LOCUS       sequence-232949-       11549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Rosa26 sgRNA, Cas9 and BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11549)
  AUTHORS   Bashir S, Dang T, Rossius J, Wolf J, Kuhn R
  TITLE     Enhancement of CRISPR-Cas9 induced precise gene editing by targeting
            histone H2A-K15 ubiquitination.
  JOURNAL   BMC Biotechnol. 2020 Oct 23;20(1):57. doi: 
            10.1186/s12896-020-00650-x.
  PUBMED    33097066
REFERENCE   2  (bases 1 to 11549)
  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: "BMC 
            Biotechnol."; date: "2020-10-23"; volume: "20(1):57. doi"; pages: " 
            10.1186/s12896-020-00650-x"
COMMENT     Sequence Label: pU6Rosa-CAG-Cas9
FEATURES             Location/Qualifiers
     source          1..11549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..18
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2..18
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        43..291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     43..63
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     214..233
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        313..388
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        423..802
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        804..1081
                     /label=chicken beta-actin promoter
     intron          1082..2098
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2022..2044
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2111..2130
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2111..2129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2134..2143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2140..6243
                     /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             2140..6243
                     /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             6247..6273
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6253..6279
                     /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="YDVPDYASL"
     CDS             6292..6312
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6319..6339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6363..6379)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6399..6623
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          6844..7782
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7739..7759
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      7817..7826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7823..7825,7826..7828,7829..8542)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     polyA_signal    8573..8684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8802..8819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8802..8818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8811..8833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(8959..9414)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9096..9117)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9308..9327
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        9441..9545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9546..9614,9615..10406)
                     /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(9764..9783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10577..11165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11066..11085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11319..11336
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11453..11474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11489..11494,11495..11512,11513..11519)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11527..11543
                     /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     join(11532..11549,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc ccccccttca ccgagggcct atttcccatg
       61 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
      121 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
      181 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
      241 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgactccagt
      301 ctttctagaa gagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
      361 acttgaaaaa gtggcaccga gtcggtgctt tttttgatcc aagggtgggg cgcgccggat
      421 tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      481 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      541 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      601 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      661 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      721 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      781 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      841 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      901 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      961 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
     1021 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1081 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     1141 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     1201 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     1261 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
     1321 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
     1381 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1441 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1501 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1561 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1621 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1681 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1741 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1801 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1861 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1921 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1981 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     2041 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagat
     2101 ccttaattaa taatacgact cactataggg gccgccacca tggacaagaa gtacagcatc
     2161 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     2221 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     2281 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     2341 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     2401 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     2461 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     2521 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     2581 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     2641 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     2701 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     2761 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     2821 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
     2881 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     2941 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     3001 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     3061 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     3121 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     3181 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     3241 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     3301 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     3361 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     3421 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     3481 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     3541 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     3601 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     3661 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     3721 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     3781 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     3841 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     3901 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     3961 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     4021 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     4081 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     4141 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     4201 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     4261 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     4321 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     4381 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     4441 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     4501 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     4561 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     4621 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     4681 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     4741 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     4801 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     4861 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     4921 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     4981 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     5041 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     5101 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     5161 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     5221 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     5281 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     5341 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     5401 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     5461 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     5521 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     5581 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     5641 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     5701 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     5761 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     5821 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     5881 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     5941 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     6001 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     6061 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     6121 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     6181 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     6241 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
     6301 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg actgaacgcg taaatgattg
     6361 cagatccact agttctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     6421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     6481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     6541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     6601 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgagatc
     6661 cactagttct agcctcgagg ctagagcggc cccggtgcct agagaaggtg gcgcggggta
     6721 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     6781 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     6841 caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc
     6901 gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
     6961 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
     7021 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     7081 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     7141 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
     7201 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     7261 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     7321 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     7381 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
     7441 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     7501 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     7561 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     7621 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     7681 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     7741 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg agcgatcgca
     7801 tcgacagtgt ttaaacgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     7861 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgaggggcga
     7921 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     7981 gctgcccgtg ccctggccca ccctcgtgac caccctgagc cacggcgtgc agtgcttcgc
     8041 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     8101 cgtccaggag cgcaccatct tcttcaagga cgacggcacc tacaagaccc gcgccgaggt
     8161 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcgtcg acttcaagga
     8221 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acatctatat
     8281 catggccgtc aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtgga
     8341 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     8401 cgtgctgctg cccgacagcc actacctgag cacccagtcc gtgctgagca aagaccccaa
     8461 cgagaagcgc gatcacatgg tcctgctgga gttccgcacc gccgccggga tcactctcgg
     8521 catggacgag ctgtacaagt gagaattcct agagctcgct gatcagcctc gactgtgcct
     8581 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     8641 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     8701 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     8761 aatagcaggc atgcgtttaa acattagtcg acagggccgc cactggccgt cgttttacaa
     8821 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     8881 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     8941 agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     9001 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     9061 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     9121 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     9181 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     9241 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     9301 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     9361 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     9421 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     9481 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     9541 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     9601 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     9661 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     9721 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     9781 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     9841 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     9901 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     9961 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
    10021 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
    10081 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
    10141 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
    10201 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
    10261 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
    10321 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
    10381 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
    10441 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
    10501 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
    10561 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
    10621 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
    10681 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
    10741 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
    10801 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
    10861 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
    10921 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
    10981 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
    11041 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
    11101 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
    11161 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
    11221 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
    11281 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
    11341 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
    11401 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
    11461 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
    11521 gtggaattgt gagcggataa caatttcac
//
