LOCUS       sequence-362763-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets TOP2A gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11691)
  AUTHORS   Hu M, Lei XY, Larson JD, McAlonis M, Ford K, McDonald D, Mach K, 
            Rusert JM, Wechsler-Reya RJ, Mali P
  TITLE     Integrated genome and tissue engineering enables screening of cancer
            vulnerabilities in physiologically relevant perfusable ex vivo 
            cultures.
  JOURNAL   Biomaterials. 2022 Jan;280:121276. doi: 
            10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2.
  PUBMED    34890975
REFERENCE   2  (bases 1 to 11691)
  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: 
            "Biomaterials."; date: "2022-01"; volume: "280:121276. doi"; pages: 
            " 10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2"
COMMENT     Sequence Label: CROP_C9_Puro_TOP2A
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..49
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        31..49
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        77..303
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             304..484
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    531..656
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1149..1382
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1567..1611
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1792..1909
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2098..2309
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2328..2337
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2334..6437
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2334..6437
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6438..6485
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6486..6509
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6519..6575
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6576..7175
                     /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(6576..6595)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7072..7092
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7191..7779
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7244..7264)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7662..7673)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7935..8183
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7935..7955
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8106..8125
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8205..8280
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8333..8513
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8591..8712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8628..8647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8682..8701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8752..8887
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8907..8926)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8908..8926)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8936..8953)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8936..8952)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8945..8967)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9094..9549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9181..9200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9391..9412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9575..9679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9680..9748,9749..10540)
                     /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(9898..9917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10711..11299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11200..11219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11453..11470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11587..11608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11623..11628,11629..11646,11647..11653)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11661..11677
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     11666..11688
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11685..11691,1..10)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11685..11691,1..10)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
       61 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
      121 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
      181 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
      241 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
      301 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      361 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      421 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
      481 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
      541 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
      601 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
      661 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
      721 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
      781 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
      841 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
      901 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
      961 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga
     1021 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1081 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1141 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1201 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1261 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1321 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1381 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     1441 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     1501 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     1561 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     1621 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     1681 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     1741 tcaccattat cgtttcagaa tcttgagaca aatggcagta ttcatccaca attttaaaag
     1801 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
     1861 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta
     1921 cagggacagc agagatccac tttggcgccg gctcgagggg gcccgggtgc aaagatggat
     1981 aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct tgaaaggagt
     2041 gggaattggc tccgtaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
     2101 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg
     2161 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2221 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2281 ttcgcaacgg gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca
     2341 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     2401 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     2461 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     2521 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     2581 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     2641 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     2701 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     2761 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     2821 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     2881 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     2941 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     3001 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     3061 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     3121 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     3181 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     3241 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     3301 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     3361 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     3421 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     3481 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     3541 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     3601 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     3661 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     3721 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     3781 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     3841 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     3901 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     3961 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     4021 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     4081 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     4141 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     4201 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     4261 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     4321 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     4381 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     4441 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     4501 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     4561 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     4621 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     4681 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     4741 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     4801 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     4861 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     4921 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     4981 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     5041 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     5101 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     5161 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     5221 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     5281 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     5341 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     5401 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     5461 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     5521 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     5581 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     5641 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     5701 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     5761 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     5821 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     5881 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     5941 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     6001 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     6061 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     6121 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     6181 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     6241 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     6301 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     6361 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     6421 ctcagctggg aggcgacaag cgacctgccg ccacaaagaa ggctggacag gctaagaaga
     6481 agaaagatta caaagacgat gacgataagg gatccggcgc aacaaacttc tctctgctga
     6541 aacaagccgg agatgtcgaa gagaatcctg gaccgatgac cgagtacaag cccacggtgc
     6601 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     6661 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     6721 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     6781 acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     6841 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     6901 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     6961 gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     7021 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     7081 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     7141 gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc
     7201 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     7261 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     7321 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     7381 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     7441 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     7501 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     7561 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     7621 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     7681 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     7741 ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac
     7801 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     7861 aattcactcc caacgaagac aagatttcac cattatcgtt tcagacccac ctcccaaccc
     7921 cgaggggacc cagagagggc ctatttccca tgattccttc atatttgcat atacgataca
     7981 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     8041 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     8101 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     8161 tcttgtggaa aggacgaaac accggccttc ctgaatttga agaggtttta gagctagaaa
     8221 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     8281 ttttttaagc ttggcgtaac tagatcttga gacactgctt tttgcttgta ctgggtctct
     8341 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     8401 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     8461 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta
     8521 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     8581 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     8641 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     8701 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     8761 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     8821 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     8881 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     8941 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     9001 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     9061 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     9121 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     9181 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     9241 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     9301 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     9361 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     9421 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     9481 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     9541 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     9601 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     9661 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     9721 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     9781 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     9841 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     9901 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     9961 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
    10021 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
    10081 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
    10141 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
    10201 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
    10261 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
    10321 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
    10381 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
    10441 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
    10501 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
    10561 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
    10621 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
    10681 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
    10741 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
    10801 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
    10861 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
    10921 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
    10981 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
    11041 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
    11101 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
    11161 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
    11221 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
    11281 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
    11341 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
    11401 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
    11461 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
    11521 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
    11581 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
    11641 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa a
//
