LOCUS       sequence-338997-       11723 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 11723)
  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 11723)
  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..11723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          128..355
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      367..376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             376..441
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             448..468
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(493..3006,3007..3009,3010..4593)
                     /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             493..4593
                     /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             6766..6813
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6829..6882
                     /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             6883..7482
                     /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(6883..6902)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7379..7399
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(7510..7527)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7516..7723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7732..7748
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7732..7748
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(7791..7866)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7887..8135)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   8144..8284
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8144..8273
                     /label=AAV2 ITR
     rep_origin      8359..8814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8446..8465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8656..8677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8831..8850)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8950..8972
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9010..9028)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9096..9200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9201..9269,9270..10061)
                     /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(9419..9438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10232..10820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10721..10740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10882..11130
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        11151..11226
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        11319..11604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
       61 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
      121 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
      181 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      241 ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg
      301 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg
      361 accggtgcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
      421 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
      481 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
      541 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
      601 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
      661 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
      721 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
      781 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
      841 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
      901 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
      961 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     1021 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     1081 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1141 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1201 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     1261 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     1321 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     1381 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     1441 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     1501 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     1561 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     1621 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     1681 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     1741 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     1801 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     1861 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     1921 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     1981 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     2041 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     2101 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2161 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     2221 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     2281 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     2341 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     2401 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     2461 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     2521 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     2581 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     2641 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     2701 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     2761 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     2821 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     2881 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     2941 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     3001 gtggacgcta tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     3061 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3121 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3181 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     3241 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     3301 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     3361 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     3421 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     3481 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     3541 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     3601 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     3661 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     3721 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     3781 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     3841 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     3901 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     3961 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     4021 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     4081 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4141 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4201 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     4261 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     4321 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     4381 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     4441 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     4501 agcacgaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     4561 acacggatcg acctgtctca gctgggaggt gactctggag gatctagcgg aggatcctct
     4621 ggcagcgaga caccaggaac aagcgagtca gcaacaccag agagcagtgg cggcagcagc
     4681 ggcggcagca gcaccctaaa tatagaagat gagtatcggc tacatgagac ctcaaaagag
     4741 ccagatgttt ctctagggtc cacatggctg tctgattttc ctcaggcctg ggcggaaacc
     4801 gggggcatgg gactggcagt tcgccaagct cctctgatca tacctctgaa agcaacctct
     4861 acccccgtgt ccataaaaca ataccccatg tcacaagaag ccagactggg gatcaagccc
     4921 cacatacaga gactgttgga ccagggaata ctggtaccct gccagtcccc ctggaacacg
     4981 cccctgctac ccgttaagaa accagggact aatgattata ggcctgtcca ggatctgaga
     5041 gaagtcaaca agcgggtgga ggacatccac cccaccgtgc ccaaccctta caacctcttg
     5101 agcgggctcc caccgtccca ccagtggtac actgtgcttg atttaaagga tgcctttttc
     5161 tgcctgagac tccaccccac cagtcagcct ctcttcgcct ttgagtggag agatccagag
     5221 atgggaatct caggacaatt gacctggacc agactcccac agggtttcaa aaacagtccc
     5281 accctgttta atgaggcact gcacagagac ctagcagact tccggatcca gcacccagac
     5341 ttgatcctgc tacagtacgt ggatgactta ctgctggccg ccacttctga gctagactgc
     5401 caacaaggta ctcgggccct gttacaaacc ctagggaacc tcgggtatcg ggcctcggcc
     5461 aagaaagccc aaatttgcca gaaacaggtc aagtatctgg ggtatcttct aaaagagggt
     5521 cagagatggc tgactgaggc cagaaaagag actgtgatgg ggcagcctac tccaaagacc
     5581 cctcgacaac taagggagtt cctagggaag gcaggcttct gtcgcctctt catccctggg
     5641 tttgcagaaa tggcagcccc cctgtaccct ctcaccaaac cggggactct gtttaattgg
     5701 ggcccagacc aacaaaaggc ctatcaagaa atcaagcaag cccttctaac tgccccagcc
     5761 ctggggttgc cagatttgac taagcccttt gaactctttg tcgacgagaa gcagggctac
     5821 gccaaaggtg tcctaacgca aaaactggga ccttggcgtc ggccggtggc ctacctgtcc
     5881 aaaaagctag acccagtagc agctgggtgg cccccttgcc tacggatggt agcagccatt
     5941 gccgtactga caaaggatgc aggcaagcta accatgggac agccactagt cattctggcc
     6001 ccccatgcag tagaggcact agtcaaacaa ccccccgacc gctggctttc caacgcccgg
     6061 atgactcact atcaggcctt gcttttggac acggaccggg tccagttcgg accggtggta
     6121 gccctgaacc cggctacgct gctcccactg cctgaggaag ggctgcaaca caactgcctt
     6181 gatatcctgg ccgaagccca cggaacccga cccgacctaa cggaccagcc gctcccagac
     6241 gccgaccaca cctggtacac ggatggaagc agtctcttac aagagggaca gcgtaaggcg
     6301 ggagctgcgg tgaccaccga gaccgaggta atctgggcta aagccctgcc agccgggaca
     6361 tccgctcagc gggctgaact gatagcactc acccaggccc taaagatggc agaaggtaag
     6421 aagctaaatg tttatactga tagccgttat gcttttgcta ctgcccatat ccatggagaa
     6481 atatacagaa ggcgtgggtg gctcacatca gaaggcaaag agatcaaaaa taaagacgag
     6541 atcttggccc tactaaaagc cctctttctg cccaaaagac ttagcataat ccattgtcca
     6601 ggacatcaaa agggacacag cgccgaggct agaggcaacc ggatggctga ccaagcggcc
     6661 cgaaaggcag ccatcacaga gactccagac acctctaccc tcctcataga aaattcatca
     6721 ccctctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagaaaag gccggcggcc
     6781 acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga
     6841 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatgaccga gtacaagccc
     6901 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     6961 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     7021 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     7081 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     7141 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     7201 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     7261 accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     7321 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     7381 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     7441 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gagaattcta actagagctc
     7501 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     7561 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     7621 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     7681 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg ctctagaact
     7741 agtggatccc ccgggctgca ggaattcgat acgaatcctg caggaaaaaa gcaccgactc
     7801 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tttctagctc
     7861 taaaacgcac cgggatactg gttgacggtg tttcgtcctt tccacaagat atataaagcc
     7921 aagaaatcga aatactttca agttacggta agcatatgat agtccatttt aaaacataat
     7981 tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat
     8041 atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt gtatcgtata
     8101 tgcaaatatg aaggaatcat gggaaatagg ccctcgcggc cgcaggaacc cctagtgatg
     8161 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     8221 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     8281 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     8341 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     8401 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     8461 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     8521 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     8581 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     8641 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     8701 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     8761 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     8821 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     8881 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     8941 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     9001 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     9061 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     9121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     9181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     9241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     9301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     9361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     9421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     9481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     9541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     9601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     9661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     9721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     9781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     9841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     9901 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     9961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
    10021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
    10081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
    10141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
    10201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
    10261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
    10321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
    10381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
    10441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
    10501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
    10561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
    10621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
    10681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
    10741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
    10801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
    10861 gctggccttt tgctcacatg tgagggccta tttcccatga ttccttcata tttgcatata
    10921 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
    10981 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
    11041 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
    11101 ttatatatct tgtggaaagg acgaaacacc ggcccagact gagcacgtga gttttagagc
    11161 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
    11221 cggtgctctg ccatcaaagc gtgctcagtc tgtttttttg ttctagtccg tttttagcgc
    11281 gtgcgccaat tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat
    11341 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata
    11401 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
    11461 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
    11521 gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac
    11581 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
    11641 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
    11701 gcgatggggg cggggggggg ggg
//
