LOCUS       sequence-339089-       11850 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 11850)
  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 11850)
  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..11850
                     /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)"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        428..713
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        715..988
                     /label=chicken beta-actin promoter
     intron          989..1216
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1228..1237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1237..1302
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1309..1329
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1354..3867,3868..3870,3871..5454)
                     /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             1354..5454
                     /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"
     CDS             7627..7674
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7690..7743
                     /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(7744..7746,7747..8457)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7744..7746,7747..8457)
                     /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(8047..8066)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8394..8415
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(8485..8502)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8491..8698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     8707..8723
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8707..8723
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(8763..8838)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(8856..9104)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   9113..9253
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   9113..9242
                     /label=AAV2 ITR
     rep_origin      9328..9783
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9415..9434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9625..9646
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9800..9819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9919..9941
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9979..9997)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10065..10169
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10170..10238,10239..11030)
                     /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(10388..10407)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11201..11789
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11690..11709
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgggtctt cgagaagacc
      361 ttttttttgt tctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
      421 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      481 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      541 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      601 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      661 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      721 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      781 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggcgcgcgcc
      841 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
      901 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      961 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
     1021 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     1081 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
     1141 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     1201 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
     1261 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
     1321 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     1381 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     1441 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     1501 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     1561 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     1621 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     1681 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     1741 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     1801 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     1861 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     1921 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     1981 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     2041 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     2101 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     2161 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     2221 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     2281 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     2341 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     2401 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     2461 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     2521 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     2581 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     2641 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     2701 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     2761 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     2821 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     2881 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     2941 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     3001 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     3061 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     3121 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     3181 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     3241 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     3301 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     3361 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     3421 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     3481 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     3541 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     3601 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     3661 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     3721 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     3781 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     3841 aaccggctgt ccgactacga tgtggacgct atcgtgcctc agagctttct gaaggacgac
     3901 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     3961 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     4021 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     4081 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     4141 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     4201 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     4261 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     4321 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     4381 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     4441 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     4501 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     4561 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     4621 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     4681 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     4741 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     4801 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     4861 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     4921 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     4981 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     5041 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     5101 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     5161 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     5221 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     5281 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     5341 atcgaccgga agaggtacac cagcacgaaa gaggtgctgg acgccaccct gatccaccag
     5401 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctgga
     5461 ggatctagcg gaggatcctc tggcagcgag acaccaggaa caagcgagtc agcaacacca
     5521 gagagcagtg gcggcagcag cggcggcagc agcaccctaa atatagaaga tgagtatcgg
     5581 ctacatgaga cctcaaaaga gccagatgtt tctctagggt ccacatggct gtctgatttt
     5641 cctcaggcct gggcggaaac cgggggcatg ggactggcag ttcgccaagc tcctctgatc
     5701 atacctctga aagcaacctc tacccccgtg tccataaaac aataccccat gtcacaagaa
     5761 gccagactgg ggatcaagcc ccacatacag agactgttgg accagggaat actggtaccc
     5821 tgccagtccc cctggaacac gcccctgcta cccgttaaga aaccagggac taatgattat
     5881 aggcctgtcc aggatctgag agaagtcaac aagcgggtgg aggacatcca ccccaccgtg
     5941 cccaaccctt acaacctctt gagcgggctc ccaccgtccc accagtggta cactgtgctt
     6001 gatttaaagg atgccttttt ctgcctgaga ctccacccca ccagtcagcc tctcttcgcc
     6061 tttgagtgga gagatccaga gatgggaatc tcaggacaat tgacctggac cagactccca
     6121 cagggtttca aaaacagtcc caccctgttt aatgaggcac tgcacagaga cctagcagac
     6181 ttccggatcc agcacccaga cttgatcctg ctacagtacg tggatgactt actgctggcc
     6241 gccacttctg agctagactg ccaacaaggt actcgggccc tgttacaaac cctagggaac
     6301 ctcgggtatc gggcctcggc caagaaagcc caaatttgcc agaaacaggt caagtatctg
     6361 gggtatcttc taaaagaggg tcagagatgg ctgactgagg ccagaaaaga gactgtgatg
     6421 gggcagccta ctccaaagac ccctcgacaa ctaagggagt tcctagggaa ggcaggcttc
     6481 tgtcgcctct tcatccctgg gtttgcagaa atggcagccc ccctgtaccc tctcaccaaa
     6541 ccggggactc tgtttaattg gggcccagac caacaaaagg cctatcaaga aatcaagcaa
     6601 gcccttctaa ctgccccagc cctggggttg ccagatttga ctaagccctt tgaactcttt
     6661 gtcgacgaga agcagggcta cgccaaaggt gtcctaacgc aaaaactggg accttggcgt
     6721 cggccggtgg cctacctgtc caaaaagcta gacccagtag cagctgggtg gcccccttgc
     6781 ctacggatgg tagcagccat tgccgtactg acaaaggatg caggcaagct aaccatggga
     6841 cagccactag tcattctggc cccccatgca gtagaggcac tagtcaaaca accccccgac
     6901 cgctggcttt ccaacgcccg gatgactcac tatcaggcct tgcttttgga cacggaccgg
     6961 gtccagttcg gaccggtggt agccctgaac ccggctacgc tgctcccact gcctgaggaa
     7021 gggctgcaac acaactgcct tgatatcctg gccgaagccc acggaacccg acccgaccta
     7081 acggaccagc cgctcccaga cgccgaccac acctggtaca cggatggaag cagtctctta
     7141 caagagggac agcgtaaggc gggagctgcg gtgaccaccg agaccgaggt aatctgggct
     7201 aaagccctgc cagccgggac atccgctcag cgggctgaac tgatagcact cacccaggcc
     7261 ctaaagatgg cagaaggtaa gaagctaaat gtttatactg atagccgtta tgcttttgct
     7321 actgcccata tccatggaga aatatacaga aggcgtgggt ggctcacatc agaaggcaaa
     7381 gagatcaaaa ataaagacga gatcttggcc ctactaaaag ccctctttct gcccaaaaga
     7441 cttagcataa tccattgtcc aggacatcaa aagggacaca gcgccgaggc tagaggcaac
     7501 cggatggctg accaagcggc ccgaaaggca gccatcacag agactccaga cacctctacc
     7561 ctcctcatag aaaattcatc accctctggc ggctcaaaaa gaaccgccga cggcagcgaa
     7621 ttcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaaggaattc
     7681 ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
     7741 ccagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     7801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     7861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     7921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     7981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     8041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     8101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     8161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     8221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     8281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     8341 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     8401 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa
     8461 ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     8521 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     8581 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     8641 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     8701 ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgataatcc tgcaggaaaa
     8761 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     8821 tatttctagc tctaaaacgt cttctcgaag accccggtgt ttcgtccttt ccacaagata
     8881 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta
     8941 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt
     9001 tgtactaata tctttgtgtt tacagtcaaa ttaattccaa ttatctctct aacagccttg
     9061 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcgcggcc gcaggaaccc
     9121 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     9181 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     9241 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     9301 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     9361 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     9421 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     9481 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     9541 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     9601 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     9661 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     9721 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     9781 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     9841 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     9901 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     9961 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
    10021 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
    10081 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
    10141 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
    10201 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
    10261 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
    10321 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
    10381 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
    10441 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
    10501 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
    10561 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
    10621 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
    10681 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
    10741 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
    10801 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
    10861 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
    10921 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
    10981 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
    11041 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
    11101 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
    11161 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
    11221 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
    11281 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
    11341 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
    11401 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
    11461 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
    11521 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    11581 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    11641 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    11701 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    11761 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
    11821 tggccttttg ctggcctttt gctcacatgt
//
