LOCUS       sequence-380118-        9142 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPR knockout of integrin alphaV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9142)
  AUTHORS   Lin FY, Li J, Xie Y, Zhu J, Huong Nguyen TT, Zhang Y, Zhu J, 
            Springer TA
  TITLE     A general chemical principle for creating closure-stabilizing 
            integrin inhibitors.
  JOURNAL   Cell. 2022 Sep 15;185(19):3533-3550.e27. doi: 
            10.1016/j.cell.2022.08.008.
  PUBMED    36113427
REFERENCE   2  (bases 1 to 9142)
  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"; doi: 
            "10.1016/j.cell.2022.08"; journalName: "Cell"; date: "2022-09-15- 
            15"; volume: "185"; issue: "19"; pages: "3533-3550.e27"
COMMENT     Sequence Label: pSpCas9n(BB)-2A-puro-aV guide B
FEATURES             Location/Qualifiers
     source          1..9142
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        80..328
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     80..100
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     251..270
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        350..425
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        522..807
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     gap             926..935
                     /estimated_length=10
     intron          1052..1279
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1291..1300
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1300..1365
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1372..1392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1417..1440,1441..1443,1444..5517)
                     /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             1417..5517
                     /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             5518..5565
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5581..5634
                     /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             5635..6234
                     /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(5635..5654)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6131..6151
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6262..6279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6268..6475
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6484..6624
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6484..6613
                     /label=AAV2 ITR
     rep_origin      6699..7154
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6786..6805)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6996..7017
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7171..7190)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7290..7312
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7350..7368)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7436..7540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7541..7609,7610..8401)
                     /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(7759..7778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8572..9142,1..18)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9061..9080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
       61 tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      121 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      181 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      241 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      301 atatatcttg tggaaaggac gaaacaccgc gaagcatgcc accaagcttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      421 ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag
      481 cgcgtgcgcc aattctgcag acaaatggct ctagaggtac ccgttacata acttacggta
      541 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca
      601 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      661 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      721 cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      781 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      841 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      901 gcagcgatgg gggcgggggg gggggnnnnn nnnnngggcg aggcggagag gtgcggcggc
      961 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     1021 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
     1081 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
     1141 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta
     1201 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc
     1261 tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga
     1321 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag
     1381 aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg
     1441 gccatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     1501 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     1561 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     1621 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     1681 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     1741 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     1801 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     1861 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     1921 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     1981 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     2041 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     2101 cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc
     2161 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     2221 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     2281 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     2341 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     2401 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     2461 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     2521 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     2581 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     2641 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     2701 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     2761 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     2821 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     2881 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     2941 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     3001 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     3061 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     3121 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     3181 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     3241 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     3301 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     3361 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     3421 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     3481 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     3541 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     3601 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     3661 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     3721 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     3781 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     3841 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     3901 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     3961 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     4021 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     4081 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     4141 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     4201 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     4261 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     4321 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     4381 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     4441 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     4501 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     4561 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     4621 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     4681 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     4741 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     4801 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     4861 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     4921 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     4981 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     5041 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     5101 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     5161 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     5221 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     5281 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     5341 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     5401 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     5461 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa
     5521 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt cggcagtgga
     5581 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccaatgacc
     5641 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     5701 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     5761 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     5821 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     5881 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     5941 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     6001 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     6061 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     6121 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     6181 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc
     6241 taactagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     6301 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     6361 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     6421 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
     6481 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     6541 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     6601 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     6661 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     6721 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     6781 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6841 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6901 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6961 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7021 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     7081 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     7141 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     7201 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     7261 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     7321 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     7381 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     7441 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     7501 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     7561 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     7621 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     7681 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     7741 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     7801 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     7861 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     7921 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     7981 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     8041 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     8101 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     8161 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     8221 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     8281 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     8341 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     8401 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     8461 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     8521 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     8581 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     8641 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     8701 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     8761 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     8821 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     8881 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     8941 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     9001 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     9061 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     9121 atttttgtga tgctcgtcag gg
//
