LOCUS       sequence-325319-        9265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  REDIT backbone for nickase Cas9n, 
            pU6-MS2-gRNA-backbone(BbsI)-CBH-SpCas9n(D10A)-T2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9265)
  AUTHORS   Wang C, Cheng JKW, Zhang Q, Hughes NW, Xia Q, Winslow MM, Cong L
  TITLE     Microbial single-strand annealing proteins enable CRISPR 
            gene-editing tools with improved knock-in efficiencies and reduced 
            off-target effects.
  JOURNAL   Nucleic Acids Res. 2021 Apr 6;49(6):e36. doi: 10.1093/nar/gkaa1264.
  PUBMED    33619540
REFERENCE   2  (bases 1 to 9265)
  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: "Nucleic 
            Acids Res."; date: "2021-04-6"; volume: "49(6):e36. doi"; pages: " 
            10.1093/nar/gkaa1264"
COMMENT     Sequence Label: pREDIT_Cas9n-MS2-BB_BbsI
FEATURES             Location/Qualifiers
     source          1..9265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        285..303
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        378..396
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        417..702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          982..1209
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1221..1230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1230..1295
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1302..1322
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1347..1370,1371..1373,1374..5447)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1347..5447
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5448..5495
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5511..5564
                     /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             join(5565..5567,5568..6278)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5565..5567,5568..6278)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5868..5887)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6215..6236
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6306..6323)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6312..6519
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6528..6668
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6528..6657
                     /label=AAV2 ITR
     rep_origin      6743..7198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6830..6849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7040..7061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7215..7234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7334..7356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7394..7412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7480..7584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7585..7653,7654..8445)
                     /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(7803..7822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8616..9204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9105..9124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtggcgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt taagagctag gccaacatga ggatcaccca
      301 tgtctgcagg gcctagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc
      361 accgagtcgg tgcgcgcaca tgaggatcac ccatgtgctt tttttctaga ggtacccgtt
      421 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      481 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      601 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
      661 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
      721 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
      781 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggsgcgc gccggggggg
      841 gggggggggg gggggggggg ggggggggsg rggsggrgrg gkgsggcggc agccaatcag
      901 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
      961 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
     1021 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
     1081 gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag
     1141 ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact
     1201 ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg
     1261 atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag aagcggaagg
     1321 tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg gccatcggca
     1381 ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca
     1441 aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt
     1501 tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca
     1561 ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg
     1621 tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc
     1681 acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc
     1741 ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc
     1801 tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg
     1861 acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca
     1921 accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt
     1981 ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga
     2041 agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca
     2101 agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg
     2161 acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg
     2221 ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca
     2281 ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga
     2341 ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg
     2401 accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct
     2461 acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc
     2521 tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc
     2581 agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc
     2641 tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg
     2701 gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca
     2761 tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg
     2821 agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc
     2881 tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg
     2941 gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt
     3001 tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg
     3061 agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca
     3121 cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg
     3181 aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg
     3241 aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc
     3301 ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca
     3361 agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact
     3421 tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg
     3481 tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca
     3541 ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc
     3601 ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg
     3661 gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca
     3721 gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc
     3781 tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc
     3841 tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg
     3901 acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg
     3961 aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta
     4021 cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata
     4081 aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac
     4141 agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag
     4201 tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt
     4261 acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg
     4321 tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact
     4381 acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta
     4441 ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg
     4501 ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg
     4561 tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga
     4621 atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca
     4681 agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg
     4741 gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca
     4801 agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca
     4861 gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg
     4921 acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa
     4981 tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg
     5041 tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg
     5101 agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga
     5161 tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg
     5221 cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt
     5281 ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc
     5341 ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca
     5401 ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg
     5461 ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag
     5521 gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc aagggcgagg
     5581 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     5641 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     5701 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5761 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5821 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5881 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     5941 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     6001 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     6061 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     6121 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     6181 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     6241 ccgccgggat cactctcggc atggacgagc tgtacaagga attctaacta gagctcgctg
     6301 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     6361 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     6421 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     6481 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     6541 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     6601 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     6661 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6721 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6781 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     6841 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6901 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     6961 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     7021 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     7081 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     7141 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     7201 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     7261 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     7321 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     7381 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     7441 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     7501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     7561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     7621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     7681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     7741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     7861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     7921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     7981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     8041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     8101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     8161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     8221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     8281 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     8341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     8401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     8461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     8521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     8641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     8701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     8761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     8821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     8881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     8941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     9001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     9061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     9121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     9181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     9241 ttttgctggc cttttgctca catgt
//
