LOCUS       sequence-440853-        9646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses Cas9-hGem and guideRNA for tagging of SMC4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9646)
  AUTHORS   Batty P, Langer CC, Takacs Z, Tang W, Blaukopf C, Peters JM, Gerlich
            DW
  TITLE     Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2
            phase.
  JOURNAL   EMBO J. 2023 Aug 15;42(16):e113475. doi: 10.15252/embj.2023113475. 
            Epub 2023 Jun 26.
  PUBMED    37357575
REFERENCE   2  (bases 1 to 9646)
  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: "EMBO J."; 
            date: "2023-08-15"; volume: "42(16):e113475. doi"; pages: " 
            10.15252/embj.2023113475. Epub 2023 Jun 26"
COMMENT     Sequence Label: sgRNA1_Cas9-hGem_Smc4
FEATURES             Location/Qualifiers
     source          1..9646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        921..996
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1093..1378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1659..1886
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1898..1907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1907..1972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1979..1999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2024..6124
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2024..6124
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6125..6172
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6185..6514
                     /codon_start=1
                     /product="degron consisting of residues 1-110 of human 
                     Geminin (Zielke and Edgar, 2015)"
                     /label=Gem1 (1-110)
                     /translation="MNPSMKQKQEEIKENIKNSSVPRRTLKMIQPSASGSLVGRENELS
                     AGLSKRKHRNDHLTSTTSSPGVIVPESSENKNLGGVTQESFDLMIKENPSSQYWKEVAE
                     KRRKAL"
     CDS             6524..6580
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      6596..6605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6602..7312
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6747..6765)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7337..7354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7343..7550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7559..7699
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7559..7688
                     /label=AAV2 ITR
     rep_origin      7774..8229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7861..7880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8071..8092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8246..8265)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8365..8387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8425..8443)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8511..8615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8616..8684,8685..9476)
                     /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(8834..8853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      901 tccaaaagaa attgcatcta gttttagagc tagaaatagc aagttaaaat aaggctagtc
      961 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata
     1021 gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc
     1081 tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1141 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1201 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1261 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1321 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1381 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1441 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1501 ggcgcgcgcc gggggggggg ggggggggsg rggggsgggg gggggsgrgg cggagaggkg
     1561 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     1621 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1681 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1741 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     1801 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     1861 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
     1921 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     1981 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     2041 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2101 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2161 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2221 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2281 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2341 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2401 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2461 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     2521 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2581 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2641 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     2701 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     2761 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     2821 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     2881 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     2941 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3001 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3061 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3121 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3181 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3241 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3301 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3361 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3421 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3481 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3541 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     3601 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3661 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     3721 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     3781 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     3841 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     3901 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3961 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4021 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4081 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4141 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4201 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4261 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4321 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4381 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4441 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4501 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     4561 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     4621 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4681 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     4741 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     4801 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     4861 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     4921 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4981 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5041 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5101 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5161 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5221 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5281 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5341 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5401 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5461 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     5521 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5581 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5641 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     5701 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     5761 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     5821 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     5881 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     5941 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6001 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6061 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6121 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccaa
     6181 gcttatgaat cccagtatga agcagaaaca agaagaaatc aaagagaata taaagaatag
     6241 ttctgtccca agaagaactc tgaagatgat tcagccttct gcatctggat ctcttgttgg
     6301 aagagaaaat gagctgtccg caggcttgtc caaaaggaaa catcggaatg accacttaac
     6361 atctacaact tccagccctg gggttattgt cccagaatct agtgaaaata aaaatcttgg
     6421 aggagtcacc caggagtcat ttgatcttat gattaaagaa aatccatcct ctcagtattg
     6481 gaaggaagtg gcagaaaaac ggagaaaggc gctgggaagc ggagctacta acttcagcct
     6541 gctgaagcag gctggagacg tggaggagaa ccctggacct gaattcatgc cggtcgccac
     6601 catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     6661 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     6721 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     6781 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     6841 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     6901 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     6961 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     7021 aatgcagaag aaaaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     7081 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     7141 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     7201 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     7261 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aggaattcct
     7321 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     7381 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     7441 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     7501 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     7561 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     7621 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     7681 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     7741 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     7801 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     7861 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     7921 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     7981 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     8041 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     8101 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     8161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     8221 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     8281 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     8341 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     8401 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     8461 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     8521 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     8581 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     8641 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     8701 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     8761 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     8821 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     8881 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     8941 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     9001 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     9061 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     9121 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     9181 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     9241 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     9301 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     9361 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     9421 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     9481 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     9541 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     9601 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
