LOCUS       sequence-358501-        9176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double cutting Cas9 plasmid with puromycin resistance and a single 
            guide RNA targeting tdTomato fluorescent protein sites 90 and 816..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9176)
  AUTHORS   Madireddy I, Pierson Smela M
  TITLE     Stably Integrating an Inducible CRISPR-Cas9 to Protect Against Viral
            Infections in Vitro.
  JOURNAL   MicroPubl Biol. 2022 Jun 16;2022. doi: 
            10.17912/micropub.biology.000590. eCollection 2022.
  PUBMED    35789697
REFERENCE   2  (bases 1 to 9176)
  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: "MicroPubl 
            Biol."; date: "2022-06-16"; volume: "2022. doi"; pages: " 
            10.17912/micropub.biology.000590. eCollection 2022"
COMMENT     Sequence Label: pX459-sg-tdTomato90/816
FEATURES             Location/Qualifiers
     source          1..9176
                     /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)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1007..1234
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1246..1255
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1255..1320
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1327..1347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1372..5472
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1372..5472
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5473..5520
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5536..5589
                     /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             5590..6189
                     /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(5590..5609)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6086..6106
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6217..6234)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6223..6430
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6439..6579
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6439..6568
                     /label=AAV2 ITR
     rep_origin      6654..7109
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6741..6760)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6951..6972
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7126..7145)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7245..7267
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7305..7323)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7391..7495
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7496..7564,7565..8356)
                     /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(7714..7733)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8527..9115
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9016..9035
                     /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 gccacgagtt cgagatcgag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggsg
      901 grgaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat
     1261 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag
     1321 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
     1381 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1441 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1501 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1561 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1621 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1681 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1741 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1801 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1861 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1921 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1981 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     2041 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     2101 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     2161 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2221 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2281 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2341 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2401 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2461 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2521 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2581 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2641 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2701 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2761 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2821 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2881 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     2941 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     3001 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     3061 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     3121 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     3181 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     3241 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3301 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3361 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3421 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3481 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3541 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3601 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3661 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3721 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3781 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3841 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3901 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3961 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     4021 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     4081 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     4141 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     4201 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     4261 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4321 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4381 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4441 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4501 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4561 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4621 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4681 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4741 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     4801 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4861 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4921 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4981 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     5041 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     5101 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     5161 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     5221 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     5281 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     5341 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     5401 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5461 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     5521 gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     5581 cctggcccaa tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     5641 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     5701 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     5761 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     5821 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     5881 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     5941 cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag
     6001 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     6061 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     6121 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     6181 ggtgcctgag aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc
     6241 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6301 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6361 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     6421 atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     6481 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     6541 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     6601 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     6661 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     6721 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6781 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6841 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     6901 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6961 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     7021 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     7081 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     7141 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     7201 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     7261 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     7321 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     7381 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     7441 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     7501 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     7561 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     7621 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     7681 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     7741 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     7801 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     7861 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     7921 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7981 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     8041 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     8101 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     8161 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     8221 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     8281 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     8341 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     8401 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     8461 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     8521 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     8581 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     8641 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     8701 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     8761 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     8821 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     8881 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     8941 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     9001 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     9061 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     9121 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
