LOCUS       sequence-197197-        9579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 with 2A-Puro and a cloning backbone for 2 custom gRNAs which 
            can be cloned in via a one-step reaction. For generation of double 
            knock-outs and large deletions in a single plasmid system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9579)
  AUTHORS   Adikusuma F, Pfitzner C, Thomas PQ
  TITLE     Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA 
            expression.
  JOURNAL   PLoS One. 2017 Dec 6;12(12):e0187236. doi: 
            10.1371/journal.pone.0187236. eCollection 2017.
  PUBMED    29211736
REFERENCE   2  (bases 1 to 9579)
  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: "PLoS One.";
            date: "2017-12-6"; volume: "12(12):e0187236. doi"; pages: " 
            10.1371/journal.pone.0187236. eCollection 2017"
COMMENT     Sequence Label: pDG459
FEATURES             Location/Qualifiers
     source          1..9579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        120..405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(502..577)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(596..844)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     rep_origin      complement(906..1494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(986..1005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1665..2456,2457..2525))
                     /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     2288..2307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2526..2630)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2698..2716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2754..2776)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2876..2895
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2912..3367)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3261..3280
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3442..3582
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3453..3582
                     /label=AAV2 ITR
     promoter        3591..3839
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        3857..3932
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3975..3991)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3975..3991)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    4000..4207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4196..4213
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4241..4840)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4324..4344)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4821..4840
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(4841..4894)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(4910..4957)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(4958..9058)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(4958..9058)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(9083..9103)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(9110..9175)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      9175..9184
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          9196..9423
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(join(9424..9579,1..118))
                     /label=chicken beta-actin promoter
ORIGIN
        1 cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa atacaaaatt
       61 gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct cacctcgacc
      121 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
      181 atgtactggg cacaatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
      241 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
      301 cattgacgtc aatggaaagt ccctattggc gttactattg acgtcaatgg gcgggggtcg
      361 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgggtac ctctagagcc
      421 atttgtctgc agaattggcg cacgcgctaa aaacggacta gccttatttt aacttgctat
      481 ttctagctct aaaacaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac
      541 tagccttatt ttaacttgct atttctagct ctaaaacagg tcttctcgaa gacccggtgt
      601 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
      661 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
      721 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa
      781 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
      841 cctcacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1321 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1561 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1621 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1681 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1741 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1801 ccgcggcttc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1861 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1921 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1981 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2041 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2101 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2161 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2221 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2281 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2341 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2401 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2461 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2521 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2641 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     2701 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     2761 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     2821 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     2881 tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa
     2941 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa
     3001 atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac
     3061 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3121 ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt
     3181 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3241 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca
     3301 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3361 ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga
     3421 gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg
     3481 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     3541 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc gagggcctat
     3601 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     3661 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     3721 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     3781 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     3841 gggtcttcga gaagacgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     3901 atcaacttga aaaagtggca ccgagtcggt gcttttttcc tgcaggattc gtatcgaatt
     3961 cctgcagccc gggggatcca ctagttctag agcggccgct ccccagcatg cctgctattc
     4021 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     4081 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     4141 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     4201 ggcacagtcg aggctgatca gcgagctcta gttagaattc tcaggcaccg ggcttgcggg
     4261 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     4321 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     4381 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     4441 ggtcgggcga gactccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     4501 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     4561 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     4621 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     4681 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg acggtgtggc
     4741 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt
     4801 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat tgggccagga ttctcctcga
     4861 cgtcaccgca tgttagcaga cttcctctgc cctctccact gccgaattcc tttttctttt
     4921 ttgcctggcc ggcctttttc gtggccgccg gccttttgtc gcctcccagc tgagacaggt
     4981 cgatccgtgt ctcgtacagg ccggtgatgc tctggtggat cagggtggcg tccagcacct
     5041 ctttggtgct ggtgtacctc ttccggtcga tggtggtgtc aaagtacttg aaggcggcag
     5101 gggctcccag attggtcagg gtaaacaggt ggatgatatt ctcggcctgc tctctgatgg
     5161 gcttatcccg gtgcttgttg taggcggaca gcactttgtc cagattagcg tcggccagga
     5221 tcactctctt ggagaactcg ctgatctgct cgatgatctc gtccaggtag tgcttgtgct
     5281 gttccacaaa cagctgtttc tgctcattat cctcggggga gcccttcagc ttctcatagt
     5341 ggctggccag gtacaggaag ttcacatatt tggagggcag ggccagttcg tttcccttct
     5401 gcagttcgcc ggcagaggcc agcattctct tccggccgtt ttccagctcg aacagggagt
     5461 acttaggcag cttgatgatc aggtcctttt tcacttcttt gtagcccttg gcttccagaa
     5521 agtcgatggg attcttctcg aagctgcttc tttccatgat ggtgatcccc agcagctctt
     5581 tcacactctt cagtttcttg gacttgccct tttccacttt ggccaccacc agcacagaat
     5641 aggccacggt ggggctgtcg aagccgccgt acttcttagg gtcccagtcc ttctttctgg
     5701 cgatcagctt atcgctgttc ctcttgggca ggatagactc tttgctgaag ccgcctgtct
     5761 gcacctcggt ctttttcacg atattcactt ggggcatgct cagcactttc cgcacggtgg
     5821 caaaatcccg gcccttatcc cacacgatct ccccggtttc gccgtttgtc tcgatcagag
     5881 gccgcttccg gatctcgccg ttggccaggg taatctcggt cttgaaaaag ttcatgatgt
     5941 tgctgtagaa gaagtacttg gcggtagcct tgccgatttc ctgctcgctc ttggcgatca
     6001 tcttccgcac gtcgtacacc ttgtagtcgc cgtacacgaa ctcgctttcc agcttagggt
     6061 actttttgat cagggcggtt cccacgacgg cgttcaggta ggcgtcgtgg gcgtggtggt
     6121 agttgttgat ctcgcgcact ttgtaaaact ggaaatcctt ccggaaatcg gacaccagct
     6181 tggacttcag ggtgatcact ttcacttccc ggatcagctt gtcattctcg tcgtacttag
     6241 tgttcatccg ggagtccagg atctgtgcca cgtgctttgt gatctgccgg gtttccacca
     6301 gctgtctctt gatgaagccg gccttatcca gttcgctcag gccgcctctc tcggccttgg
     6361 tcagattgtc gaactttctc tgggtaatca gcttggcgtt cagcagctgc cgccagtagt
     6421 tcttcatctt cttcacgacc tcttcggagg gcacgttgtc gctcttgccc cggttcttgt
     6481 cgcttctggt cagcaccttg ttgtcgatgg agtcgtcctt cagaaagctc tgaggcacga
     6541 tatggtccac atcgtagtcg gacagccggt tgatgtccag ttcctggtcc acgtacatat
     6601 cccgcccatt ctgcaggtag tacaggtaca gcttctcgtt ctgcagctgg gtgttttcca
     6661 cggggtgttc tttcaggatc tggctgccca gctctttgat gccctcttcg atccgcttca
     6721 ttctctcgcg gctgttcttc tgtcccttct gggtggtctg gttctctctg gccatttcga
     6781 tcacgatgtt ctcgggcttg tgccggccca tcactttcac gagctcgtcc accaccttca
     6841 ctgtctgcag gatgcccttc ttaatggcgg ggctgccggc cagattggca atgtgctcgt
     6901 gcaggctatc gccctggccg gacacctggg ctttctggat gtcctcttta aaggtcaggc
     6961 tgtcgtcgtg gatcagctgc atgaagtttc tgttggcgaa gccgtcggac ttcaggaaat
     7021 ccaggattgt cttgccggac tgcttgtccc ggatgccgtt gatcagcttc cggctcagcc
     7081 tgccccagcc ggtgtatctc cgccgcttca gctgcttcat cactttgtcg tcgaacaggt
     7141 gggcataggt tttcagccgt tcctcgatca tctctctgtc ctcaaacagt gtcagggtca
     7201 gcacgatatc ttccagaatg tcctcgtttt cctcattgtc caggaagtcc ttgtccttga
     7261 taattttcag cagatcgtgg tatgtgccca gggaggcgtt gaaccgatct tccacgccgg
     7321 agatttccac ggagtcgaag cactcgattt tcttgaagta gtcctctttc agctgcttca
     7381 cggtcacttt ccggttggtc ttgaacagca ggtccacgat ggcctttttc tgctcgccgc
     7441 tcaggaaggc gggctttctc attccctcgg tcacgtattt cactttggtc agctcgttat
     7501 acacggtgaa gtactcgtac agcaggctgt gcttgggcag caccttctcg ttgggcaggt
     7561 tcttatcgaa gttggtcatc cgctcgatga agctctgggc ggaagcgccc ttgtccacca
     7621 cttcctcgaa gttccagggg gtgatggttt cctcgctctt tctggtcatc caggcgaatc
     7681 tgctgtttcc cctggccaga gggcccacgt agtaggggat gcggaaggtc aggatcttct
     7741 cgatcttttc ccggttgtcc ttcaggaatg ggtaaaaatc ttcctgccgc cgcagaatgg
     7801 cgtgcagctc tcccaggtgg atctggtggg ggatgctgcc gttgtcgaag gtccgctgct
     7861 tccgcagcag gtcctctctg ttcagcttca cgagcagttc ctcggtgccg tccatctttt
     7921 ccaggatggg cttgatgaac ttgtagaact cttcctggct ggctccgccg tcaatgtagc
     7981 cggcgtagcc gttcttgctc tggtcgaaga aaatctcttt gtacttctca ggcagctgct
     8041 gccgcacgag agctttcagc agggtcaggt cctggtggtg ctcgtcgtat ctcttgatca
     8101 tagaggcgct caggggggcc ttggtgatct cggtgttcac tctcaggatg tcgctcagca
     8161 ggatggcgtc ggacaggttc ttggcggcca gaaacaggtc ggcgtactgg tcgccgatct
     8221 gggccagcag gttgtccagg tcgtcgtcgt aggtgtcctt gctcagctgc agtttggcat
     8281 cctcggccag gtcgaagttg ctcttgaagt tgggggtcag gcccaggctc agggcaatca
     8341 ggtttccgaa caggccattc ttcttctcgc cgggcagctg ggcgatcaga ttttccagcc
     8401 gtctgctctt gctcagtctg gcagacagga tggccttggc gtccacgccg ctggcgttga
     8461 tggggttttc ctcgaacagc tggttgtagg tctgcaccag ctggatgaac agcttgtcca
     8521 cgtcgctgtt gtcggggttc aggtcgccct cgatcaggaa gtggccccgg aacttgatca
     8581 tgtgggccag ggccagatag atcagccgca ggtcggcctt gtcggtgctg tccaccagtt
     8641 tctttctcag gtggtagatg gtggggtact tctcgtggta ggccacctcg tccacgatgt
     8701 tgccgaagat ggggtgccgc tcgtgcttct tatcctcttc caccaggaag gactcttcca
     8761 gtctgtggaa gaagctgtcg tccaccttgg ccatctcgtt gctgaagatc tcttgcagat
     8821 agcagatccg gttcttccgt ctggtgtatc ttcttctggc ggttctcttc agccgggtgg
     8881 cctcggctgt ttcgccgctg tcgaacagca gggctccgat caggttcttc ttgatgctgt
     8941 gccggtcggt gttgcccagc accttgaatt tcttgctggg caccttgtac tcgtcggtga
     9001 tcacggccca gcccacagag ttggtgccga tgtccaggcc gatgctgtac ttcttgtcgg
     9061 ctgctgggac tccgtggata ccgaccttcc gcttcttctt tggggccatc ttatcgtcat
     9121 cgtctttgta atcaatatca tgatccttgt agtctccgtc gtggtcctta tagtccatgg
     9181 tggcaccggt ccaacctgaa aaaaagtgat ttcaggcagg tgctccaggt aattaaacat
     9241 taatacccca ccaaccaacc atcccttaaa cccttacctc ttgctcagct aattacagcc
     9301 cggaggagaa gggccgtccc gcccgctcac ctgtgggagt aacgcggtca gtcagagccg
     9361 gggcgggcgg cgcgaggcgg cggcggagcg gggcacgggg cgaaggcagc gcgcagcgac
     9421 tcccgcccgc cgcgcgcttc gctttttata gggccgccgc cgccgccgcc tcgccataaa
     9481 aggaaacttt cggagcgcgc cgctctgatt ggctgccgcc gcacctctcc gcctcgcccc
     9541 gccccgcccc tcgccccgcc ccgccccgcc tggcgcgcg
//
