LOCUS       sequence-362803-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets TYMS 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_TYMS
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..53
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        35..53
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        81..307
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             308..488
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    535..660
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1153..1386
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1571..1615
                     /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    1796..1913
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2102..2313
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2332..2341
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2338..6441
                     /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             2338..6441
                     /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             6442..6489
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6490..6513
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6523..6579
                     /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             6580..7179
                     /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(6580..6599)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7076..7096
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7195..7783
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7248..7268)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7666..7677)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7939..8187
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7939..7959
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8110..8129
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8209..8284
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8337..8517
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8595..8716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8632..8651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8686..8705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8756..8891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8911..8930)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8912..8930)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8940..8957)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8940..8956)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8949..8971)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9098..9553
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9185..9204)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9395..9416
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9579..9683
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9684..9752,9753..10544)
                     /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(9902..9921)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10715..11303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11204..11223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11457..11474
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11591..11612
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11627..11632,11633..11650,11651..11657)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11665..11681
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     join(11670..11691,1)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11689..11691,1..14)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11689..11691,1..14)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
       61 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
      121 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
      181 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
      241 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
      301 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
      361 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
      421 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
      481 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
      541 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
      601 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
      661 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
      721 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
      781 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
      841 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
      901 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
      961 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     1021 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     1081 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     1141 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     1201 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     1261 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     1321 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     1381 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     1441 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     1501 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     1561 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     1621 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     1681 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     1741 atattcacca ttatcgtttc agaatcttga gacaaatggc agtattcatc cacaatttta
     1801 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     1861 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     1921 attacaggga cagcagagat ccactttggc gccggctcga gggggcccgg gtgcaaagat
     1981 ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag
     2041 gagtgggaat tggctccgta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     2101 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2161 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2221 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2281 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
     2341 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     2401 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     2461 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     2521 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     2581 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     2641 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     2701 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     2761 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     2821 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     2881 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     2941 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     3001 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     3061 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     3121 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     3181 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     3241 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     3301 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     3361 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     3421 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     3481 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     3541 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     3601 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     3661 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     3721 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     3781 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     3841 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     3901 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     3961 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     4021 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     4081 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     4141 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     4201 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     4261 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     4321 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     4381 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     4441 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     4501 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     4561 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     4621 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     4681 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     4741 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     4801 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     4861 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     4921 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     4981 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     5041 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     5101 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     5161 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     5221 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     5281 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     5341 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     5401 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     5461 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     5521 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     5581 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     5641 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     5701 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     5761 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     5821 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     5881 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     5941 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     6001 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     6061 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     6121 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     6181 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     6241 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     6301 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     6361 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     6421 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
     6481 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
     6541 ctgaaacaag ccggagatgt cgaagagaat cctggaccga tgaccgagta caagcccacg
     6601 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6661 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     6721 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6781 gacgacggcg ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6841 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     6901 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     6961 gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     7021 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     7081 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     7141 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa
     7201 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     7261 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     7321 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     7381 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     7441 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     7501 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     7561 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     7621 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     7681 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     7741 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     7801 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     7861 ggctaattca ctcccaacga agacaagatt tcaccattat cgtttcagac ccacctccca
     7921 accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga
     7981 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     8041 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     8101 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     8161 tatatcttgt ggaaaggacg aaacaccgcc tgcatgccga ataccgacgt tttagagcta
     8221 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     8281 gtgctttttt aagcttggcg taactagatc ttgagacact gctttttgct tgtactgggt
     8341 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8401 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     8461 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     8521 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     8581 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     8641 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     8701 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     8761 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     8821 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     8881 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     8941 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     9001 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     9061 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     9121 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     9181 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     9241 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     9301 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     9361 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     9421 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     9481 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     9541 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     9601 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     9661 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     9721 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     9781 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     9841 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     9901 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     9961 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
    10021 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
    10081 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
    10141 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
    10201 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
    10261 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
    10321 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
    10381 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
    10441 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
    10501 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
    10561 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
    10621 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
    10681 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
    10741 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
    10801 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
    10861 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
    10921 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
    10981 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
    11041 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
    11101 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
    11161 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
    11221 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
    11281 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
    11341 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
    11401 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
    11461 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
    11521 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
    11581 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
    11641 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca g
//
