LOCUS       sequence-82408-e        8271 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Fok1 nuclease domain fused to catalytically inactive Cas9 
            DNA-binding domain in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8271)
  AUTHORS   Guilinger JP, Thompson DB, Liu DR
  TITLE     Fusion of catalytically inactive Cas9 to FokI nuclease improves the 
            specificity of genome modification.
  JOURNAL   Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2909.
  PUBMED    24770324
REFERENCE   2  (bases 1 to 8271)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2909."
COMMENT     Sequence Label: FokI-dCas9
FEATURES             Location/Qualifiers
     source          1..8271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             412..477
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             484..504
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             541..1128
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             join(1177..1200,1201..1203,1204..3690,3691..3693,
                     3694..5280)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1177..4575,4576..4578,4579..5175,5176,5177..5179,
                     5180..5181,5182..5184,5185..5277)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5286..5303
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5326..5343)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5332..5556
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5627..5643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5627..5643)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5640..5662)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5651..5667
                     /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(5675..5681,5682..5699,5700..5705))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5720..5741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5858..5875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6029..6617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6109..6128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6788..7579,7580..7648))
                     /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     7411..7430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7649..7753)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7832..7851)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8023..8271,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggactacaaa
      421 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagatg
      481 gcccccaaga agaagaggaa ggtgggcatt caccgcgggg tacctggagg ttctggatcc
      541 caactagtca aaagtgaact ggaggagaag aaatctgaac ttcgtcataa attgaaatat
      601 gtgcctcatg aatatattga attaattgaa attgccagaa attccactca ggatagaatt
      661 cttgaaatga aggtaatgga attttttatg aaagtttatg gatatagagg taaacatttg
      721 ggtggatcaa ggaaaccgga cggagcaatt tatactgtcg gatctcctat tgattacggt
      781 gtgatcgtgg atactaaagc ttatagcgga ggttataatc tgccaattgg ccaagcagat
      841 gaaatgcaac gatatgtcga agaaaatcaa acacgaaaca aacatatcaa ccctaatgaa
      901 tggtggaaag tctatccatc ttctgtaacg gaatttaagt ttttatttgt gagtggtcac
      961 tttaaaggaa actacaaagc tcagcttaca cgattaaatc atatcactaa ttgtaatgga
     1021 gctgttctta gtgtagaaga gcttttaatt ggtggagaaa tgattaaagc cggcacatta
     1081 accttagagg aagtcagacg gaaatttaat aacggcgaga taaactttag cggcagcgag
     1141 actcccggga cctcagagtc cgccacaccc gaaagtgata aaaagtattc tattggttta
     1201 gctatcggca ctaattccgt tggatgggct gtcataaccg atgaatacaa agtaccttca
     1261 aagaaattta aggtgttggg gaacacagac cgtcattcga ttaaaaagaa tcttatcggt
     1321 gccctcctat tcgatagtgg cgaaacggca gaggcgactc gcctgaaacg aaccgctcgg
     1381 agaaggtata cacgtcgcaa gaaccgaata tgttacttac aagaaatttt tagcaatgag
     1441 atggccaaag ttgacgattc tttctttcac cgtttggaag agtccttcct tgtcgaagag
     1501 gacaagaaac atgaacggca ccccatcttt ggaaacatag tagatgaggt ggcatatcat
     1561 gaaaagtacc caacgattta tcacctcaga aaaaagctag ttgactcaac tgataaagcg
     1621 gacctgaggt taatctactt ggctcttgcc catatgataa agttccgtgg gcactttctc
     1681 attgagggtg atctaaatcc ggacaactcg gatgtcgaca aactgttcat ccagttagta
     1741 caaacctata atcagttgtt tgaagagaac cctataaatg caagtggcgt ggatgcgaag
     1801 gctattctta gcgcccgcct ctctaaatcc cgacggctag aaaacctgat cgcacaatta
     1861 cccggagaga agaaaaatgg gttgttcggt aaccttatag cgctctcact aggcctgaca
     1921 ccaaatttta agtcgaactt cgacttagct gaagatgcca aattgcagct tagtaaggac
     1981 acgtacgatg acgatctcga caatctactg gcacaaattg gagatcagta tgcggactta
     2041 tttttggctg ccaaaaacct tagcgatgca atcctcctat ctgacatact gagagttaat
     2101 actgagatta ccaaggcgcc gttatccgct tcaatgatca aaaggtacga tgaacatcac
     2161 caagacttga cacttctcaa ggccctagtc cgtcagcaac tgcctgagaa atataaggaa
     2221 atattctttg atcagtcgaa aaacgggtac gcaggttata ttgacggcgg agcgagtcaa
     2281 gaggaattct acaagtttat caaacccata ttagagaaga tggatgggac ggaagagttg
     2341 cttgtaaaac tcaatcgcga agatctactg cgaaagcagc ggactttcga caacggtagc
     2401 attccacatc aaatccactt aggcgaattg catgctatac ttagaaggca ggaggatttt
     2461 tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa tcctaacctt tcgcatacct
     2521 tactatgtgg gacccctggc ccgagggaac tctcggttcg catggatgac aagaaagtcc
     2581 gaagaaacga ttactccatg gaattttgag gaagttgtcg ataaaggtgc gtcagctcaa
     2641 tcgttcatcg agaggatgac caactttgac aagaatttac cgaacgaaaa agtattgcct
     2701 aagcacagtt tactttacga gtatttcaca gtgtacaatg aactcacgaa agttaagtat
     2761 gtcactgagg gcatgcgtaa acccgccttt ctaagcggag aacagaagaa agcaatagta
     2821 gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aattgaaaga ggactacttt
     2881 aagaaaattg aatgcttcga ttctgtcgag atctccgggg tagaagatcg atttaatgcg
     2941 tcacttggta cgtatcatga cctcctaaag ataattaaag ataaggactt cctggataac
     3001 gaagagaatg aagatatctt agaagatata gtgttgactc ttaccctctt tgaagatcgg
     3061 gaaatgattg aggaaagact aaaaacatac gctcacctgt tcgacgataa ggttatgaaa
     3121 cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt cgcggaaact tatcaacggg
     3181 ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga cggcttcgcc
     3241 aataggaact ttatgcagct gatccatgat gactctttaa ccttcaaaga ggatatacaa
     3301 aaggcacagg tttccggaca aggggactca ttgcacgaac atattgcgaa tcttgctggt
     3361 tcgccagcca tcaaaaaggg catactccag acagtcaaag tagtggatga gctagttaag
     3421 gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga tggcacgcga aaatcaaacg
     3481 actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga gaatagaaga gggtattaaa
     3541 gaactgggca gccagatctt aaaggagcat cctgtggaaa atacccaatt gcagaacgag
     3601 aaactttacc tctattacct acaaaatgga agggacatgt atgttgatca ggaactggac
     3661 ataaaccgtt tatctgatta cgacgtcgat gccattgtac cccaatcctt tttgaaggac
     3721 gattcaatcg acaataaagt gcttacacgc tcggataaga accgagggaa aagtgacaat
     3781 gttccaagcg aggaagtcgt aaagaaaatg aagaactatt ggcggcagct cctaaatgcg
     3841 aaactgataa cgcaaagaaa gttcgataac ttaactaaag ctgagagggg tggcttgtct
     3901 gaacttgaca aggccggatt tattaaacgt cagctcgtgg aaacccgcca aatcacaaag
     3961 catgttgcac agatactaga ttcccgaatg aatacgaaat acgacgagaa cgataagctg
     4021 attcgggaag tcaaagtaat cactttaaag tcaaaattgg tgtcggactt cagaaaggat
     4081 tttcaattct ataaagttag ggagataaat aactaccacc atgcgcacga cgcttatctt
     4141 aatgccgtcg tagggaccgc actcattaag aaatacccga agctagaaag tgagtttgtg
     4201 tatggtgatt acaaagttta tgacgtccgt aagatgatcg cgaaaagcga acaggagata
     4261 ggcaaggcta cagccaaata cttcttttat tctaacatta tgaatttctt taagacggaa
     4321 atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa tggggagaca
     4381 ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg tgagaaaagt tttgtccatg
     4441 ccccaagtca acatagtaaa gaaaactgag gtgcagaccg gagggttttc aaaggaatcg
     4501 attcttccaa aaaggaatag tgataagctc atcgctcgta aaaaggactg ggacccgaaa
     4561 aagtacggtg gcttcgatag ccctacagtt gcctattctg tcctagtagt ggcaaaagtt
     4621 gagaagggaa aatccaagaa actgaagtca gtcaaagaat tattggggat aacgattatg
     4681 gagcgctcgt cttttgaaaa gaaccccatc gacttccttg aggcgaaagg ttacaaggaa
     4741 gtaaaaaagg atctcataat taaactacca aagtatagtc tgtttgagtt agaaaatggc
     4801 cgaaaacgga tgttggctag cgccggagag cttcaaaagg ggaacgaact cgcactaccg
     4861 tctaaatacg tgaatttcct gtatttagcg tcccattacg agaagttgaa aggttcacct
     4921 gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct cgacgaaatc
     4981 atagagcaaa tttcggaatt cagtaagaga gtcatcctag ctgatgccaa tctggacaaa
     5041 gtattaagcg catacaacaa gcacagggat aaacccatac gtgagcaggc ggaaaatatt
     5101 atccatttgt ttactcttac caacctcggc gctccagccg cattcaagta ttttgacaca
     5161 acgatagatc gcaaacgata cacttctacc aaggaggtgc tagacgcgac actgattcac
     5221 caatccatca cgggattata tgaaactcgg atagatttgt cacagcttgg gggtgactga
     5281 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5341 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5401 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5461 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5521 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5581 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5701 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5761 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5821 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5881 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5941 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6001 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6061 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6121 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6181 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     6241 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6301 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6361 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6421 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6481 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6541 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6601 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6661 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6721 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6781 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6841 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6901 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6961 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7021 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7081 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7141 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7201 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7261 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7321 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7381 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7441 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7501 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7561 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7621 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7681 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7741 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7801 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     7861 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     7921 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     7981 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8041 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8101 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8161 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8221 gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat c
//
