LOCUS       sequence-362620-       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..23
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     20..36
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     20..36
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     55..75
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        57..75
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        103..329
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             330..510
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    557..682
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1175..1408
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1593..1637
                     /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    1818..1935
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2124..2335
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2354..2363
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2360..6463
                     /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             2360..6463
                     /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             6464..6511
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6512..6535
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6545..6601
                     /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             6602..7201
                     /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(6602..6621)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7098..7118
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7217..7805
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7270..7290)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7688..7699)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7961..8209
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7961..7981
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8132..8151
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8231..8306
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8359..8539
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8617..8738
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8654..8673)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8708..8727
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8778..8913
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8933..8952)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8934..8952)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8962..8979)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8962..8978)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8971..8993)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9120..9575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9207..9226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9417..9438
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9601..9705
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9706..9774,9775..10566)
                     /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(9924..9943)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10737..11325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11226..11245
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11479..11496
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11613..11634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11649..11654,11655..11672,11673..11679)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11687..11691,1..12)
                     /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 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
       61 aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac
      121 tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa
      181 agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc
      241 aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg
      301 tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct
      361 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
      421 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
      481 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
      541 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
      601 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
      661 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
      721 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
      781 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
      841 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
      901 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
      961 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1021 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1081 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1141 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1201 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1261 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1321 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1381 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1441 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     1501 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     1561 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     1621 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     1681 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     1741 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagaatctt gagacaaatg
     1801 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     1861 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     1921 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc
     1981 gagggggccc gggtgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa
     2041 tggaccttct aggtcttgaa aggagtggga attggctccg taggtcttga aaggagtggg
     2101 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     2161 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
     2221 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     2281 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg
     2341 gttctagagc gctgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa
     2401 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     2461 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     2521 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
     2581 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     2641 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     2701 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     2761 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     2821 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     2881 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     2941 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     3001 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     3061 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     3121 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     3181 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     3241 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     3301 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     3361 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     3421 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     3481 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     3541 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     3601 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     3661 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     3721 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     3781 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     3841 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     3901 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     3961 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     4021 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     4081 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     4141 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     4201 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     4261 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     4321 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     4381 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     4441 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     4501 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     4561 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     4621 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     4681 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     4741 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     4801 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     4861 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     4921 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     4981 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     5041 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     5101 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     5161 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     5221 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     5281 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     5341 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     5401 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     5461 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     5521 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     5581 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     5641 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     5701 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     5761 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     5821 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     5881 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     5941 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     6001 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     6061 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     6121 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     6181 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     6241 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     6301 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     6361 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     6421 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac
     6481 aaagaaggct ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc
     6541 cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc
     6601 gatgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     6661 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     6721 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     6781 catcggcaag gtgtgggtcg cggacgacgg cgccgccgtg gcggtctgga ccacgccgga
     6841 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     6901 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     6961 gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg
     7021 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     7081 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     7141 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     7201 aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     7261 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     7321 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     7381 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     7441 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     7501 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     7561 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     7621 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     7681 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     7741 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     7801 cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     7861 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tttcaccatt
     7921 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     7981 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     8041 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     8101 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     8161 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gatcgagtct
     8221 tccctcccgc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     8281 ttgaaaaagt ggcaccgagt cggtgctttt ttaagcttgg cgtaactaga tcttgagaca
     8341 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     8401 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     8461 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     8521 gtgtggaaaa tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta
     8581 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     8641 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     8701 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     8761 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     8821 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     8881 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     8941 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     9001 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     9061 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     9121 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     9181 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     9241 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     9301 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     9361 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     9421 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     9481 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     9541 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     9601 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     9661 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     9721 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     9781 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     9841 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     9901 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     9961 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
    10021 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
    10081 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
    10141 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
    10201 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
    10261 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
    10321 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
    10381 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
    10441 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
    10501 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
    10561 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
    10621 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
    10681 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
    10741 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
    10801 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
    10861 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
    10921 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
    10981 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
    11041 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
    11101 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
    11161 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
    11221 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
    11281 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
    11341 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
    11401 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
    11461 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
    11521 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
    11581 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
    11641 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt g
//
