LOCUS       sequence-362777-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets VEGFB 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_VEGFB
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..36
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        18..36
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        64..290
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             291..471
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    518..643
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1136..1369
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1554..1598
                     /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    1779..1896
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2085..2296
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2315..2324
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2321..6424
                     /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             2321..6424
                     /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             6425..6472
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6473..6496
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6506..6562
                     /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             6563..7162
                     /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(6563..6582)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7059..7079
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7178..7766
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7231..7251)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7649..7660)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7922..8170
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7922..7942
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8093..8112
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8192..8267
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8320..8500
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8578..8699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8615..8634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8669..8688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8739..8874
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8894..8913)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8895..8913)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8923..8940)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8923..8939)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8932..8954)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9081..9536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9168..9187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9378..9399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9562..9666
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9667..9735,9736..10527)
                     /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(9885..9904)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10698..11286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11187..11206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11440..11457
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11574..11595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11610..11615,11616..11633,11634..11640)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11648..11664
                     /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     11653..11675
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11672..11688
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11672..11688
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
       61 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      121 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
      181 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
      241 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
      301 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
      361 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      421 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
      481 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
      541 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      601 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      661 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      721 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      781 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
      841 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
      901 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
      961 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1021 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1081 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1141 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1201 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1261 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1321 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1381 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1441 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1501 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1561 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1621 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1681 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     1741 ttcagaatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     1801 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     1861 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     1921 gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa gttttaaaca
     1981 gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc
     2041 gtaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     2101 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgatccggtg cctagagaag
     2161 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2221 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2281 tgccgccaga acacaggacc ggttctagag cgctgccacc atggacaaga agtacagcat
     2341 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2401 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2461 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2521 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2581 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2641 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2701 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2761 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     2821 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2881 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2941 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3001 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     3061 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3121 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3181 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     3241 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3301 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3361 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3421 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3481 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3541 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3601 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3661 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3721 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3781 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3841 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     3901 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3961 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4021 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4081 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4141 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4201 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     4261 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4321 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4381 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4441 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4501 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4561 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4621 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4681 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4741 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4801 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     4861 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     4921 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4981 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5041 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5101 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5161 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     5221 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     5281 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5341 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5401 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5461 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5521 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5581 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5641 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5701 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5761 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     5821 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5881 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5941 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6001 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6061 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6121 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6181 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     6241 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6301 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6361 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6421 cgacaagcga cctgccgcca caaagaaggc tggacaggct aagaagaaga aagattacaa
     6481 agacgatgac gataagggat ccggcgcaac aaacttctct ctgctgaaac aagccggaga
     6541 tgtcgaagag aatcctggac cgatgaccga gtacaagccc acggtgcgcc tcgccacccg
     6601 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     6661 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     6721 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgccgt
     6781 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     6841 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     6901 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     6961 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     7021 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     7081 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     7141 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     7201 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     7261 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     7321 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     7381 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     7441 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     7501 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     7561 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     7621 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     7681 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     7741 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     7801 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     7861 cgaagacaag atttcaccat tatcgtttca gacccacctc ccaaccccga ggggacccag
     7921 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     7981 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     8041 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     8101 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     8161 acgaaacacc ggcagcaggc ggcggagcag agttttagag ctagaaatag caagttaaaa
     8221 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttaagcttg
     8281 gcgtaactag atcttgagac actgcttttt gcttgtactg ggtctctctg gttagaccag
     8341 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     8401 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     8461 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     8521 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     8581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     8641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     8701 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     8761 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     8821 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     8881 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     8941 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     9001 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     9061 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     9121 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     9181 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     9241 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     9301 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     9361 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     9421 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     9481 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     9541 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     9601 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     9661 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     9721 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     9781 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     9841 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     9901 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     9961 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
    10021 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
    10081 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
    10141 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
    10201 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
    10261 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
    10321 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
    10381 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
    10441 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
    10501 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
    10561 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
    10621 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
    10681 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
    10741 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
    10801 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
    10861 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
    10921 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
    10981 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
    11041 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
    11101 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
    11161 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
    11221 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
    11281 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
    11341 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
    11401 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
    11461 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
    11521 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
    11581 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
    11641 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca t
//
