LOCUS       sequence-490173-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets BRD4 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_BRD4
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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    2773..2890
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3079..3290
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3309..3318
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3315..7418
                     /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             3315..7418
                     /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             7419..7466
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7467..7490
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7500..7556
                     /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             7557..8156
                     /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(7557..7576)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8053..8073
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8172..8760
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8225..8245)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8643..8654)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        8916..9164
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8916..8936
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9087..9106
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9186..9261
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             9314..9494
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    9572..9693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9609..9628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9663..9682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9733..9868
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9888..9907)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9889..9907)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9917..9934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9917..9933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9926..9948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10075..10530
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10162..10181)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10372..10393
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10556..10660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10661..10729,10730..11521)
                     /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(10879..10898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga atcttgagac aaatggcagt
     2761 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2821 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2881 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg
     2941 ggcccgggtg caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac
     3001 cttctaggtc ttgaaaggag tgggaattgg ctccgtaggt cttgaaagga gtgggaattg
     3061 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     3121 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     3181 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     3241 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggttct
     3301 agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg
     3361 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     3421 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     3481 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     3541 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     3601 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     3661 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     3721 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     3781 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     3841 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     3901 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     3961 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     4021 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     4081 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     4141 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     4201 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     4261 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     4321 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     4381 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     4441 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     4501 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     4561 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     4621 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     4681 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     4741 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     4801 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     4861 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     4921 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     4981 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     5041 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     5101 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     5161 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     5221 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     5281 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     5341 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     5401 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     5461 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     5521 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     5581 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     5641 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     5701 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     5761 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     5821 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     5881 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     5941 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     6001 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     6061 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     6121 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     6181 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     6241 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     6301 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     6361 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     6421 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     6481 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     6541 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     6601 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     6661 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     6721 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     6781 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     6841 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     6901 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     6961 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     7021 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     7081 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     7141 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     7201 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     7261 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     7321 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     7381 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     7441 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggcg
     7501 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatga
     7561 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     7621 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     7681 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     7741 gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt ctggaccacg ccggagagcg
     7801 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     7861 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     7921 cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg
     7981 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     8041 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     8101 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc
     8161 gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     8221 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     8281 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     8341 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     8401 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     8461 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     8521 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     8581 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     8641 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     8701 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     8761 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     8821 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatttca ccattatcgt
     8881 ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt
     8941 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     9001 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     9061 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     9121 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgccaga cccctgtcat
     9181 gacaggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     9241 aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatcttg agacactgct
     9301 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     9361 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     9421 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     9481 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     9541 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     9601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     9661 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     9721 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     9781 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     9841 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     9901 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     9961 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
    10021 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
    10081 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
    10141 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
    10201 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
    10261 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
    10321 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
    10381 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
    10441 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
    10501 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
    10561 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
    10621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
    10681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
    10741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
    10801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
    10861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
    10921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
    10981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
    11041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
    11101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
    11161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
    11221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
    11281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
    11341 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
    11401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
    11461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
    11521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
    11581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
    11641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
