LOCUS       sequence-190184-        8893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 with 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 8893)
  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 8893)
  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: pDG330
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..983
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          984..1211
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1223..1232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1232..1297
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1304..1324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1349..5449
                     /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             1349..5449
                     /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             5450..5497
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5525..5542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5531..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5747..5763
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5747..5763
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(5806..5881)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5899..6147)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   6156..6296
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6156..6285
                     /label=AAV2 ITR
     rep_origin      6371..6826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6458..6477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6668..6689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6843..6862)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6962..6984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7022..7040)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7108..7212
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7213..7281,7282..8073)
                     /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     complement(7431..7450)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8244..8832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8733..8752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
      901 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      961 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1021 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1081 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     1141 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     1201 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     1261 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1321 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1381 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1441 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1501 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1561 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1621 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1681 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1741 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1801 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1861 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1921 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1981 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2041 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     2101 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     2161 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     2221 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2281 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2341 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2401 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2461 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2521 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2581 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2641 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2701 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2761 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2821 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2881 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2941 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3001 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3061 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     3121 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     3181 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     3241 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     3301 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     3361 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     3421 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     3481 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     3541 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     3601 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3661 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3721 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3781 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3841 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     3901 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3961 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     4021 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     4081 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     4141 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     4201 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     4261 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     4321 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     4381 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     4441 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     4501 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     4561 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4621 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     4681 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     4741 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4801 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     4861 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     4921 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     4981 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     5041 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     5101 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     5161 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     5221 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     5281 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     5341 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     5401 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc
     5461 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga
     5521 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5581 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5641 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5701 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgctcta gaactagtgg
     5761 atcccccggg ctgcaggaat tcgatacgaa tcctgcagga aaaaagcacc gactcggtgc
     5821 cactttttca agttgataac ggactagcct tattttaact tgctatttct agctctaaaa
     5881 cgtcttctcg aagaccccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
     5941 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
     6001 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
     6061 gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta tatgcaaata
     6121 tgaaggaatc atgggaaata ggccctcgcg gccgcaggaa cccctagtga tggagttggc
     6181 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     6241 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     6301 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     6361 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     6421 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     6481 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     6541 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     6601 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     6661 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     6721 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     6781 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     6841 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     6901 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     6961 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     7021 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     7081 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     7141 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     7201 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     7261 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     7321 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     7381 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     7441 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     7501 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     7561 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     7621 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     7681 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     7741 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     7801 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     7861 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     7921 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     7981 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     8041 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     8101 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     8161 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     8221 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     8281 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     8341 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     8401 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     8461 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     8521 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     8581 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     8641 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     8701 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     8761 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     8821 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     8881 tttgctcaca tgt
//
