacag
      721 caccgacaag gccgacctgc gcctgatcta cctggccctg gcccacatga tcaagttccg
      781 cggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
      841 catccagctg gtgcagacct acaaccagct gttcgaggag aaccccatca acgccagcgg
      901 cgtggacgcc aaggccatcc tgagcgcccg cctgagcaag agccgccgcc tggagaacct
      961 gatcgcccag ctgcccggcg agaagaagaa cggcctgttc ggcaacctga tcgccctgag
     1021 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggacg ccaagctgca
     1081 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     1141 gtacgccgac ctgttcctgg ccgccaagaa cctgagcgac gccatcctgc tgagcgacat
     1201 cctgcgcgtg aacaccgaga tcaccaaggc ccccctgagc gccagcatga tcaagcgcta
     1261 cgacgagcac caccaggacc tgaccctgct gaaggccctg gtgcgccagc agctgcccga
     1321 gaagtacaag gagatcttct tcgaccagag caagaacggc tacgccggct acatcgacgg
     1381 cggcgccagc caggaggagt tctacaagtt catcaagccc atcctggaga agatggacgg
     1441 caccgaggag ctgctggtga agctgaaccg cgaggacctg ctgcgcaagc agcgcacctt
     1501 cgacaacggc agcatccccc accagatcca cctgggcgag ctgcacgcca tcctgcgccg
     1561 ccaggaggac ttctacccct tcctgaagga caaccgcgag aagatcgaga agatcctgac
     1621 cttccgcatc ccctactacg tgggccccct ggcccgcggc aacagccgct tcgcctggat
     1681 gacccgcaag agcgaggaga ccatcacccc ctggaacttc gaggaggtgg tggacaaggg
     1741 cgccagcgcc cagagcttca tcgagcgcat gaccaacttc gacaagaacc tgcccaacga
     1801 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac
     1861 caaggtgaag tacgtgaccg agggcatgcg caagcccgcc ttcctgagcg gcgagcagaa
     1921 gaaggccatc gtggacctgc tgttcaagac caaccgcaag gtgaccgtga agcagctgaa
     1981 ggaggactac ttcaagaaga tcgagtgctt cgacagcgtg gagatcagcg gcgtggagga
     2041 ccgcttcaac gccagcctgg gcacctacca cgacctgctg aagatcatca aggacaagga
     2101 cttcctggac aacgaggaga acgaggacat cctggaggac atcgtgctga ccctgaccct
     2161 gttcgaggac cgcgagatga tcgaggagcg cctgaagacc tacgcccacc tgttcgacga
     2221 caaggtgatg aagcagctga agcgccgccg ctacaccggc tggggccgcc tgagccgcaa
     2281 gcttatcaac ggcatccgcg acaagcagag cggcaagacc atcctggact tcctgaagag
     2341 cgacggcttc gccaaccgca acttcatgca gctgatccac gacgacagcc tgaccttcaa
     2401 ggaggacatc cagaaggccc aggtgagcgg ccagggcgac agcctgcacg agcacatcgc
     2461 caacctggcc ggcagccccg ccatcaagaa gggcatcctg cagaccgtga aggtggtgga
     2521 cgagctggtg aaggtgatgg gccgccacaa gcccgagaac atcgtgatcg agatggcccg
     2581 cgagaaccag accacccaga agggccagaa gaacagccgc gagcgcatga agcgcatcga
     2641 ggagggcatc aaggagctgg gcagccagat cctgaaggag caccccgtgg agaacaccca
     2701 gctgcagaac gagaagctgt acctgtacta cctgcagaac ggccgcgaca tgtacgtgga
     2761 ccaggagctg gacatcaacc gcctgagcga ctacgacgtg gaccacatcg tgccccagag
     2821 cttcctgaag gacgacagca tcgacaacaa ggtgctgacc cgcagcgaca agaaccgcgg
     2881 caagagcgac aacgtgccca gcgaggaggt ggtgaagaag atgaagaact actggcgcca
     2941 gctgctgaac gccaagctga tcacccagcg caagttcgac aacctgacca aggccgagcg
     3001 cggcggcctg agcgagctgg acaaggccgg cttcatcaag cgccagctgg tggagacccg
     3061 ccagatcacc aagcacgtgg cccagatcct ggacagccgc atgaacacca agtacgacga
     3121 gaacgacaag ctgatccgcg aggtgaaggt gatcaccctg aagagcaagc tggtgagcga
     3181 cttccgcaag gacttccagt tctacaaggt gcgcgagatc aacaactacc accacgccca
     3241 cgacgcctac ctgaacgccg tggtgggcac cgccctgatc aagaagtacc ccaagctgga
     3301 gagcgagttc gtgtacggcg actacaaggt gtacgacgtg cgcaagatga tcgccaagag
     3361 cgagcaggag atcggcaagg ccaccgccaa gtacttcttc tacagcaaca tcatgaactt
     3421 cttcaagacc gagatcaccc tggccaacgg cgagatccgc aagcgccccc tgatcgagac
     3481 caacggcgag accggcgaga tcgtgtggga caagggccgc gacttcgcca ccgtgcgcaa
     3541 ggtgctgagc atgccccagg tgaacatcgt gaagaagacc gaggtgcaga ccggcggctt
     3601 cagcaaggag agcatcctgc ccaagcgcaa cagcgacaag ctgatcgccc gcaagaagga
     3661 ctgggacccc aagaagtacg gcggcttcga cagccccacc gtggcctaca gcgtgctggt
     3721 ggtggccaag gtggagaagg gcaagagcaa gaagctgaag agcgtgaagg agctgctggg
     3781 catcaccatc atggagcgca gcagcttcga gaagaacccc atcgacttcc tggaggccaa
     3841 gggctacaag gaggtgaaga aggacctgat catcaagctg cccaagtaca gcctgttcga
     3901 gctggagaac ggccgcaagc gcatgctggc cagcgccggc gagctgcaga agggcaacga
     3961 gctggccctg cccagcaagt acgtgaactt cctgtacctg gccagccact acgagaagct
     4021 gaagggcagc cccgaggaca acgagcagaa gcagctgttc gtggagcagc acaagcacta
     4081 cctggacgag atcatcgagc agatcagcga gttcagcaag cgcgtgatcc tggccgacgc
     4141 caacctggac aaggtgctga gcgcctacaa caagcaccgc gacaagccca tccgcgagca
     4201 ggccgagaac atcatccacc tgttcaccct gaccaacctg ggcgcccccg ccgccttcaa
     4261 gtacttcgac accaccatcg accgcaagcg ctacaccagc accaaggagg tgctggacgc
     4321 caccctgatc caccagagca tcaccggtct gtacgagacc cgcatcgacc tgagccagct
     4381 gggcggcgac ggcggctccg gacctccaaa gaaaaagaga aaagtatacc cctacgacgt
     4441 gcccgactac gccgaattcg gaagcgggca gtgcaccaac tacgccctgc tgaagctggc
     4501 cggcgacgtg gagagcaacc ccggccccgg atccatggtg tctaagggcg aagagctgat
     4561 taaggagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc accacttcaa
     4621 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
     4681 ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg
     4741 cagcagaacc ttcatcaacc acacccaggg catccccgac ttctttaagc agtccttccc
     4801 tgagggcttc acatgggaga gagtcaccac atacgaggac gggggcgtgc tgaccgctac
     4861 ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa
     4921 cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccaacaccga
     4981 gatgctgtac cccgctgacg gcggcctgga aggcagaagc gacatggccc tgaagctcgt
     5041 gggcgggggc cacctgatct gcaacttcaa gaccacatac agatccaaga aacccgctaa
     5101 gaacctcaag atgcccggcg tctactatgt ggaccacaga ctggaaagaa tcaaggaggc
     5161 cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc
     5221 tagcaaactg gggcacaaac ttaattgagc ggccgctcga gtctagaggg cccgtttaaa
     5281 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     5341 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     5401 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     5461 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     5521 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     5581 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5641 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5701 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5761 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     5821 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     5881 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     5941 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     6001 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     6061 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     6121 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     6181 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     6241 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     6301 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     6361 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     6421 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     6481 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     6541 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     6601 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     6661 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     6721 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     6781 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     6841 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     6901 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     6961 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     7021 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     7081 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     7141 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     7201 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     7261 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     7321 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     7381 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     7441 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     7501 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     7561 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     7621 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     7681 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     7741 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     7801 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     7861 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     7921 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     7981 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     8041 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     8101 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     8161 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     8221 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     8281 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     8341 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     8401 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     8461 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     8521 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     8581 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     8641 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     8701 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     8761 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     8821 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     8881 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     8941 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     9001 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     9061 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     9121 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     9181 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     9241 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     9301 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     9361 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     9421 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     9481 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     9541 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     9601 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     9661 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     9721 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     9781 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     9841 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     9901 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatg
//
