LOCUS       sequence-340552-       11853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivers all prime editing nickase components in a single, GFP 
            selectable plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11853)
  AUTHORS   Adikusuma F, Lushington C, Arudkumar J, Godahewa GI, Chey YCJ, 
            Gierus L, Piltz S, Geiger A, Jain Y, Reti D, Wilson LOW, Bauer DC, 
            Thomas PQ
  TITLE     Optimized nickase- and nuclease-based prime editing in human and 
            mouse cells.
  JOURNAL   Nucleic Acids Res. 2021 Sep 17. pii: 6371974. doi: 
            10.1093/nar/gkab792.
  PUBMED    34534334
REFERENCE   2  (bases 1 to 11853)
  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. 2021 Sep 17. pii: 6371974. doi: 10.1093/nar/gkab792."
COMMENT     Sequence Label: PEA1-GFP
FEATURES             Location/Qualifiers
     source          1..11853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3743..3790
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3806..3859
                     /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(3860..3862,3863..4573)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3860..3862,3863..4573)
                     /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(4163..4182)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4510..4531
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4601..4618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4607..4814
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4823..4839
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4823..4839
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(4879..4954)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4972..5220)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   5229..5369
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5229..5358
                     /label=AAV2 ITR
     rep_origin      5444..5899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5531..5550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5741..5762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5916..5935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6035..6057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6095..6113)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6181..6285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6286..6354,6355..7146)
                     /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(6504..6523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7317..7905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7806..7825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7967..8215
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8234..8309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8394..8679
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          8958..9185
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      9197..9206
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9206..9271
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             9278..9298
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(9323..11836,11837..11839,11840..11853,1..1570)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     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(9323..11853,1..1570)
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
       61 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
      121 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
      181 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
      241 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
      301 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
      361 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
      421 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
      481 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
      541 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
      601 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
      661 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
      721 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
      781 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
      841 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
      901 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
      961 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     1021 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     1081 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     1141 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     1201 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     1261 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     1321 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     1381 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     1441 gtactttgac accaccatcg accggaagag gtacaccagc acgaaagagg tgctggacgc
     1501 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     1561 gggaggtgac tctggaggat ctagcggagg atcctctggc agcgagacac caggaacaag
     1621 cgagtcagca acaccagaga gcagtggcgg cagcagcggc ggcagcagca ccctaaatat
     1681 agaagatgag tatcggctac atgagacctc aaaagagcca gatgtttctc tagggtccac
     1741 atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac tggcagttcg
     1801 ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca taaaacaata
     1861 ccccatgtca caagaagcca gactggggat caagccccac atacagagac tgttggacca
     1921 gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg ttaagaaacc
     1981 agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc gggtggagga
     2041 catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac cgtcccacca
     2101 gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc accccaccag
     2161 tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag gacaattgac
     2221 ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttaatg aggcactgca
     2281 cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac agtacgtgga
     2341 tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc gggccctgtt
     2401 acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa tttgccagaa
     2461 acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga ctgaggccag
     2521 aaaagagact gtgatggggc agcctactcc aaagacccct cgacaactaa gggagttcct
     2581 agggaaggca ggcttctgtc gcctcttcat ccctgggttt gcagaaatgg cagcccccct
     2641 gtaccctctc accaaaccgg ggactctgtt taattggggc ccagaccaac aaaaggccta
     2701 tcaagaaatc aagcaagccc ttctaactgc cccagccctg gggttgccag atttgactaa
     2761 gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc taacgcaaaa
     2821 actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc cagtagcagc
     2881 tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa aggatgcagg
     2941 caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag aggcactagt
     3001 caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc aggccttgct
     3061 tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg ctacgctgct
     3121 cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg aagcccacgg
     3181 aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct ggtacacgga
     3241 tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga ccaccgagac
     3301 cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg ctgaactgat
     3361 agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt atactgatag
     3421 ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc gtgggtggct
     3481 cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac taaaagccct
     3541 ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg gacacagcgc
     3601 cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca tcacagagac
     3661 tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tctggcggct caaaaagaac
     3721 cgccgacggc agcgaattcg agaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     3781 aaagaaaaag gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     3841 cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3901 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3961 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     4021 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     4081 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     4141 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     4201 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     4261 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4321 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4381 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4441 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4501 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4561 cgagctgtac aaggaattct aactagagct cgctgatcag cctcgactgt gccttctagt
     4621 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4681 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4741 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     4801 aggcatgctg gggagcggcc gctctagaac tagtggatcc cccgggctgc aggaattcga
     4861 taatcctgca ggaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag
     4921 ccttatttta acttgctatt tctagctcta aaacgtcttc tcgaagaccc cggtgtttcg
     4981 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     5041 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     5101 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     5161 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     5221 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     5281 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     5341 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5401 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5521 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5641 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5701 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5761 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5821 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5881 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5941 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     6001 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     6061 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     6121 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     6181 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6241 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6301 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6361 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6421 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6481 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6541 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6601 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6661 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6721 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6781 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6841 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6901 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6961 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     7021 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     7081 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7141 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     7201 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7261 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7321 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7381 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7441 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7501 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7561 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7621 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7681 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7741 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7801 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7861 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7921 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc
     7981 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     8041 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     8101 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     8161 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc
     8221 ttcgagaaga cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     8281 aacttgaaaa agtggcaccg agtcggtgcg ggtcttcgag aagacctttt ttttgttcta
     8341 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca
     8401 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     8461 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     8521 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     8581 gacggtaaat ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact
     8641 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     8701 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     8761 taattatttt gtgcagcgat gggggcgggg gggggggggg nnnsgcgcsr ggsggggsgg
     8821 ggsggggsgr ggggsggggs ggggsgrggs ggrgrggkgs ggcggcagcc aatcagagcg
     8881 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     8941 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     9001 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     9061 gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat
     9121 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt
     9181 ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact acaaggatca
     9241 tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
     9301 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa
     9361 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     9421 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     9481 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     9541 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     9601 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     9661 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     9721 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     9781 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     9841 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     9901 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     9961 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
    10021 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
    10081 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
    10141 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
    10201 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
    10261 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
    10321 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
    10381 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
    10441 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
    10501 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
    10561 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
    10621 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
    10681 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
    10741 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
    10801 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
    10861 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
    10921 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
    10981 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
    11041 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
    11101 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
    11161 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
    11221 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
    11281 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
    11341 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
    11401 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
    11461 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
    11521 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
    11581 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
    11641 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
    11701 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
    11761 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
    11821 cgactacgat gtggacgcta tcgtgcctca gag
//
