LOCUS       sequence-362782-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets TOP1 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_TOP1
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..24
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     21..37
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     21..37
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     56..76
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        58..76
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        104..330
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             331..511
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    558..683
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1176..1409
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1594..1638
                     /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    1819..1936
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2125..2336
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2355..2364
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2361..6464
                     /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             2361..6464
                     /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             6465..6512
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6513..6536
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6546..6602
                     /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             6603..7202
                     /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(6603..6622)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7099..7119
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7218..7806
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7271..7291)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7689..7700)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7962..8210
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7962..7982
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8133..8152
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8232..8307
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8360..8540
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8618..8739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8655..8674)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8709..8728
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8779..8914
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8934..8953)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8935..8953)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8963..8980)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8963..8979)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8972..8994)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9121..9576
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9208..9227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9418..9439
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9602..9706
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9707..9775,9776..10567)
                     /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(9925..9944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10738..11326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11227..11246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11480..11497
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11614..11635
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11650..11655,11656..11673,11674..11680)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11688..11691,1..13)
                     /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)."
ORIGIN
        1 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
       61 taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata
      121 ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa
      181 aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg
      241 caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt
      301 gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc
      361 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
      421 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
      481 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga
      541 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa
      601 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg
      661 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa
      721 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa
      781 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta
      841 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat
      901 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca
      961 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag
     1021 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1081 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1141 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     1201 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     1261 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     1321 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     1381 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     1441 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     1501 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     1561 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     1621 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     1681 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     1741 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaatct tgagacaaat
     1801 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     1861 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     1921 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct
     1981 cgagggggcc cgggtgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta
     2041 atggaccttc taggtcttga aaggagtggg aattggctcc gtaggtcttg aaaggagtgg
     2101 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2161 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
     2221 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2281 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
     2341 ggttctagag cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca
     2401 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     2461 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     2521 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     2581 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     2641 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     2701 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     2761 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     2821 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     2881 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     2941 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     3001 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     3061 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     3121 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     3181 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     3241 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     3301 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     3361 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     3421 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     3481 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     3541 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     3601 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     3661 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     3721 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     3781 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     3841 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     3901 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     3961 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     4021 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     4081 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     4141 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     4201 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     4261 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     4321 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     4381 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     4441 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     4501 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     4561 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     4621 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     4681 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     4741 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     4801 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     4861 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     4921 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     4981 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     5041 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     5101 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     5161 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     5221 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     5281 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     5341 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     5401 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     5461 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     5521 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     5581 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     5641 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     5701 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     5761 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     5821 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     5881 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     5941 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     6001 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     6061 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     6121 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     6181 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     6241 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     6301 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     6361 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     6421 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca
     6481 caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat
     6541 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     6601 cgatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     6661 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     6721 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     6781 acatcggcaa ggtgtgggtc gcggacgacg gcgccgccgt ggcggtctgg accacgccgg
     6841 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     6901 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     6961 agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg
     7021 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     7081 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     7141 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     7201 gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     7261 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     7321 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     7381 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     7441 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     7501 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     7561 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     7621 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     7681 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     7741 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     7801 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     7861 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atttcaccat
     7921 tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga
     7981 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
     8041 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     8101 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     8161 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gttatggata
     8221 accacaaaga ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     8281 cttgaaaaag tggcaccgag tcggtgcttt tttaagcttg gcgtaactag atcttgagac
     8341 actgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     8401 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     8461 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     8521 agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt
     8581 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     8641 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     8701 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     8761 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     8821 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     8881 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     8941 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     9001 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     9061 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     9121 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     9181 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     9241 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     9301 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     9361 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     9421 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     9481 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     9541 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     9601 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     9661 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     9721 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     9781 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     9841 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     9901 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     9961 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
    10021 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
    10081 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
    10141 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
    10201 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
    10261 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
    10321 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
    10381 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
    10441 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
    10501 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
    10561 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
    10621 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
    10681 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
    10741 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
    10801 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
    10861 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
    10921 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
    10981 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
    11041 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
    11101 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
    11161 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
    11221 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
    11281 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
    11341 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
    11401 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
    11461 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
    11521 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
    11581 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
    11641 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg t
//
