LOCUS       sequence-462176-       12053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For packaging spCas9, dTomato mRNA into SARS-CoV-2 virus-like 
            particles (VLPs) with a 'PS9' packaging signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12053)
  AUTHORS   Yang Q, Davidson BA, Pajic P, Chen X, Gokcumen O, Gao M, Neelamegham
            S
  TITLE     Tuning the tropism and infectivity of SARS-CoV-2 virus-like 
            particles for mRNA delivery.
  JOURNAL   Nucleic Acids Res. 2025 Feb 27;53(5):gkaf133. doi: 
            10.1093/nar/gkaf133.
  PUBMED    40037714
REFERENCE   2  (bases 1 to 12053)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2025-02-27"; volume: "53(5):gkaf133. doi"; 
            pages: " 10.1093/nar/gkaf133"
COMMENT     Sequence Label: pcDNA3.1 Cas9-P2A-dTomato-PS9
FEATURES             Location/Qualifiers
     source          1..12053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        395..598
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     549..569
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     643..662
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        643..661
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             674..4777
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             674..4777
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4778..4825
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4841..4897
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4904..5605
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGKGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(5037..5055)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5305..5324
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5499..5522
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    6767..7355
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6820..6840)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7238..7249)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7424..7441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7430..7654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7700..8128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7787..7806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7997..8018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8137..8157)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8142..8471
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8322..8457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8384..8403
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             8538..9332
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(8592..8611)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9202..9221
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    9506..9627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9543..9562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9597..9616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9676..9692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9676..9692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9689..9711)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9700..9716
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9724..9730,9731..9748,9749..9754))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9769..9790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9907..9924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10078..10666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10158..10177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10837..11628,11629..11697))
                     /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     11460..11479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11698..11802)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(11877..11896)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      421 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      481 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      541 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      601 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
      661 ggagaccgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
      721 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
      781 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
      841 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
      901 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
      961 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     1021 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     1081 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     1141 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     1201 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     1261 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     1321 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     1381 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     1441 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     1501 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     1561 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     1621 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     1681 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     1741 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     1801 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     1861 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     1921 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     1981 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     2041 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     2101 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     2161 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     2221 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     2281 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     2341 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     2401 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     2461 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     2521 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     2581 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     2641 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     2701 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     2761 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     2821 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     2881 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     2941 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     3001 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     3061 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     3121 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     3181 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     3241 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     3301 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     3361 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     3421 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     3481 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     3541 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     3601 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     3661 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     3721 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     3781 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     3841 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     3901 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     3961 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     4021 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     4081 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     4141 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     4201 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     4261 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     4321 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     4381 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     4441 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     4501 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     4561 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     4621 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     4681 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     4741 cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgtcctgctg ctactaagaa
     4801 agctggtcaa gctaagaaaa agaaagctag cggcagcggc gccaccaact tcagcctgct
     4861 gaagcaggcc ggcgacgtgg aggagaaccc cggccccgtt aacatggtga gcaagggcga
     4921 ggaggtcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca
     4981 cgagttcgag atcgagggca agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     5041 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     5101 catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt acaagaagct
     5161 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt
     5221 gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg tgaagatgcg
     5281 cggcaccaac ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     5341 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc accaggccct
     5401 gaagctgaag gacggcggcc actacctggt ggagttcaag accatctaca tggccaagaa
     5461 gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca cctcccacaa
     5521 cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc acctgttcct
     5581 gtacggcatg gacgagctgt acaagtaagg agatatagat ctctgtaggt cccaaacaag
     5641 ctagtcttaa tggagtcaca ttaattggag aagccgtaaa aacacagttc aattattata
     5701 agaaagttga tggtgttgtc caacaattac ctgaaactta ctttactcag agtagaaatt
     5761 tacaagaatt taaacccagg agtcaaatgg aaattgattt cttagaatta gctatggatg
     5821 aattcattga acggtataaa ttagaaggct atgccttcga acatatcgtt tatggagatt
     5881 ttagtcatag tcagttaggt ggtttacatc tactgattgg actagctaaa cgttttaagg
     5941 aatcaccttt tgaattagaa gattttattc ctatggacag tacagttaaa aactatttca
     6001 taacagatgc gcaaacaggt tcatctaagt gtgtgtgttc tgttattgat ttattacttg
     6061 atgattttgt tgaaataata aaatcccaag atttatctgt agtttctaag gttgtcaaag
     6121 tgactattga ctatacagaa atttcattta tgctttggtg taaagatggc catgtagaaa
     6181 cattttaccc aaaattacaa tctagtcaag cgtggcaacc gggtgttgct atgcctaatc
     6241 tttacaaaat gcaaagaatg ctattagaaa agtgtgacct tcaaaattat ggtgatagtg
     6301 caacattacc taaaggcata atgatgaatg tcgcaaaata tactcaactg tgtcaatatt
     6361 taaacacatt aacattagct gtaccctata atatgagagt tatacatttt ggtgctggtt
     6421 ctgataaagg agttgcacca ggtacagctg ttttaagaca gtggttgcct acgggtacgc
     6481 tgcttgtcga ttcagatctt aatgactttg tctctgatgc agattcaact ttgattggtg
     6541 attgtgcaac tgtacataca gctaataaat gggatctcat tattagtgat atgtacgacc
     6601 ctaagactaa aaatgttaca aaagaaaatg actctaaaga gggttttttc acttacattt
     6661 gtgggtttat acaacaaaag ctagctcttg gaggttccgt ggctataaag atagatccca
     6721 actccataag tgtgtcgaca tataaggatc cactcgacga ctcgacaatc aacctctgga
     6781 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     6841 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     6901 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     6961 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     7021 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     7081 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     7141 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     7201 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     7261 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg catcttcgcc ttcgccctca
     7321 gacgagtcgg atctcccttt gggccgcctc cccgcctgga attaattcga gctcggtacc
     7381 ttgattctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     7441 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     7501 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     7561 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     7621 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     7681 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     7741 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     7801 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     7861 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     7921 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     7981 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     8041 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     8101 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     8161 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     8221 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     8281 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     8341 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     8401 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     8461 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     8521 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     8581 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     8641 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     8701 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     8761 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     8821 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     8881 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     8941 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     9001 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     9061 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     9121 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     9181 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     9241 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     9301 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     9361 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     9421 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     9481 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     9541 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     9601 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccctccacct ctagctagag
     9661 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     9721 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     9781 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     9841 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     9901 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     9961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
    10021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
    10081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
    10141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
    10201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
    10261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
    10321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
    10381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
    10441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
    10501 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
    10561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
    10621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
    10681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
    10741 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
    10801 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
    10861 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
    10921 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
    10981 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
    11041 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
    11101 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
    11161 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
    11221 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
    11281 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
    11341 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
    11401 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
    11461 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
    11521 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
    11581 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
    11641 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
    11701 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
    11761 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
    11821 gccacctgac ctccacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
    11881 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
    11941 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
    12001 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggc
//
