LOCUS       sequence-127249-       10271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  human codon optimized Csy4 and FokI-dCas9 expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10271)
  AUTHORS   Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin
            MJ, Aryee MJ, Joung JK
  TITLE     Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome 
            editing.
  JOURNAL   Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908.
  PUBMED    24770325
REFERENCE   2  (bases 1 to 10271)
  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: "Nat 
            Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908."
COMMENT     Sequence Label: pSQT1601
FEATURES             Location/Qualifiers
     source          1..10271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          638..1654
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1578..1600
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1662..1681
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1747..1756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2317..2370
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2374..2394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2401..2988
                     /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(3004..6402,6403..6405,6406..7002,7003,7004..7006,
                     7007..7008,7009..7011,7012..7104)
                     /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             join(3004..3027,3028..3030,3031..5517,5518..5520,
                     5521..7104)
                     /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             7111..7131
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7138..7203
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(7293..7312)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7358..7413
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7412..7431)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7774..7790)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7774..7790)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7787..7809)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7798..7814
                     /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(7822..7828,7829..7846,7847..7852))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7867..7888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7947..8142
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7993..8128
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    8148..8282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8185..8204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8239..8258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8350..8367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8521..9109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8601..8620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9280..10071,10072..10140))
                     /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     9903..9922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10141..10245)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcga ggggcggggc ggggcgaggc ggagaggtgc
      541 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
      601 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
      661 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      721 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
      781 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg
      841 ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
      901 cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg
      961 tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg
     1021 gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg
     1081 tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca
     1141 cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg
     1201 cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg
     1261 ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg
     1321 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
     1381 ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa
     1441 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
     1501 ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg
     1561 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc
     1621 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt
     1681 gctgtctcat cattttggca aagaattctg cagtcgacgg taccgcgggc ccgggatcca
     1741 ccggtcgcca ccatgggtga tcattatctg gatattcggc tgaggcctga tccagagttc
     1801 ccacctgcgc agctgatgtc tgtccttttt ggcaaacttc atcaggccct ggttgcccag
     1861 ggcggagatc ggataggggt aagctttcca gacctcgacg aaagccggag ccgcctggga
     1921 gaacgcctgc ggatccacgc ttctgccgac gatctgagag ccttgctggc aaggccatgg
     1981 cttgaggggc tccgggatca cctgcagttt ggcgaacccg ccgttgttcc ccacccaacc
     2041 ccttatcggc aggtgtctag agtgcaggcc aaatctaatc cagaacggct gcgacggcga
     2101 ctcatgcggc gacatgatct tagcgaggaa gaggcccgaa aaagaatccc tgataccgtg
     2161 gcccgcgccc ttgacttgcc ttttgtcaca ctgcggtccc agagtacggg gcagcatttc
     2221 agacttttca ttcgacacgg gccactgcaa gttaccgccg aagaaggagg ctttacttgt
     2281 tatggactct ccaagggagg tttcgtgccc tggtttgagg gcagaggaag tctgttaaca
     2341 tgcggtgacg tcgaggagaa tcctggccca atgcctaaga agaagcggaa ggtgagcagc
     2401 caacttgtga agtctgaact cgaggagaaa aaatcagagt tgagacacaa gttgaagtac
     2461 gtgccacacg aatacatcga gcttatcgag atcgccagaa acagtaccca ggataggatc
     2521 cttgagatga aagtcatgga gttctttatg aaggtctacg gttatagagg aaagcacctt
     2581 ggcggtagca gaaagcccga tggcgccatc tatactgtcg gatctcctat cgattatggg
     2641 gtgatcgtgg ataccaaagc ttactcaggc gggtacaact tgcccatagg acaagccgac
     2701 gagatgcagc ggtatgtcga agagaaccag acgcgcaaca agcacatcaa ccccaatgaa
     2761 tggtggaaag tgtacccaag tagtgtgact gagttcaagt tcctgtttgt ctccggccac
     2821 tttaagggca attataaagc tcagctcact agactcaatc acatcacaaa ctgcaacgga
     2881 gctgtgttgt cagtggagga gctcctgatt ggaggcgaga tgatcaaagc cggcaccctt
     2941 acactggagg aggtgcggcg gaagttcaac aatggagaga tcaacttcgg tggcggtgga
     3001 tccgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc
     3061 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt
     3121 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag
     3181 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt
     3241 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt
     3301 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga
     3361 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa
     3421 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat
     3481 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat
     3541 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct
     3601 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga
     3661 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac
     3721 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa
     3781 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca
     3841 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc
     3901 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca
     3961 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt
     4021 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca
     4081 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta
     4141 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga
     4201 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat
     4261 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt
     4321 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct
     4381 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa
     4441 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag
     4501 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg
     4561 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta
     4621 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca
     4681 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc
     4741 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata
     4801 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg
     4861 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct
     4921 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga
     4981 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc
     5041 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac
     5101 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg
     5161 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca
     5221 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta
     5281 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg
     5341 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct
     5401 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg
     5461 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatgcc
     5521 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg
     5581 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag
     5641 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta
     5701 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag
     5761 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat
     5821 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca
     5881 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac
     5941 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa
     6001 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag
     6061 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct
     6121 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga
     6181 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc
     6241 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg
     6301 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc
     6361 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc
     6421 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc
     6481 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac
     6541 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag
     6601 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt
     6661 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc
     6721 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag
     6781 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc
     6841 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa
     6901 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct
     6961 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag
     7021 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata
     7081 gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt ctcgagcgac
     7141 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
     7201 aagtgaagcg gccgcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     7261 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     7321 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     7381 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     7441 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     7501 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     7561 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     7621 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     7681 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     7741 ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     7801 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     7861 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     7921 gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc
     7981 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     8041 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     8101 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg
     8161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     8221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     8281 tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     8341 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     8401 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     8461 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     8521 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     8581 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     8641 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     8701 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     8761 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     8821 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     8881 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     8941 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     9001 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     9061 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     9121 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     9181 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     9241 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     9301 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     9361 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     9421 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     9481 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     9541 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     9601 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     9661 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     9721 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     9781 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     9841 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     9901 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     9961 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
    10021 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
    10081 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
    10141 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
    10201 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
    10261 agtgccacct g
//
