LOCUS       sequence-437116-        9441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The backbone dual-CRISPR plasmid containing Cas9 that is used for 
            making dual-CRISPR plasmids..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9441)
  AUTHORS   Li Y, Tan M, Akkari-Henic A, Zhang L, Kip M, Sun S, Sepers JJ, Xu N,
            Ariyurek Y, Kloet SL, Davis RP, Mikkers H, Gruber JJ, Snyder MP, Li 
            X, Pang B
  TITLE     Genome-wide Cas9-mediated screening of essential non-coding 
            regulatory elements via libraries of paired single-guide RNAs.
  JOURNAL   Nat Biomed Eng. 2024 May 22. doi: 10.1038/s41551-024-01204-8.
  PUBMED    38778183
REFERENCE   2  (bases 1 to 9441)
  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: "Nat Biomed 
            Eng. 2024 May 22. doi: 10.1038/s41551-024-01204-8."
COMMENT     Sequence Label: Dual-CRISPR-Cas9-U6-H1 (pBP48)
FEATURES             Location/Qualifiers
     source          1..9441
                     /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)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        complement(384..598)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(444..463)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     enhancer        689..974
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1254..1481
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1496..1585
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             1592..1612
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1637..5737
                     /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             1637..5737
                     /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             5738..5785
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5801..5854
                     /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             5855..6454
                     /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(5855..5874)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6351..6371
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6482..6499)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6488..6695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6704..6844
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6704..6833
                     /label=AAV2 ITR
     rep_origin      6919..7374
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7006..7025)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7216..7237
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7391..7410)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7510..7532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7570..7588)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7656..7760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7761..7829,7830..8621)
                     /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(7979..7998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8792..9380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9281..9300
                     /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 ggtcttcgaa aaggatccat gagcaaaata aaaaaacgct agttttagta
      301 actcgcgttg ttttcttcac ctttaataat agctactcca ccacttgttc ctaagcggat
      361 ccagctagaa gactcgggaa agagtggtct catacagaac ttataagatt cccaaatcca
      421 aagacatttc acgtttatgg tgatttccca gaacacatag cgacatgcaa atattgcagg
      481 gcgccactcc cctgtccctc acagccatct tcctgccagg gcgcacgcgc gctgggtgtt
      541 cccgcctagt gacactgggc ccgcgattcc ttggagcggg ttgatgacgt cagcgttcgt
      601 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      661 tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
      721 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
      781 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      841 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      901 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      961 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     1021 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     1081 cggggggggg gggggggcgc gcgccrggsg gggsggggsg gggsgggggg sggggsgggg
     1141 sgrggsggrg rggkgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1201 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1261 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
     1321 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
     1381 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
     1441 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtatgtaccc
     1501 atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatcgta
     1561 tccttatgac gttccagatt acgctatggc cccaaagaag aagcggaagg tcggtatcca
     1621 cggagtccca gcagccgaca agaagtacag catcggcctg gacatcggca ccaactctgt
     1681 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     1741 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     1801 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     1861 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     1921 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     1981 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     2041 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     2101 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     2161 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     2221 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     2281 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     2341 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     2401 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     2461 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     2521 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     2581 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     2641 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     2701 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     2761 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     2821 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     2881 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     2941 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     3001 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     3061 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     3121 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     3181 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     3241 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     3301 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     3361 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     3421 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     3481 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     3541 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     3601 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     3661 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     3721 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     3781 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     3841 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     3901 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     3961 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     4021 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     4081 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     4141 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     4201 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     4261 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     4321 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     4381 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     4441 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     4501 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     4561 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     4621 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     4681 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     4741 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     4801 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     4861 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     4921 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     4981 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     5041 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     5101 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     5161 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     5221 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     5281 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     5341 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     5401 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     5461 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     5521 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     5581 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     5641 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     5701 cgagacacgg atcgacctgt ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa
     5761 ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga gagggcagag gaagtctgct
     5821 aacatgcggt gacgtcgagg agaatcctgg cccaatgacc gagtacaagc ccacggtgcg
     5881 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     5941 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     6001 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     6061 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     6121 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     6181 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     6241 agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     6301 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     6361 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     6421 cacctggtgc atgacccgca agcccggtgc ctgagaattc taactagagc tcgctgatca
     6481 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     6541 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     6601 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     6661 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     6721 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     6781 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     6841 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     6901 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     6961 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     7021 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     7081 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     7141 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     7201 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     7261 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     7321 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     7381 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7441 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7501 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     7561 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     7621 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     7681 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     7741 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     7801 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     7861 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     7921 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     7981 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     8041 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     8101 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     8161 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     8221 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     8281 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     8341 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     8401 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     8461 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     8521 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     8581 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     8641 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     8701 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     8761 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     8821 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     8881 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     8941 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     9001 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     9061 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     9121 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     9181 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     9241 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     9301 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     9361 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     9421 gctggccttt tgctcacatg t
//
