LOCUS       sequence-355352-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets DNMT3B 
            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_DNMT3B
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     36..56
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        38..56
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        84..310
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             311..491
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    538..663
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1156..1389
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1574..1618
                     /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    1799..1916
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2105..2316
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2341..6444
                     /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             2341..6444
                     /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             6445..6492
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6493..6516
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6526..6582
                     /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             6583..7182
                     /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(6583..6602)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7079..7099
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7198..7786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7251..7271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7669..7680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7942..8190
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7942..7962
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8113..8132
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8212..8287
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8340..8520
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8598..8719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8635..8654)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8689..8708
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8759..8894
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8914..8933)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8915..8933)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8943..8960)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8943..8959)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8952..8974)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9101..9556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9188..9207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9398..9419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9582..9686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9687..9755,9756..10547)
                     /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(9905..9924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10718..11306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11207..11226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11460..11477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11594..11615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11630..11635,11636..11653,11654..11660)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11668..11684
                     /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     join(11673..11691,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
       61 aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat
      121 ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt
      181 ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg
      241 gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg
      301 atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      361 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      421 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      481 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      541 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      601 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      661 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      721 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
      781 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
      841 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
      901 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
      961 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     1021 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     1081 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     1141 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     1201 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     1261 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     1321 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     1381 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     1441 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     1501 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     1561 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     1621 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     1681 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     1741 gggatattca ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt
     1801 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     1861 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     1921 tttattacag ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa
     1981 gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
     2041 aaggagtggg aattggctcc gtaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     2101 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     2161 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     2221 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     2281 gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc
     2341 atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     2401 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     2461 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     2521 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     2581 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     2641 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     2701 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     2761 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     2821 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     2881 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     2941 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     3001 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     3061 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     3121 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     3181 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     3241 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     3301 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     3361 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     3421 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     3481 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     3541 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     3601 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     3661 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     3721 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     3781 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     3841 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     3901 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     3961 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     4021 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     4081 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     4141 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     4201 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     4261 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     4321 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     4381 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     4441 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     4501 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     4561 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     4621 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     4681 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     4741 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     4801 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     4861 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     4921 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     4981 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     5041 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     5101 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     5161 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     5221 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     5281 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     5341 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     5401 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     5461 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     5521 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     5581 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     5641 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     5701 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     5761 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     5821 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     5881 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     5941 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     6001 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     6061 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     6121 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     6181 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     6241 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     6301 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     6361 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     6421 gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct
     6481 aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac aaacttctct
     6541 ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatgaccga gtacaagccc
     6601 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     6661 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     6721 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     6781 gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     6841 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     6901 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     6961 accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     7021 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     7081 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     7141 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat
     7201 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     7261 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     7321 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     7381 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     7441 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     7501 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     7561 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     7621 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     7681 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     7741 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     7801 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     7861 aagggctaat tcactcccaa cgaagacaag atttcaccat tatcgtttca gacccacctc
     7921 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     7981 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     8041 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     8101 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     8161 ttatatatct tgtggaaagg acgaaacacc ggatcgagtc ttccctcccg cgttttagag
     8221 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     8281 tcggtgcttt tttaagcttg gcgtaactag atcttgagac actgcttttt gcttgtactg
     8341 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     8401 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     8461 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     8521 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     8581 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     8641 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     8701 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     8761 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     8821 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     8881 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     8941 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     9001 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     9061 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     9121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     9181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     9241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     9301 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     9361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     9421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     9481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     9541 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     9601 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     9661 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     9721 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     9781 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     9841 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     9901 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     9961 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
    10021 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
    10081 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
    10141 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
    10201 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
    10261 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
    10321 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
    10381 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
    10441 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
    10501 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
    10561 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
    10621 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
    10681 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
    10741 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
    10801 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    10861 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    10921 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    10981 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    11041 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    11101 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    11161 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    11221 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    11281 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    11341 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
    11401 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
    11461 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
    11521 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
    11581 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
    11641 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac a
//
