LOCUS       sequence-362609-       11691 bp    DNA     circular SYN 15-APR-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets BTK 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 Apr 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_BTK
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..27
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11..27
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     46..66
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        48..66
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        94..320
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             321..501
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    548..673
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1166..1399
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1584..1628
                     /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    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2115..2326
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2345..2354
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2351..6454
                     /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             2351..6454
                     /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             6455..6502
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6503..6526
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6536..6592
                     /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             6593..7192
                     /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(6593..6612)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7089..7109
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7208..7796
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7261..7281)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7679..7690)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7952..8200
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7952..7972
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8123..8142
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8222..8297
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8350..8530
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8608..8729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8645..8664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8699..8718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8769..8904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8924..8943)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8925..8943)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8953..8970)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8953..8969)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8962..8984)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9111..9566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9198..9217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9408..9429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9592..9696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9697..9765,9766..10557)
                     /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(9915..9934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10728..11316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11217..11236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11470..11487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11604..11625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11640..11645,11646..11663,11664..11670)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11678..11691,1..3)
                     /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(11683..11691,1..14)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
       61 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
      121 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
      181 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
      241 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
      301 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
      361 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      421 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      481 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      541 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      601 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      661 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      721 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      781 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      841 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
      901 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
      961 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     1021 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     1081 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     1141 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     1201 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     1261 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     1321 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     1381 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     1441 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     1501 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     1561 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     1621 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     1681 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     1741 gagttaggca gggatattca ccattatcgt ttcagaatct tgagacaaat ggcagtattc
     1801 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct cgagggggcc
     1981 cgggtgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta atggaccttc
     2041 taggtcttga aaggagtggg aattggctcc gtaggtcttg aaaggagtgg gaattggctc
     2101 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2161 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2221 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2281 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     2341 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     2401 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     2461 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     2521 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     2581 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     2641 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     2701 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     2761 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     2821 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     2881 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     2941 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     3001 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     3061 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     3121 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     3181 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     3241 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     3301 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     3361 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     3421 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     3481 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     3541 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     3601 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     3661 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     3721 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     3781 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     3841 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     3901 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     3961 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     4021 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     4081 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     4141 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     4201 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     4261 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     4321 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     4381 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     4441 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     4501 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     4561 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     4621 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     4681 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     4741 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     4801 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     4861 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     4921 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     4981 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     5041 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     5101 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     5161 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     5221 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     5281 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     5341 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     5401 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     5461 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     5521 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     5581 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     5641 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
     5701 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
     5761 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     5821 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     5881 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     5941 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     6001 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     6061 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     6121 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     6181 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     6241 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     6301 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
     6361 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     6421 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
     6481 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac
     6541 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgatgaccga
     6601 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     6661 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     6721 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     6781 ggtgtgggtc gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga
     6841 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     6901 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     6961 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     7021 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     7081 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     7141 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     7201 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     7261 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     7321 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     7381 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     7441 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     7501 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     7561 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     7621 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     7681 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     7741 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     7801 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     7861 gggggactgg aagggctaat tcactcccaa cgaagacaag atttcaccat tatcgtttca
     7921 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     7981 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     8041 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     8101 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     8161 tttcttggct ttatatatct tgtggaaagg acgaaacacc gctgtgtttg ctaaatccac
     8221 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     8281 tggcaccgag tcggtgcttt tttaagcttg gcgtaactag atcttgagac actgcttttt
     8341 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     8401 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     8461 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     8521 atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca
     8581 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     8641 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     8701 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     8761 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     8821 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     8881 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     8941 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     9001 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     9061 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     9121 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     9181 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     9241 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     9301 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     9361 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     9421 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     9481 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     9541 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     9601 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     9661 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     9721 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     9781 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     9841 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     9901 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     9961 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
    10021 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
    10081 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
    10141 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
    10201 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
    10261 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
    10321 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
    10381 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
    10441 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
    10501 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
    10561 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
    10621 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
    10681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
    10741 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
    10801 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
    10861 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
    10921 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
    10981 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
    11041 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
    11101 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
    11161 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
    11221 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
    11281 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
    11341 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
    11401 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
    11461 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
    11521 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
    11581 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
    11641 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata a
//
