LOCUS       sequence-362642-       11689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets KDR gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11689)
  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 11689)
  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_KDR
FEATURES             Location/Qualifiers
     source          1..11689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(255..299)
                     /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    484..717
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1210..1335
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1382..1562
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1563..1789)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(1817..1837)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1817..1835)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1856..1872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1856..1872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1869..1891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1880..1896
                     /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)."
     promoter        complement(join(1904..1910,1911..1928,1929..1934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1949..1970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2087..2104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2258..2846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2338..2357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3017..3808,3809..3877))
                     /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     3640..3659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3878..3982)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4008..4463)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4145..4166)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4357..4376
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4590..4612
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4604..4621
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4605..4621
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4631..4650
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4631..4649
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      4670..4805
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4845..4966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4856..4875)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4910..4929
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             5044..5224
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_RNA        complement(5277..5352)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5380..5620)
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     complement(5430..5449)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5600..5620)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    5776..6364
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5882..5893
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6291..6311
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6380..6979)
                     /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(6463..6483)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6960..6979
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(6980..7036)
                     /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             complement(7046..7069)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(7070..7117)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(7118..11221)
                     /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             complement(7118..11221)
                     /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"
     regulatory      11218..11227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(11246..11457)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    join(11646..11689,1..74)
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 gtatgtctgt tgctattatg tctactattc tttcccctgc actgtacccc ccaatccccc
       61 cttttctttt aaaattgtgg atgaatactg ccatttgtct caagattctg aaacgataat
      121 ggtgaatatc cctgcctaac tctattcact atagaaagta cagcaaaaac tattcttaaa
      181 cctaccaagc ctcctactat cattatgaat aattttatat accacagcca atttgttatg
      241 ttaaaccaat tccacaaact tgcccattta tctaattcca ataattcttg ttcattcttt
      301 tcttgctggt tttgcgattc ttcaattaag gagtgtatta agcttgtgta attgttaatt
      361 tctctgtccc actccatcca ggtcgtgtga ttccaaatct gttccagaga tttattactc
      421 caactagcat tccaaggcac agcagtggtg caaatgagtt ttccagagca accccaaatc
      481 cccaggagct gttgatcctt taggtatctt tccacagcca ggattcttgc ctggagctgc
      541 ttgatgcccc agactgtgag ttgcaacaga tgctgttgcg cctcaatagc cctcagcaaa
      601 ttgttctgct gctgcactat accagacaat aattgtctgg cctgtaccgt cagcgtcatt
      661 gacgctgcgc ccatagtgct tcctgctgct cccaagaacc caaggaacaa agctcctatt
      721 cccactgctc ttttttctct ctgcaccact cttctctttg ccttggtggg tgctactcct
      781 aatggttcaa tttttactac tttatattta tataattcac ttctccaatt gtccctcata
      841 tctcctcctc caggtctgaa gatcagcggc cgcttgctgt gcggtggtct tacttttgtt
      901 ttgctcttcc tctatcttgt ctaaagcttc cttggtgtct tttatctcta tcctttgatg
      961 cacacaatag agggttgcta ctgtattata taatgatcta agttcttctg atcctgtctg
     1021 aagggatggt tgtagctgtc ccagtatttg tctacagcct tctgatgttt ctaacaggcc
     1081 aggattaact gcgaatcgtt ctagctccct gcttgcccat actatatgtt ttaatttata
     1141 ttttttcttt ccccctggcc ttaaccgaat tttttcccat cgcgatctaa ttctcccccg
     1201 cttaatactg acgctctcgc acccatctct ctccttctag cctccgctag tcaaaatttt
     1261 tggcgtactc accagtcgcc gcccctcgcc tcttgccgtg cgcgcttcag caagccgagt
     1321 cctgcgtcga gagagctcct ctggtttccc tttcgctttc aagtccctgt tcgggcgcca
     1381 ctgctagaga ttttccacac tgactaaaag ggtctgaggg atctctagtt accagagtca
     1441 cacaacagac gggcacacac tacttgaagc actcaaggca agctttattg aggcttaagc
     1501 agtgggttcc ctagttagcc agagagctcc caggctcaga tctggtctaa ccagagagac
     1561 ccgtttatgt atcgagctag gcacttaaat acaatatctc tgcaatgcgg caattcagtg
     1621 gttcgtccaa tccatgtcag acccgtctgt tgccttccta ataaggcacg atcgtaccac
     1681 cttacttcca ccaatcggca tgcacggtgc tttttctctc cttgtaaggc atgttgctaa
     1741 ctcatcgtta ccatgttgca agactacaag agtattgcat aagactacat taagcttgca
     1801 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     1861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3121 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     4021 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4081 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4141 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4201 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4261 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4321 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4381 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4441 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     4501 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     4561 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4621 tgagcgcgcg taatacgact cactataggg cgaattgggt acgtccctag gcctccaaaa
     4681 aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa
     4741 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     4801 gggatgggcg gagttagggg cgggatagct agagccagac atgataagat acattgatga
     4861 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4921 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttcctc tcactctctg
     4981 atattcattt ctttgcaagt tataaatact gaataataag atgacatgaa ctactatacg
     5041 tactgctaga gattttccac actgactaaa agggtctgag ggatctctag ttaccagagt
     5101 cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat tgaggcttaa
     5161 gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct aaccagagag
     5221 acccagtaca agcaaaaagc agtgtctcaa gatctagtta cgccaagctt aaaaaagcac
     5281 cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc
     5341 tagctctaaa acactggatg ctgcacaggt gtcggtttcg tcctttccac aagatatata
     5401 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
     5461 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
     5521 ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc
     5581 gtatatgcaa atatgaagga atcatgggaa ataggccctc tctgggtccc ctcggggttg
     5641 ggaggtgggt ctgaaacgat aatggtgaaa tcttgtcttc gttgggagtg aattagccct
     5701 tccagtcccc ccttttcttt taaaaagtgg ctaagatcta cagctgcctt gtaagtcatt
     5761 ggtcttaaag tcgacgcggg gaggcggccc aaagggagat ccgactcgtc tgagggcgaa
     5821 ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc gcgggaagga aggtccgctg
     5881 gattgagggc cgaagggacg tagcagaagg acgtcccgcg cagaatccag gtggcaacac
     5941 aggcgagcag ccaaggaaag gacgatgatt tccccgacaa caccacggaa ttgtcagtgc
     6001 ccaacagccg agcccctgtc cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg
     6061 caatagggag ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc
     6121 aaccagtggg ggttgcgtca gcaaacacag tgcacaccac gccacgttgc ctgacaacgg
     6181 gccacaactc ctcataaaga gacagcaacc aggatttata caaggaggag aaaatgaaag
     6241 ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca catagcgtaa
     6301 aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa tccagaggtt
     6361 gattgtcgac ttaacgcgtt caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc
     6421 cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt
     6481 tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc gcctccactc
     6541 cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgag actccgacgg
     6601 tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct tccatctgtt
     6661 gctgcgcggc cagccgggaa ccgctcaact cggccatgcg cgggccgatc tcggcgaaca
     6721 ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc cacggcggcg ccgtcgtccg
     6781 cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg
     6841 tgacccgctc gatgtggcgg tccggatcga cggtgtggcg cgtggcgggg tagtcggcga
     6901 acgcggcggc gagggtgcgt acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg
     6961 tgggcttgta ctcggtcatc ggtccaggat tctcttcgac atctccggct tgtttcagca
     7021 gagagaagtt tgttgcgccg gatcccttat cgtcatcgtc tttgtaatct ttcttcttct
     7081 tagcctgtcc agccttcttt gtggcggcag gtcgcttgtc gcctcccagc tgagacaggt
     7141 cgatccgtgt ctcgtacagg ccggtgatgc tctggtggat cagggtggcg tccagcacct
     7201 ctttggtgct ggtgtacctc ttccggtcga tggtggtgtc aaagtacttg aaggcggcag
     7261 gggctcccag attggtcagg gtaaacaggt ggatgatatt ctcggcctgc tctctgatgg
     7321 gcttatcccg gtgcttgttg taggcggaca gcactttgtc cagattagcg tcggccagga
     7381 tcactctctt ggagaactcg ctgatctgct cgatgatctc gtccaggtag tgcttgtgct
     7441 gttccacaaa cagctgtttc tgctcattat cctcggggga gcccttcagc ttctcatagt
     7501 ggctggccag gtacaggaag ttcacatatt tggagggcag ggccagttcg tttcccttct
     7561 gcagttcgcc ggcagaggcc agcattctct tccggccgtt ttccagctcg aacagggagt
     7621 acttaggcag cttgatgatc aggtcctttt tcacttcttt gtagcccttg gcttccagaa
     7681 agtcgatggg attcttctcg aagctgcttc tttccatgat ggtgatcccc agcagctctt
     7741 tcacactctt cagtttcttg gacttgccct tttccacttt ggccaccacc agcacagaat
     7801 aggccacggt ggggctgtcg aagccgccgt acttcttagg gtcccagtcc ttctttctgg
     7861 cgatcagctt atcgctgttc ctcttgggca ggatagactc tttgctgaag ccgcctgtct
     7921 gcacctcggt ctttttcacg atattcactt ggggcatgct cagcactttc cgcacggtgg
     7981 caaaatcccg gcccttatcc cacacgatct ccccggtttc gccgtttgtc tcgatcagag
     8041 gccgcttccg gatctcgccg ttggccaggg taatctcggt cttgaaaaag ttcatgatgt
     8101 tgctgtagaa gaagtacttg gcggtagcct tgccgatttc ctgctcgctc ttggcgatca
     8161 tcttccgcac gtcgtacacc ttgtagtcgc cgtacacgaa ctcgctttcc agcttagggt
     8221 actttttgat cagggcggtt cccacgacgg cgttcaggta ggcgtcgtgg gcgtggtggt
     8281 agttgttgat ctcgcgcact ttgtaaaact ggaaatcctt ccggaaatcg gacaccagct
     8341 tggacttcag ggtgatcact ttcacttccc ggatcagctt gtcattctcg tcgtacttag
     8401 tgttcatccg ggagtccagg atctgtgcca cgtgctttgt gatctgccgg gtttccacca
     8461 gctgtctctt gatgaagccg gccttatcca gttcgctcag gccgcctctc tcggccttgg
     8521 tcagattgtc gaactttctc tgggtaatca gcttggcgtt cagcagctgc cgccagtagt
     8581 tcttcatctt cttcacgacc tcttcggagg gcacgttgtc gctcttgccc cggttcttgt
     8641 cgcttctggt cagcaccttg ttgtcgatgg agtcgtcctt cagaaagctc tgaggcacga
     8701 tatggtccac atcgtagtcg gacagccggt tgatgtccag ttcctggtcc acgtacatat
     8761 cccgcccatt ctgcaggtag tacaggtaca gcttctcgtt ctgcagctgg gtgttttcca
     8821 cggggtgttc tttcaggatc tggctgccca gctctttgat gccctcttcg atccgcttca
     8881 ttctctcgcg gctgttcttc tgtcccttct gggtggtctg gttctctctg gccatttcga
     8941 tcacgatgtt ctcgggcttg tgccggccca tcactttcac gagctcgtcc accaccttca
     9001 ctgtctgcag gatgcccttc ttaatggcgg ggctgccggc cagattggca atgtgctcgt
     9061 gcaggctatc gccctggccg gacacctggg ctttctggat gtcctcttta aaggtcaggc
     9121 tgtcgtcgtg gatcagctgc atgaagtttc tgttggcgaa gccgtcggac ttcaggaaat
     9181 ccaggattgt cttgccggac tgcttgtccc ggatgccgtt gatcagcttc cggctcagcc
     9241 tgccccagcc ggtgtatctc cgccgcttca gctgcttcat cactttgtcg tcgaacaggt
     9301 gggcataggt tttcagccgt tcctcgatca tctctctgtc ctcaaacagt gtcagggtca
     9361 gcacgatatc ttccagaatg tcctcgtttt cctcattgtc caggaagtcc ttgtccttga
     9421 taattttcag cagatcgtgg tatgtgccca gggaggcgtt gaaccgatct tccacgccgg
     9481 agatttccac ggagtcgaag cactcgattt tcttgaagta gtcctctttc agctgcttca
     9541 cggtcacttt ccggttggtc ttgaacagca ggtccacgat ggcctttttc tgctcgccgc
     9601 tcaggaaggc gggctttctc attccctcgg tcacgtattt cactttggtc agctcgttat
     9661 acacggtgaa gtactcgtac agcaggctgt gcttgggcag caccttctcg ttgggcaggt
     9721 tcttatcgaa gttggtcatc cgctcgatga agctctgggc ggaagcgccc ttgtccacca
     9781 cttcctcgaa gttccagggg gtgatggttt cctcgctctt tctggtcatc caggcgaatc
     9841 tgctgtttcc cctggccaga gggcccacgt agtaggggat gcggaaggtc aggatcttct
     9901 cgatcttttc ccggttgtcc ttcaggaatg ggtaaaaatc ttcctgccgc cgcagaatgg
     9961 cgtgcagctc tcccaggtgg atctggtggg ggatgctgcc gttgtcgaag gtccgctgct
    10021 tccgcagcag gtcctctctg ttcagcttca cgagcagttc ctcggtgccg tccatctttt
    10081 ccaggatggg cttgatgaac ttgtagaact cttcctggct ggctccgccg tcaatgtagc
    10141 cggcgtagcc gttcttgctc tggtcgaaga aaatctcttt gtacttctca ggcagctgct
    10201 gccgcacgag agctttcagc agggtcaggt cctggtggtg ctcgtcgtat ctcttgatca
    10261 tagaggcgct caggggggcc ttggtgatct cggtgttcac tctcaggatg tcgctcagca
    10321 ggatggcgtc ggacaggttc ttggcggcca gaaacaggtc ggcgtactgg tcgccgatct
    10381 gggccagcag gttgtccagg tcgtcgtcgt aggtgtcctt gctcagctgc agtttggcat
    10441 cctcggccag gtcgaagttg ctcttgaagt tgggggtcag gcccaggctc agggcaatca
    10501 ggtttccgaa caggccattc ttcttctcgc cgggcagctg ggcgatcaga ttttccagcc
    10561 gtctgctctt gctcagtctg gcagacagga tggccttggc gtccacgccg ctggcgttga
    10621 tggggttttc ctcgaacagc tggttgtagg tctgcaccag ctggatgaac agcttgtcca
    10681 cgtcgctgtt gtcggggttc aggtcgccct cgatcaggaa gtggccccgg aacttgatca
    10741 tgtgggccag ggccagatag atcagccgca ggtcggcctt gtcggtgctg tccaccagtt
    10801 tctttctcag gtggtagatg gtggggtact tctcgtggta ggccacctcg tccacgatgt
    10861 tgccgaagat ggggtgccgc tcgtgcttct tatcctcttc caccaggaag gactcttcca
    10921 gtctgtggaa gaagctgtcg tccaccttgg ccatctcgtt gctgaagatc tcttgcagat
    10981 agcagatccg gttcttccgt ctggtgtatc ttcttctggc ggttctcttc agccgggtgg
    11041 cctcggctgt ttcgccgctg tcgaacagca gggctccgat caggttcttc ttgatgctgt
    11101 gccggtcggt gttgcccagc accttgaatt tcttgctggg caccttgtac tcgtcggtga
    11161 tcacggccca gcccacagag ttggtgccga tgtccaggcc gatgctgtac ttcttgtcca
    11221 tggtggcagc gctctagaac cggtcctgtg ttctggcggc aaacccgttg cgaaaaagaa
    11281 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga
    11341 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggatc
    11401 aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact
    11461 gacgggcacc ggagccaatt cccactcctt tcaagaccta cggagccaat tcccactcct
    11521 ttcaagacct agaaggtcca ttagctgcaa agattcctct ctgtttaaaa ctttatccat
    11581 ctttgcaccc gggccccctc gagccggcgc caaagtggat ctctgctgtc cctgtaataa
    11641 acccgaaaat tttgaatttt tgtaatttgt ttttgtaatt ctttagttt
//
