LOCUS       sequence-362606-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets AR 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_AR
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..26
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10..26
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     45..65
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        47..65
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        93..319
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             320..500
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    547..672
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1165..1398
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1583..1627
                     /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    1808..1925
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2114..2325
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2344..2353
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2350..6453
                     /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             2350..6453
                     /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             6454..6501
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6502..6525
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6535..6591
                     /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             6592..7191
                     /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(6592..6611)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7088..7108
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7207..7795
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7260..7280)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7678..7689)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7951..8199
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7951..7971
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8122..8141
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8221..8296
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8349..8529
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8607..8728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8644..8663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8698..8717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8768..8903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8923..8942)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8924..8942)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8952..8969)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8952..8968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8961..8983)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9110..9565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9197..9216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9407..9428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9591..9695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9696..9764,9765..10556)
                     /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(9914..9933)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10727..11315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11216..11235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11469..11486
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11603..11624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11639..11644,11645..11662,11663..11669)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11677..11691,1..2)
                     /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(11682..11691,1..13)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
       61 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
      121 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
      181 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
      241 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
      301 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
      361 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      421 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      481 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
      541 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
      601 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
      661 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
      721 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
      781 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
      841 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
      901 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
      961 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     1021 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1081 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1141 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1201 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1261 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1321 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1381 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1441 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1501 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     1561 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     1621 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     1681 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     1741 agttaggcag ggatattcac cattatcgtt tcagaatctt gagacaaatg gcagtattca
     1801 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     1861 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     1921 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc
     1981 gggtgcaaag atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct
     2041 aggtcttgaa aggagtggga attggctccg taggtcttga aaggagtggg aattggctcc
     2101 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2161 gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2221 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2281 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc
     2341 gctgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     2401 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     2461 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2521 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     2581 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     2641 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     2701 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     2761 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     2821 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     2881 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     2941 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     3001 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     3061 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     3121 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     3181 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     3241 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     3301 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     3361 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     3421 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3481 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3541 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     3601 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     3661 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     3721 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     3781 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     3841 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     3901 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     3961 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     4021 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     4081 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     4141 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     4201 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     4261 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     4321 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     4381 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     4441 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     4501 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     4561 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     4621 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     4681 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     4741 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     4801 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     4861 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     4921 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     4981 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     5041 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     5101 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     5161 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     5221 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     5281 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     5341 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     5401 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     5461 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     5521 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     5581 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     5641 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     5701 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     5761 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     5821 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     5881 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     5941 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     6001 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     6061 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     6121 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     6181 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     6241 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     6301 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     6361 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     6421 acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct
     6481 ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca
     6541 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gatgaccgag
     6601 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6661 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6721 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     6781 gtgtgggtcg cggacgacgg cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa
     6841 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     6901 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6961 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     7021 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     7081 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     7141 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     7201 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     7261 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     7321 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     7381 gagtygtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     7441 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     7501 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     7561 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     7621 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     7681 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     7741 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     7801 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     7861 ggggactgga agggctaatt cactcccaac gaagacaaga tttcaccatt atcgtttcag
     7921 acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat tccttcatat
     7981 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     8041 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     8101 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     8161 ttcttggctt tatatatctt gtggaaagga cgaaacaccg agggtaccac acatcaggtg
     8221 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     8281 ggcaccgagt cggtgctttt ttaagcttgg cgtaactaga tcttgagaca ctgctttttg
     8341 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     8401 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     8461 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     8521 tctctagcag tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa
     8581 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat
     8641 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     8701 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac
     8761 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     8821 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     8881 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat
     8941 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     9001 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     9061 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     9121 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     9181 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     9241 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     9301 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     9361 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     9421 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     9481 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     9541 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     9601 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     9661 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     9721 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     9781 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     9841 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     9901 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     9961 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
    10021 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
    10081 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
    10141 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
    10201 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
    10261 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
    10321 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
    10381 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
    10441 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
    10501 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
    10561 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
    10621 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
    10681 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
    10741 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
    10801 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
    10861 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
    10921 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
    10981 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
    11041 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
    11101 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
    11161 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
    11221 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
    11281 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
    11341 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
    11401 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
    11461 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
    11521 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
    11581 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
    11641 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa c
//
