LOCUS       sequence-447632-        8612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCBh Human expression plasmid for SpCas9 with with C-term 
            BPNLS-P2A-mTagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8612)
  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 8612)
  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: pCBh-T7-SpCas9-P2A-mTagBFP2(RAS3575)
FEATURES             Location/Qualifiers
     source          1..8612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(190..981,982..1050))
                     /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     813..832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1051..1155)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1234..1253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1439..1724
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2004..2231
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2243..2252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2249..6352
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2249..6352
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6407..6427
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6440..6505
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6524..6580
                     /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             6581..7291
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             7300..7317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7340..7357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7346..7570
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7641..7657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7641..7657)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7654..7676)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7665..7681
                     /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(7689..7695,7696..7713,7714..7719))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7734..7755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7872..7889)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8043..8612,1..19))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8123..8142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
       61 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      121 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      181 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      301 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1141 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     1201 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     1261 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     1321 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     1381 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtctagatc agggtacccg
     1441 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     1501 cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1561 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1621 tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc
     1681 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     1741 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     1801 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggggc gccccggggg
     1861 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggcccaac
     1921 aagggggggg gccccgaaag gttttttttt ttgtgggggg gggggggggg ggggccccta
     1981 aaaaacaaga agcggggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     2041 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     2101 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     2161 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     2221 ctttttttca ggttggaccg gcgccaccat ggacaagaag tacagcatcg gcctggacat
     2281 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2341 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     2401 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     2461 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     2521 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     2581 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     2641 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     2701 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     2761 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     2821 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     2881 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     2941 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3001 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3061 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3121 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3181 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3241 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3301 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     3361 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3421 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     3481 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     3541 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     3601 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     3661 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     3721 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     3781 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     3841 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     3901 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     3961 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4021 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4081 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4141 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4201 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4261 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4321 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4381 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4441 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     4501 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     4561 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     4621 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     4681 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     4741 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     4801 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     4861 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     4921 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     4981 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5041 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5101 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5161 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5221 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5281 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5341 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5401 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     5461 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     5521 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     5581 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     5641 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     5701 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     5761 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     5821 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     5881 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     5941 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6001 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6061 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6121 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6181 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6241 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6301 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acggatccgg
     6361 cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag
     6421 gaaagtctcg agcggaggcg actacaaaga ccatgacggt gattataaag atcatgacat
     6481 cgattacaag gatgacgatg acaagggagg cggtggaagc ggcgcaacaa acttctctct
     6541 gctgaaacaa gccggagatg tcgaagagaa tcctggaccg atggtgtcta agggcgaaga
     6601 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg acaaccatca
     6661 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     6721 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ctagcttcct
     6781 ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct tcaagcagtc
     6841 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     6901 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     6961 ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctt
     7021 caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca tggccctgaa
     7081 gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc acatatagat ccaagaaacc
     7141 cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg aaagaatcaa
     7201 ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca gatactgcga
     7261 cctccctagc aaactggggc acaaacttaa ttgaccggtc atcatcacca tcaccattga
     7321 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     7381 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     7441 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     7501 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     7561 ggctctatgg cttctgaggc ggaaagaacc agctggggct cgataccgtc gacctctagc
     7621 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     7681 attccacaca acatacgagc cggaagcata aagtgtaaag cctagggtgc ctaatgagtg
     7741 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     7801 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     7861 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     7921 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     7981 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     8041 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     8101 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     8161 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     8221 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     8281 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     8341 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     8401 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     8461 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     8521 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     8581 ggtttttttg tttgcaagca gcagattacg cg
//
