LOCUS       sequence-254486-       13045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Opti-SpCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13045)
  AUTHORS   Choi GCG, Zhou P, Yuen CTL, Chan BKC, Xu F, Bao S, Chu HY, Thean D, 
            Tan K, Wong KH, Zheng Z, Wong ASL
  TITLE     Combinatorial mutagenesis en masse optimizes the genome editing 
            activities of SpCas9.
  JOURNAL   Nat Methods. 2019 Aug;16(8):722-730. doi: 10.1038/s41592-019-0473-0.
            Epub 2019 Jul 15.
  PUBMED    31308554
REFERENCE   2  (bases 1 to 13045)
  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"; doi: 
            "10.1038/s41592-019-0473-0"; journalName: "Nat Methods"; date: 
            "2019-08"; volume: "16"; issue: "8"; pages: "722-730"
COMMENT     Sequence Label: pAWp63-clone32 (Opti-SpCas9)
FEATURES             Location/Qualifiers
     source          1..13045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    24..612
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(77..97)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(495..506)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(615..631)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(616..632)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             1137..1317
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1343..1360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1349..1573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1619..2047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1706..1725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1916..1937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2056..2076)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2061..2390
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2241..2376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2303..2322
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2438..2485
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2504..2878
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3008..3129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3045..3064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3099..3118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3178..3194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3178..3194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3191..3213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3202..3218
                     /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(3226..3232,3233..3250,3251..3256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3271..3292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3409..3426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3580..4168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3660..3679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4339..5130,5131..5199))
                     /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     4962..4981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5200..5304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5379..5398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5570..5949
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5951..6149
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6104..6124
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6167..6347
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6394..6519
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7012..7245
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7430..7474
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7772..7889
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7959..8170
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8194..8203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8203..8268
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8275..8295
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8320..12420
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPHLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             12421..12468
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12484..12540
                     /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             12541..12915
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
ORIGIN
        1 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
       61 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
      121 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
      181 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
      241 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
      301 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
      361 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
      421 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
      481 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
      541 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
      601 ccgcctcccc gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa
      661 gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg
      721 aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg
      781 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
      841 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc
      901 agaactacac accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc
      961 tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt
     1021 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt
     1081 ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt
     1141 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     1201 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     1261 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg
     1321 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     1381 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     1441 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     1501 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     1561 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     1621 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1681 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1741 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1801 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1861 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1921 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1981 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2041 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2101 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2161 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2221 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2281 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2341 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2401 gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata
     2461 gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag
     2521 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg
     2581 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt
     2641 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt
     2701 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt
     2761 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt
     2821 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca
     2881 cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     2941 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     3001 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3061 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3121 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     3181 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3241 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3301 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3361 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3421 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3481 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     4381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     4441 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     4501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     4561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     4621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     4681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     4741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     5041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     5101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     5161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     5221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     5281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     5341 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
     5401 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     5461 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     5521 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     5581 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     5641 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     5701 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     5761 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     5821 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     5881 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     5941 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     6001 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     6061 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     6121 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
     6181 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6241 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6301 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     6361 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     6421 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     6481 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     6541 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     6601 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     6661 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     6721 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     6781 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     6841 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     6901 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     6961 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     7021 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     7081 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     7141 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     7201 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     7261 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     7321 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     7381 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     7441 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     7501 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     7561 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     7621 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     7681 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
     7741 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     7801 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     7861 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     7921 tttggttaat taatcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     7981 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     8041 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     8101 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     8161 cagaacacag gtgtcgtgac gcgggatcta gaggccacca tggactataa ggaccacgac
     8221 ggagactaca aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag
     8281 aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc
     8341 ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
     8401 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
     8461 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
     8521 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     8581 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     8641 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     8701 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     8761 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     8821 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     8881 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     8941 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     9001 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     9061 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     9121 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     9181 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     9241 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac
     9301 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     9361 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     9421 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     9481 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     9541 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat
     9601 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     9661 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     9721 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     9781 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     9841 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     9901 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     9961 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
    10021 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
    10081 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
    10141 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
    10201 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
    10261 aagcagctga agcggcggag atacaccggc tggggcgcgc tgagccggaa gctgatcaac
    10321 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
    10381 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
    10441 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
    10501 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
    10561 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
    10621 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
    10681 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
    10741 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
    10801 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
    10861 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
    10921 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
    10981 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
    11041 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
    11101 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
    11161 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
    11221 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
    11281 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctcacctgga aagcgagttc
    11341 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
    11401 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
    11461 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
    11521 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
    11581 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
    11641 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
    11701 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
    11761 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc
    11821 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
    11881 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
    11941 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg
    12001 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
    12061 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
    12121 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
    12181 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
    12241 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
    12301 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc
    12361 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
    12421 aagcgtcctg ctgctactaa gaaagctggt caagctaaga aaaagaaagc tagcggcagc
    12481 ggcgccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc
    12541 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
    12601 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
    12661 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
    12721 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
    12781 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
    12841 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
    12901 gaggagcagg actgataggg ataacagggt aatcgatatc aagcttatcg ataatcaacc
    12961 cgatctacac ggaaccgcaa cggtattctt cactcgcttg cagacaatct cttgcaaccg
    13021 acatcttgca aaaaccacct tgcag
//
