LOCUS       sequence-362636-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets HDAC3 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_HDAC3
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(15..20,21..38,39..45)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    53..69
                     /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     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     112..132
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        114..132
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        160..386
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             387..567
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    614..739
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1232..1465
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1650..1694
                     /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    1875..1992
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2181..2392
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2411..2420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2417..6520
                     /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             2417..6520
                     /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             6521..6568
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6569..6592
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6602..6658
                     /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             6659..7258
                     /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(6659..6678)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7155..7175
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7274..7862
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7327..7347)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7745..7756)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8018..8266
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8018..8038
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8189..8208
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8288..8363
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8416..8596
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8674..8795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8711..8730)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8765..8784
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8835..8970
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8990..9009)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8991..9009)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9019..9036)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9019..9035)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9028..9050)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9177..9632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9264..9283)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9474..9495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9658..9762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9763..9831,9832..10623)
                     /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(9981..10000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10794..11382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11283..11302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11536..11553
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11670..11691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      121 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      181 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      241 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      301 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      361 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      421 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      481 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      541 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      601 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      661 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      721 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
      781 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
      841 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
      901 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
      961 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1021 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1081 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1141 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1201 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1261 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1321 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1381 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1441 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1501 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1561 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1621 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1681 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1741 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     1801 tgaatagagt taggcaggga tattcaccat tatcgtttca gaatcttgag acaaatggca
     1861 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     1921 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     1981 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     2041 ggggcccggg tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg
     2101 accttctagg tcttgaaagg agtgggaatt ggctccgtag gtcttgaaag gagtgggaat
     2161 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2221 gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2281 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2341 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggaccggtt
     2401 ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaactc
     2461 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     2521 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     2581 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     2641 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     2701 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     2761 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     2821 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     2881 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     2941 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     3001 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     3061 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     3121 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     3181 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     3241 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     3301 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     3361 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     3421 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     3481 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     3541 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     3601 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     3661 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     3721 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     3781 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     3841 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     3901 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     3961 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     4021 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     4081 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     4141 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     4201 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     4261 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     4321 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     4381 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     4441 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     4501 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     4561 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     4621 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     4681 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     4741 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     4801 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     4861 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     4921 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     4981 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     5041 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     5101 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     5161 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     5221 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     5281 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     5341 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     5401 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     5461 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     5521 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     5581 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     5641 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     5701 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     5761 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     5821 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     5881 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     5941 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     6001 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     6061 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     6121 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     6181 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     6241 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     6301 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     6361 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     6421 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     6481 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg ccgccacaaa
     6541 gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata agggatccgg
     6601 cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat
     6661 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     6721 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     6781 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     6841 cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag
     6901 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     6961 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     7021 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag
     7081 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     7141 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     7201 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     7261 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     7321 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     7381 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     7441 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     7501 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     7561 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     7621 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     7681 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     7741 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     7801 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     7861 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     7921 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagattt caccattatc
     7981 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     8041 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     8101 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     8161 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     8221 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgggg gtcgtagaaa
     8281 taggccagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     8341 aaaaagtggc accgagtcgg tgctttttta agcttggcgt aactagatct tgagacactg
     8401 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     8461 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     8521 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     8581 tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa
     8641 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt
     8701 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     8761 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc
     8821 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     8881 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     8941 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga
     9001 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     9061 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     9121 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     9181 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     9241 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     9301 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     9361 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     9421 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     9481 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     9541 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     9601 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     9661 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     9721 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     9781 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     9841 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     9901 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     9961 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
    10021 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
    10081 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
    10141 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
    10201 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
    10261 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
    10321 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
    10381 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
    10441 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
    10501 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
    10561 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
    10621 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
    10681 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
    10741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
    10801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
    10861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
    10921 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
    10981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
    11041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
    11101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
    11161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
    11221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
    11281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    11341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    11401 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
    11461 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
    11521 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
    11581 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
    11641 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca t
//
