LOCUS       sequence-224965-       11545 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 11545)
  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 11545)
  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..11545
                     /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        44..292
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     44..64
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     215..234
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        314..389
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        424..803
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        805..1082
                     /label=chicken beta-actin promoter
     intron          1083..2094
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2018..2040
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2107..2126
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2107..2125
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2130..2139
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2136..6239
                     /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             2136..6239
                     /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             6243..6269
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6249..6275
                     /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             6288..6308
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6315..6335
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6359..6375)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    6395..6619
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          6840..7778
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7735..7755
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      7813..7822
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7819..7821,7822..7824,7825..8538)
                     /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    8569..8680
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8798..8815)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8798..8814)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8807..8829)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8956..9411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9043..9062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9253..9274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9437..9541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9542..9610,9611..10402)
                     /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(9760..9779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10573..11161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11062..11081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11315..11332
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11449..11470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11485..11490,11491..11508,11509..11515)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11523..11539
                     /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(11528..11545,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc cccccccttc accgagggcc tatttcccat
       61 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
      121 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      181 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
      241 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgactccag
      301 tctttctaga agagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      361 aacttgaaaa agtggcaccg agtcggtgct ttttttgatc caagggtggg gcgcgccgga
      421 ttcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      481 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      541 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      601 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      661 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      721 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      781 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      841 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      901 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      961 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1021 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1081 cgggagtcgc tgcgtcgcgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1141 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1201 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
     1261 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
     1321 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
     1381 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1441 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1501 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1561 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
     1621 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1681 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
     1741 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1801 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
     1861 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1921 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
     1981 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     2041 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagatcctt
     2101 aattaataat acgactcact ataggggccg ccaccatgga caagaagtac agcatcggcc
     2161 tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     2221 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     2281 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     2341 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     2401 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     2461 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     2521 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     2581 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     2641 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     2701 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     2761 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     2821 tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga
     2881 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     2941 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     3001 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     3061 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     3121 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     3181 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     3241 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     3301 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     3361 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     3421 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     3481 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     3541 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     3601 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     3661 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     3721 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
     3781 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     3841 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     3901 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     3961 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     4021 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     4081 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     4141 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     4201 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     4261 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     4321 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     4381 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     4441 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     4501 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     4561 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     4621 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg
     4681 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
     4741 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
     4801 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     4861 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     4921 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
     4981 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     5041 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     5101 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     5161 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     5221 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     5281 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     5341 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
     5401 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     5461 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
     5521 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     5581 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     5641 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     5701 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     5761 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     5821 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     5881 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
     5941 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     6001 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
     6061 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     6121 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     6181 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacg
     6241 cctatcccta tgacgtgccc gattatgcca gcctgggcag cggctccccc aagaaaaaac
     6301 gcaaggtgga agatcctaag aaaaagcgga aagtggactg aacgcgtaaa tgattgcaga
     6361 tccactagtt ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     6421 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6481 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6541 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     6601 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gagatccact
     6661 agttctagcc tcgaggctag agcggccccg gtgcctagag aaggtggcgc ggggtaaact
     6721 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     6781 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     6841 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
     6901 cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa
     6961 gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg
     7021 aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc
     7081 tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt
     7141 ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt
     7201 ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg
     7261 gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc
     7321 tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt
     7381 cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa
     7441 aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg
     7501 cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc
     7561 acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt
     7621 atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca gcttggcact
     7681 tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc
     7741 ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgagcg atcgcatcga
     7801 cagtgtttaa acgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     7861 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgag gggcgagggc
     7921 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     7981 cccgtgccct ggcccaccct cgtgaccacc ctgagccacg gcgtgcagtg cttcgcccgc
     8041 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     8101 caggagcgca ccatcttctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag
     8161 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcgtcgactt caaggaggac
     8221 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacat ctatatcatg
     8281 gccgtcaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa cgtggaggac
     8341 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     8401 ctgctgcccg acagccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag
     8461 aagcgcgatc acatggtcct gctggagttc cgcaccgccg ccgggatcac tctcggcatg
     8521 gacgagctgt acaagtgaga attcctagag ctcgctgatc agcctcgact gtgccttcta
     8581 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     8641 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     8701 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata
     8761 gcaggcatgc gtttaaacat tagtcgacag ggccgccact ggccgtcgtt ttacaacgtc
     8821 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     8881 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     8941 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     9001 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     9061 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     9121 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     9181 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     9241 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     9301 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     9361 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     9421 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     9481 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     9541 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     9601 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     9661 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     9721 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     9781 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     9841 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     9901 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     9961 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
    10021 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
    10081 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
    10141 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
    10201 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
    10261 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
    10321 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
    10381 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
    10441 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
    10501 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
    10561 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
    10621 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
    10681 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
    10741 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
    10801 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
    10861 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
    10921 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
    10981 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
    11041 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
    11101 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
    11161 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
    11221 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
    11281 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
    11341 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
    11401 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
    11461 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
    11521 aattgtgagc ggataacaat ttcac
//
