LOCUS       sequence-389146-        9773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9-T2A-EGFP with dual sgRNA cloning backbones.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9773)
  AUTHORS   Yoshinaga N, Zhou JK, Xu C, Quek CH, Zhu Y, Tang D, Hung LY, Najjar 
            SA, Shiu CYA, Margolis KG, Lao YH, Leong KW
  TITLE     Phenylboronic Acid-Functionalized Polyplexes Tailored to Oral CRISPR
            Delivery.
  JOURNAL   Nano Lett. 2023 Jan 17. doi: 10.1021/acs.nanolett.2c02306.
  PUBMED    36648291
REFERENCE   2  (bases 1 to 9773)
  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: "Nano Lett. 
            2023 Jan 17. doi: 10.1021/acs.nanolett.2c02306."
COMMENT     Sequence Label: PX333-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..9773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        70..318
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        337..412
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        503..751
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        778..853
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        943..1228
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1508..1735
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1747..1756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1756..1821
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1828..1848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1873..5973
                     /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             1873..5973
                     /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             5974..6021
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6037..6090
                     /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             join(6091..6093,6094..6807)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6091..6093,6094..6804)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6394..6413)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6741..6762
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6832..6849)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6838..7045
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7054..7194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7054..7183
                     /label=AAV2 ITR
     rep_origin      7269..7724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7356..7375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7566..7587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7741..7760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7860..7882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7920..7938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8006..8110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8111..8179,8180..8971)
                     /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(8329..8348)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9142..9730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9631..9650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tcacatgcgc cattctcgag catgcgccat tctcgagcat gctcgagaat
       61 ggcgcatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
      121 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
      181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
      241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
      301 tggaaaggac gaaacaccgg gtcttcgaga agacctgttt tagagctaga aatagcaagt
      361 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      481 tctgcagaca aatggctcta gagagggcct atttcccatg attccttcat atttgcatat
      541 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      601 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      661 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      721 tttatatatc ttgtggaaag gacgaaacac cggagaccta ttcgccttaa ggtctcggtt
      781 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      841 accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc
      901 gtttttagcg cgtgcgccaa ttctgcagac aaatggggta cccgttacat aacttacggt
      961 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc
     1021 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1081 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1141 gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1201 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     1261 ccccatctcc cccccctccc cacccccaat tttgtattta tttattttkk rakkattttg
     1321 tgcrgcgrtg ggggsggggg gggggggggg gcgggsgccg gggggggsgg ggsggggcga
     1381 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
     1441 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
     1501 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
     1561 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
     1621 ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg
     1681 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg
     1741 accggtgcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
     1801 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     1861 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     1921 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     1981 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2041 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     2101 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     2161 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     2221 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     2281 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     2341 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     2401 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     2461 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     2521 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     2581 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     2641 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     2701 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     2761 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     2821 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     2881 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     2941 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3001 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3061 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     3121 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     3181 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     3241 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     3301 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     3361 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     3421 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     3481 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     3541 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     3601 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     3661 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     3721 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     3781 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     3841 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     3901 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     3961 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     4021 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     4081 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     4141 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     4201 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     4261 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     4321 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     4381 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     4441 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     4501 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     4561 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     4621 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     4681 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     4741 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     4801 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     4861 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     4921 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     4981 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     5041 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     5101 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     5161 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     5221 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     5281 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     5341 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     5401 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     5461 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     5521 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     5581 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     5641 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     5701 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     5761 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     5821 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     5881 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     5941 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     6001 ggccaggcaa aaaagaaaaa gttattcggc agtggagagg gcagaggaag tctgctaaca
     6061 tgcggtgacg tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg
     6121 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     6181 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     6241 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     6301 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     6361 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     6421 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     6481 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     6541 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     6601 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     6661 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     6721 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     6781 ctcggcatgg acgagctgta caagtaagaa ttcctagagc tcgctgatca gcctcgactg
     6841 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     6901 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     6961 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     7021 aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca
     7081 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     7141 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     7201 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     7261 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     7321 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     7381 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     7441 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     7501 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     7561 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     7621 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     7681 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     7741 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     7801 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     7861 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     7921 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     7981 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     8041 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     8101 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     8161 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     8221 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     8281 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     8341 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     8401 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     8461 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     8521 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     8581 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     8641 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     8701 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     8761 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     8821 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     8881 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     8941 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     9001 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     9061 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     9121 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     9181 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     9241 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     9301 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     9361 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     9421 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     9481 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     9541 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     9601 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     9661 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     9721 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gct
//
