LOCUS       sequence-462174-       13103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For packaging spCas9, dTomato mRNA into SARS-CoV-2 virus-like 
            particles (VLPs) with a 'T20' packaging signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13103)
  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 13103)
  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-T20
FEATURES             Location/Qualifiers
     source          1..13103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        137..516
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        517..720
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     671..691
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     765..784
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        765..783
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      790..799
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             796..4899
                     /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             796..4899
                     /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             4900..4947
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4963..5019
                     /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             5026..5727
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(5159..5177)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5427..5446
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5621..5644
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    7939..8527
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7992..8012)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8410..8421)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8596..8613)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8602..8826
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8872..9300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8959..8978)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9169..9190
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9309..9329)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9314..9643
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9494..9629
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9556..9575
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             9710..10504
                     /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(9764..9783)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     10374..10393
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    10678..10799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10715..10734)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10769..10788
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10848..10864)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10848..10864)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10861..10883)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10872..10888
                     /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(10896..10902,10903..10920,10921..10926))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10941..10962
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11079..11096)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11250..11838)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11330..11349)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12009..12800,12801..12869))
                     /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     12632..12651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12870..12974)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13049..13068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
       61 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
      121 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      181 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      241 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      301 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      361 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      421 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      481 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      541 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      601 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      661 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      721 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
      781 agggagaccg ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct
      841 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
      901 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
      961 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     1021 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     1081 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     1141 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     1201 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     1261 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     1321 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     1381 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     1441 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     1501 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     1561 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     1621 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     1681 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     1741 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     1801 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     1861 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     1921 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     1981 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     2041 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     2101 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     2161 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     2221 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     2281 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     2341 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     2401 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     2461 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     2521 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     2581 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     2641 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     2701 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     2761 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     2821 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     2881 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     2941 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     3001 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     3061 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     3121 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     3181 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     3241 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     3301 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     3361 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     3421 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     3481 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     3541 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     3601 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     3661 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     3721 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     3781 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     3841 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     3901 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     3961 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     4021 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     4081 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     4141 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     4201 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     4261 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     4321 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     4381 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     4441 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     4501 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     4561 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     4621 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     4681 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     4741 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     4801 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     4861 tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgtcctgc tgctactaag
     4921 aaagctggtc aagctaagaa aaagaaagct agcggcagcg gcgccaccaa cttcagcctg
     4981 ctgaagcagg ccggcgacgt ggaggagaac cccggccccg ttaacatggt gagcaagggc
     5041 gaggaggtca tcaaagagtt catgcgcttc aaggtgcgca tggagggctc catgaacggc
     5101 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     5161 aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct gtccccccag
     5221 ttcatgtacg gctccaaggc gtacgtgaag caccccgccg acatccccga ttacaagaag
     5281 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggtctg
     5341 gtgaccgtga cccaggactc ctccctgcag gacggcacgc tgatctacaa ggtgaagatg
     5401 cgcggcacca acttcccccc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     5461 gcctccaccg agcgcctgta cccccgcgac ggcgtgctga agggcgagat ccaccaggcc
     5521 ctgaagctga aggacggcgg ccactacctg gtggagttca agaccatcta catggccaag
     5581 aagcccgtgc aactgcccgg ctactactac gtggacacca agctggacat cacctcccac
     5641 aacgaggact acaccatcgt ggaacagtac gagcgctccg agggccgcca ccacctgttc
     5701 ctgtacggca tggacgagct gtacaagtaa ggagatatag atctctgtag gtcccaaaca
     5761 agctagtctt aatggagtca cattaattgg agaagccgta aaaacacagt tcaattatta
     5821 taagaaagtt gatggtgttg tccaacaatt acctgaaact tactttactc agagtagaaa
     5881 tttacaagaa tttaaaccca ggagtcaaat ggaaattgat ttcttagaat tagctatgga
     5941 tgaattcatt gaacggtata aattagaagg ctatgccttc gaacatatcg tttatggaga
     6001 ttttagtcat agtcagttag gtggtttaca tctactgatt ggactagcta aacgttttaa
     6061 ggaatcacct tttgaattag aagattttat tcctatggac agtacagtta aaaactattt
     6121 cataacagat gcgcaaacag gttcatctaa gtgtgtgtgt tctgttattg atttattact
     6181 tgatgatttt gttgaaataa taaaatccca agatttatct gtagtttcta aggttgtcaa
     6241 agtgactatt gactatacag aaatttcatt tatgctttgg tgtaaagatg gccatgtaga
     6301 aacattttac ccaaaattac aatctagtca agcgtggcaa ccgggtgttg ctatgcctaa
     6361 tctttacaaa atgcaaagaa tgctattaga aaagtgtgac cttcaaaatt atggtgatag
     6421 tgcaacatta cctaaaggca taatgatgaa tgtcgcaaaa tatactcaac tgtgtcaata
     6481 tttaaacaca ttaacattag ctgtacccta taatatgaga gttatacatt ttggtgctgg
     6541 ttctgataaa ggagttgcac caggtacagc tgttttaaga cagtggttgc ctacgggtac
     6601 gctgcttgtc gattcagatc ttaatgactt tgtctctgat gcagattcaa ctttgattgg
     6661 tgattgtgca actgtacata cagctaataa atgggatctc attattagtg atatgtacga
     6721 ccctaagact aaaaatgtta caaaagaaaa tgactctaaa gagggttttt tcacttacat
     6781 ttgtgggttt atacaacaaa agctagctct tggaggttcc gtggctataa agataacaga
     6841 acattcttgg aatgctgatc tttataagct catgggacac ttcgcatggt ggacagcctt
     6901 tgttactaat gtgaatgcgt catcatctga agcattttta attggatgta attatcttgg
     6961 caaaccacgc gaacaaatag atggttatgt catgcatgca aattacatat tttggaggaa
     7021 tacaaatcca attcagttgt cttcctattc tttatttgac atgagtaaat ttccccttaa
     7081 attaaggggt actgctgtta tgtctttaaa agaaggtcaa atcaatgata tgattttatc
     7141 tcttcttagt aaaggtagac ttataattag agaaaacaac agagttgtta tttctagtga
     7201 tgttcttgtt aacaactaaa cgaacaatgt ttgtttttct tgttttattg ccactagtct
     7261 ctagtcagtg tgttaatctt acaaccagaa ctcaattacc ccctgcatac actaattctt
     7321 tcacacgtgg tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc
     7381 aggacttgtt cttacctttc ttttccaatg ttacttggtt ccatgctata catgtctctg
     7441 ggaccaaggg tactaagagg tttgataacc ctgtcctacc atttaatgat ggtgtttatt
     7501 ttgcttccac tgagaagtct aacataataa gaggctggat ttttggtact actttagatt
     7561 cgaagaccca gtccctactt attgttaata acgctactaa tgttgttatt aaagtctgtg
     7621 aatttcaatt ttgtaatgat ccatttttgg gtgtttatta ccacaaaaac aacaaaagtt
     7681 ggatggaaag tgagttcaga gtttattcta gtgcgaataa ttgcactttt gaatatgtct
     7741 ctcagccttt tcttatggac cttgaaggaa aacagggtaa tttcaaaaat cttagggaat
     7801 ttgtgtttaa gaatattgat ggttatttta aaatatattc taagcacacg cctattaatt
     7861 tagtgcgtga tctccctcag ggtttgatcc caactccata agtgtgtcga catataagga
     7921 tccactcgac gactcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     7981 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     8041 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     8101 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     8161 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     8221 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     8281 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     8341 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     8401 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     8461 cggcctcttc cgcatcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     8521 tccccgcctg gaattaattc gagctcggta ccttgattct cgagtctaga gggcccgttt
     8581 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     8641 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     8701 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     8761 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     8821 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     8881 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     8941 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     9001 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     9061 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     9121 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     9181 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     9241 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     9301 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     9361 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     9421 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     9481 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     9541 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     9601 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     9661 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     9721 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     9781 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     9841 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     9901 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     9961 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
    10021 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
    10081 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
    10141 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
    10201 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
    10261 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
    10321 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
    10381 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
    10441 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
    10501 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
    10561 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
    10621 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
    10681 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
    10741 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
    10801 catgtctgta taccctccac ctctagctag agcttggcgt aatcatggtc atagctgttt
    10861 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
    10921 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    10981 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    11041 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    11101 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    11161 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    11221 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    11281 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    11341 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    11401 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    11461 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    11521 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    11581 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    11641 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    11701 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    11761 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    11821 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    11881 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    11941 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    12001 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    12061 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    12121 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    12181 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    12241 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    12301 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    12361 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    12421 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    12481 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    12541 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    12601 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    12661 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    12721 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    12781 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    12841 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    12901 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    12961 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acctccacgg atcgggagat
    13021 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
    13081 agtatctgct ccctgcttgt gtg
//
