LOCUS       sequence-103232-        9896 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Vector for Cas9n (D10A) mRNA synthesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9896)
  AUTHORS   Ohtsuka M
  TITLE     Ohtsuka lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9896)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBHD
FEATURES             Location/Qualifiers
     source          1..9896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             965..991
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             995..1015
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1025..1048,1049..1051,1052..5125)
                     /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             1025..5125
                     /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             5129..5149
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5399..5428
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5444..5461
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5484..5501)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5490..5714
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5760..6188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5847..5866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6057..6078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6197..6217)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6202..6532
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6383..6518
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6445..6464
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6599..7393
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6653..6672)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7263..7282
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7569..7690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7606..7625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7660..7679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7739..7755)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7739..7755)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7752..7774)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7763..7779
                     /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(7787..7793,7794..7811,7812..7817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7832..7853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7970..7987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8141..8729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8221..8240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8900..9691,9692..9760))
                     /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     9523..9542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9761..9865)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattgca ccggtgccac
      961 catgtaccca tacgatgttc cagattacgc ttcgccgaag aaaaagcgca aggtcgaagc
     1021 gtccgacaag aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt
     1081 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
     1141 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
     1201 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
     1261 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
     1321 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
     1381 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     1441 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     1501 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     1561 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     1621 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     1681 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggcaa
     1741 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     1801 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     1861 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     1921 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     1981 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     2041 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     2101 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     2161 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     2221 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     2281 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     2341 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     2401 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     2461 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     2521 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     2581 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     2641 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     2701 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     2761 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     2821 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     2881 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     2941 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     3001 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     3061 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     3121 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     3181 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     3241 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     3301 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     3361 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     3421 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     3481 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca
     3541 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     3601 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     3661 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     3721 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     3781 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     3841 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     3901 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     3961 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     4021 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     4081 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     4141 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     4201 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     4261 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     4321 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     4381 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     4441 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     4501 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     4561 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     4621 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     4681 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     4741 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     4801 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     4861 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     4921 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     4981 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     5041 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     5101 cgacctgtct cagctgggag gcgacagccc caagaagaag agaaaggtgg aggccagcta
     5161 agaattctgc agatatccag cacagtggcg gccgctcgac tatgggcacc aaagaacctg
     5221 taaacgttat ctttttaaat tgaatgtgca caaataaaag tttggaaaag aaaaaaaaaa
     5281 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5341 aaaaaaaaaa aaatacgcat catacgctgt ggcgtacctc gagtctagag ggcccttcga
     5401 acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca
     5461 ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     5521 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5581 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5641 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     5701 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     5761 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5821 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5881 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     5941 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     6001 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     6061 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     6121 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6181 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     6241 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     6301 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     6361 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     6421 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     6481 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     6541 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     6601 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     6661 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     6721 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     6781 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     6841 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     6901 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     6961 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     7021 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     7081 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     7141 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     7201 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     7261 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     7321 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     7381 cgagttcttc tgagcgggac tctggggttc gcgaaatgac cgaccaagcg acgcccaacc
     7441 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     7501 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     7561 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     7621 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     7681 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     7741 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     7801 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     7861 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     7921 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     7981 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     8041 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     8101 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     8161 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     8221 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     8281 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     8341 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     8401 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     8461 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     8521 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     8581 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     8641 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     8701 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     8761 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     8821 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     8881 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     8941 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     9001 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     9061 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     9121 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     9181 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     9241 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     9301 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     9361 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     9421 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     9481 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     9541 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     9601 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     9661 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     9721 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     9781 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     9841 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
