LOCUS       sequence-447704-        9591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCMV and pT7 Human expression plasmid for TadCBEd-SpCas9 with C-term
            BPNLS-P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9591)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 9591)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pCMV-T7-TadCBEd-SpCas9-P2A-EGFP (BKS327)
FEATURES             Location/Qualifiers
     source          1..9591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     730..749
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        730..748
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             798..818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1413..1436,1437..1439,1440..5513)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1413..5513
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5544..5792
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5823..6071
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6114..6134
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6144..6200
                     /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             join(6201..6203,6204..6206,6207..6917)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6201..6203,6204..6206,6207..6917)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6507..6526)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6854..6875
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6930..6950
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6959..6976
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6999..7016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7005..7229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7300..7316)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7300..7316)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7313..7335)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7324..7340
                     /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(7348..7354,7355..7372,7373..7378))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7393..7414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7531..7548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7702..8290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7782..7801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8461..9252,9253..9321))
                     /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     9084..9103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9322..9426)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9505..9524)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
      721 cgcggccgct aatacgactc actataggga gagccgccac catgaaacgg acagccgacg
      781 gaagcgagtt cgagtcacca aagaagaagc ggaaagtctc tgaggtggag ttttcccacg
      841 agtactggat gagacatgcc ctgaccctgg ccaagagggc acgcgatgag aggaaggccc
      901 ctgtgggagc cgtgctggtg ctgaacaata gagtgatcgg cgagggctgg aacagagcca
      961 tcggcctgca cgacccaaca gcccatgccg aaattatcgc cctgagacag ggcggcctgg
     1021 tcatgcagaa ctacagactg attgacgcca ccctgtacgt gacattcgag ccttgcgtga
     1081 tgtgcgccgg cgccatgatc aactctagga tcggccgcgt ggtgtttggc gtgaggaaca
     1141 gcaaaagagg cgccgcaggc tccctgatga acgtgctgaa ctaccccggc atgaatcacc
     1201 gcgtcgaaat taccgaggga atcctggcag atgaatgtgc cgccctgctg tgcgatttct
     1261 accggatgcc tagacaggtg ttcaatgctc agaagaaggc ccagagctcc atcaactccg
     1321 gaggatctag cggaggctcc tctggctctg agacacctgg cacaagcgag agcgcaacac
     1381 ctgaaagcag cgggggcagc agcggggggt cagacaagaa gtacagcatc ggcctggcca
     1441 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1501 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1561 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1621 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1681 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1741 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1801 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1861 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1921 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1981 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     2041 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     2101 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     2161 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     2221 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     2281 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     2341 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     2401 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     2461 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2521 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2581 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2641 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2701 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2761 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     2821 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2881 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2941 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     3001 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     3061 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     3121 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     3181 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     3241 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     3301 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     3361 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     3421 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3481 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3541 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3601 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3661 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3721 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3781 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3841 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3901 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3961 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     4021 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     4081 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     4141 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     4201 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     4261 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     4321 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     4381 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     4441 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4501 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4561 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4621 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4681 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4741 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4801 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4861 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4921 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4981 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     5041 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     5101 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     5161 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     5221 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     5281 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     5341 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     5401 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     5461 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt gacagcggcg
     5521 ggagcggcgg gagcgggggg agcactaatc tgagcgacat cattgagaag gagactggga
     5581 aacagctggt cattcaggag tccatcctga tgctgcctga ggaggtggag gaagtgatcg
     5641 gcaacaagcc agagtctgac atcctggtgc acaccgccta cgacgagtcc acagatgaga
     5701 atgtgatgct gctgacctct gacgcccccg agtataagcc ttgggccctg gtcatccagg
     5761 attctaacgg cgagaataag atcaagatgc tgagcggagg atccggagga tctggaggca
     5821 gcaccaacct gtctgacatc atcgagaagg agacaggcaa gcagctggtc atccaggaga
     5881 gcatcctgat gctgcccgaa gaagtcgaag aagtgatcgg aaacaagcct gagagcgata
     5941 tcctggtcca taccgcctac gacgagagta ccgacgaaaa tgtgatgctg ctgacatccg
     6001 acgccccaga gtataagccc tgggctctgg tcatccagga ttccaacgga gagaacaaaa
     6061 tcaaaatgct gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga
     6121 agaagaggaa agtcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     6181 tggaggagaa ccctggacct atggtgagca agggcgagga gctgttcacc ggggtggtgc
     6241 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     6301 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     6361 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta tggagtgcag tgcttcagcc
     6421 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     6481 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     6541 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     6601 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     6661 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     6721 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     6781 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     6841 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     6901 tggacgagct gtacaagtct ggtggttctc ccaagaagaa gaggaaagtc taaccggtca
     6961 tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     7021 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     7081 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     7141 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     7201 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     7261 gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     7321 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     7381 ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     7441 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     7501 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     7561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     7921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     8041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     8101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     8161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     8221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     8281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacactcagt ggaacgaaaa
     8341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     8401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     8461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     8521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     8581 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     8641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     8701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     8761 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     9001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     9061 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     9121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     9181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     9241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     9301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     9361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     9421 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc
     9481 cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     9541 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa t
//
