LOCUS       sequence-339092-       11752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Delivers all prime editing (nickase) components targeting the HEK3 
            site for a CTT inserrtion, in a single, puromycin selectable 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11752)
  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 11752)
  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-Puro-HEK3 CTT ins
FEATURES             Location/Qualifiers
     source          1..11752
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        438..723
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        725..998
                     /label=chicken beta-actin promoter
     intron          999..1226
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1238..1247
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1247..1312
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1319..1339
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1364..3877,3878..3880,3881..5464)
                     /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             1364..5464
                     /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             7637..7684
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7700..7753
                     /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             7754..8353
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7754..7773)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8250..8270
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(8381..8398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8387..8594
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     8603..8619
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8603..8619
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(8662..8737)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(8758..9006)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   9015..9155
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   9015..9144
                     /label=AAV2 ITR
     rep_origin      9230..9685
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9317..9336)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9527..9548
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9702..9721)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9821..9843
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9881..9899)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9967..10071
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10072..10140,10141..10932)
                     /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(10290..10309)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11103..11691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11592..11611
                     /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 gcccagactg agcacgtgag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgctctgc catcaaagcg
      361 tgctcagtct gtttttttgt tctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa
      421 atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      481 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa
      541 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      601 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac
      661 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      721 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
      781 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg
      841 ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg
      901 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      961 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1021 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1081 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     1141 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     1201 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
     1261 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     1321 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     1381 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     1441 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     1501 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     1561 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     1621 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     1681 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     1741 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     1801 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1861 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     1921 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     1981 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     2041 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     2101 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     2161 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     2221 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     2281 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     2341 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     2401 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     2461 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     2521 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     2581 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     2641 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     2701 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     2761 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     2821 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     2881 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     2941 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3001 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     3061 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     3121 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     3181 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     3241 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3301 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     3361 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     3421 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     3481 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     3541 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     3601 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     3661 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     3721 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     3781 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     3841 actggacatc aaccggctgt ccgactacga tgtggacgct atcgtgcctc agagctttct
     3901 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     3961 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4021 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     4081 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     4141 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     4201 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     4261 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4321 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     4381 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     4441 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     4501 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     4561 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     4621 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     4681 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     4741 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     4801 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     4861 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     4921 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     4981 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     5041 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     5101 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     5161 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     5221 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     5281 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     5341 tgacaccacc atcgaccgga agaggtacac cagcacgaaa gaggtgctgg acgccaccct
     5401 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     5461 tgactctgga ggatctagcg gaggatcctc tggcagcgag acaccaggaa caagcgagtc
     5521 agcaacacca gagagcagtg gcggcagcag cggcggcagc agcaccctaa atatagaaga
     5581 tgagtatcgg ctacatgaga cctcaaaaga gccagatgtt tctctagggt ccacatggct
     5641 gtctgatttt cctcaggcct gggcggaaac cgggggcatg ggactggcag ttcgccaagc
     5701 tcctctgatc atacctctga aagcaacctc tacccccgtg tccataaaac aataccccat
     5761 gtcacaagaa gccagactgg ggatcaagcc ccacatacag agactgttgg accagggaat
     5821 actggtaccc tgccagtccc cctggaacac gcccctgcta cccgttaaga aaccagggac
     5881 taatgattat aggcctgtcc aggatctgag agaagtcaac aagcgggtgg aggacatcca
     5941 ccccaccgtg cccaaccctt acaacctctt gagcgggctc ccaccgtccc accagtggta
     6001 cactgtgctt gatttaaagg atgccttttt ctgcctgaga ctccacccca ccagtcagcc
     6061 tctcttcgcc tttgagtgga gagatccaga gatgggaatc tcaggacaat tgacctggac
     6121 cagactccca cagggtttca aaaacagtcc caccctgttt aatgaggcac tgcacagaga
     6181 cctagcagac ttccggatcc agcacccaga cttgatcctg ctacagtacg tggatgactt
     6241 actgctggcc gccacttctg agctagactg ccaacaaggt actcgggccc tgttacaaac
     6301 cctagggaac ctcgggtatc gggcctcggc caagaaagcc caaatttgcc agaaacaggt
     6361 caagtatctg gggtatcttc taaaagaggg tcagagatgg ctgactgagg ccagaaaaga
     6421 gactgtgatg gggcagccta ctccaaagac ccctcgacaa ctaagggagt tcctagggaa
     6481 ggcaggcttc tgtcgcctct tcatccctgg gtttgcagaa atggcagccc ccctgtaccc
     6541 tctcaccaaa ccggggactc tgtttaattg gggcccagac caacaaaagg cctatcaaga
     6601 aatcaagcaa gcccttctaa ctgccccagc cctggggttg ccagatttga ctaagccctt
     6661 tgaactcttt gtcgacgaga agcagggcta cgccaaaggt gtcctaacgc aaaaactggg
     6721 accttggcgt cggccggtgg cctacctgtc caaaaagcta gacccagtag cagctgggtg
     6781 gcccccttgc ctacggatgg tagcagccat tgccgtactg acaaaggatg caggcaagct
     6841 aaccatggga cagccactag tcattctggc cccccatgca gtagaggcac tagtcaaaca
     6901 accccccgac cgctggcttt ccaacgcccg gatgactcac tatcaggcct tgcttttgga
     6961 cacggaccgg gtccagttcg gaccggtggt agccctgaac ccggctacgc tgctcccact
     7021 gcctgaggaa gggctgcaac acaactgcct tgatatcctg gccgaagccc acggaacccg
     7081 acccgaccta acggaccagc cgctcccaga cgccgaccac acctggtaca cggatggaag
     7141 cagtctctta caagagggac agcgtaaggc gggagctgcg gtgaccaccg agaccgaggt
     7201 aatctgggct aaagccctgc cagccgggac atccgctcag cgggctgaac tgatagcact
     7261 cacccaggcc ctaaagatgg cagaaggtaa gaagctaaat gtttatactg atagccgtta
     7321 tgcttttgct actgcccata tccatggaga aatatacaga aggcgtgggt ggctcacatc
     7381 agaaggcaaa gagatcaaaa ataaagacga gatcttggcc ctactaaaag ccctctttct
     7441 gcccaaaaga cttagcataa tccattgtcc aggacatcaa aagggacaca gcgccgaggc
     7501 tagaggcaac cggatggctg accaagcggc ccgaaaggca gccatcacag agactccaga
     7561 cacctctacc ctcctcatag aaaattcatc accctctggc ggctcaaaaa gaaccgccga
     7621 cggcagcgaa ttcgagaaaa ggccggcggc cacgaaaaag gccggccagg caaaaaagaa
     7681 aaaggaattc ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
     7741 gaatcctggc ccaatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     7801 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     7861 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     7921 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     7981 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     8041 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     8101 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     8161 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     8221 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     8281 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     8341 gcccggtgcc tgagaattct aactagagct cgctgatcag cctcgactgt gccttctagt
     8401 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     8461 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     8521 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     8581 aggcatgctg gggagcggcc gctctagaac tagtggatcc cccgggctgc aggaattcga
     8641 tacgaatcct gcaggaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac
     8701 tagccttatt ttaacttgct atttctagct ctaaaacgca ccgggatact ggttgacggt
     8761 gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
     8821 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
     8881 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc
     8941 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
     9001 gccctcgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     9061 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     9121 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     9181 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     9241 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     9301 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     9361 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     9421 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     9481 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     9541 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     9601 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     9661 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     9721 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     9781 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     9841 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     9901 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     9961 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
    10021 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
    10081 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
    10141 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
    10201 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
    10261 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
    10321 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
    10381 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
    10441 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
    10501 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
    10561 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
    10621 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
    10681 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
    10741 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
    10801 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
    10861 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
    10921 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
    10981 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
    11041 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
    11101 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
    11161 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
    11221 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
    11281 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
    11341 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
    11401 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
    11461 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
    11521 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
    11581 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
    11641 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
    11701 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
