LOCUS       sequence-362656-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets PDGFRA 
            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_PDGFRA
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..24
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6..24
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        52..278
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             279..459
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    506..631
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1124..1357
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1542..1586
                     /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    1767..1884
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2073..2284
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2303..2312
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2309..6412
                     /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             2309..6412
                     /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             6413..6460
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6461..6484
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6494..6550
                     /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             6551..7150
                     /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(6551..6570)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7047..7067
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7166..7754
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7219..7239)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7637..7648)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7910..8158
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7910..7930
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8081..8100
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8180..8255
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8308..8488
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8566..8687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8603..8622)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8657..8676
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8727..8862
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8882..8901)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8883..8901)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8911..8928)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8911..8927)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8920..8942)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9069..9524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9156..9175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9366..9387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9550..9654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9655..9723,9724..10515)
                     /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(9873..9892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10686..11274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11175..11194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11428..11445
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11562..11583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11598..11603,11604..11621,11622..11628)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11636..11652
                     /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     11641..11663
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11660..11676
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11660..11676
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
       61 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
      121 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
      181 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
      241 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
      301 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      361 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      421 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
      481 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
      541 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
      601 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
      661 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
      721 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
      781 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
      841 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
      901 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
      961 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1021 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1081 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1141 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1201 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1261 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1321 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1381 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1441 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1501 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1561 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1621 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1681 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagaatcttg
     1741 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1801 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1861 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     1921 cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga gaggaatctt
     1981 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgt aggtcttgaa
     2041 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2101 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     2161 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2221 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2281 acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat
     2341 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2401 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     2461 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     2521 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     2581 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     2641 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     2701 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     2761 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     2821 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     2881 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     2941 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3001 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3061 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3121 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3181 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3241 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3301 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3361 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     3421 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3481 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     3541 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     3601 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     3661 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     3721 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     3781 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     3841 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     3901 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     3961 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4021 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4081 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4141 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4201 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4261 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4321 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4381 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4441 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4501 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     4561 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     4621 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     4681 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     4741 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     4801 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     4861 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     4921 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     4981 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5041 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5101 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5161 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5221 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5281 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5341 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5401 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5461 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     5521 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     5581 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     5641 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     5701 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     5761 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     5821 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     5881 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     5941 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6001 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6061 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6121 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6181 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6241 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6301 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6361 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc
     6421 tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga
     6481 taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     6541 tcctggaccg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     6601 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     6661 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     6721 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg cggtctggac
     6781 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     6841 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     6901 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa
     6961 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     7021 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     7081 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     7141 cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     7201 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     7261 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     7321 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     7381 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     7441 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     7501 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     7561 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     7621 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     7681 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     7741 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     7801 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     7861 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     7921 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     7981 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     8041 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     8101 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
     8161 taaccttaca caacagtgag ttttagagct agaaatagca agttaaaata aggctagtcc
     8221 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc gtaactagat
     8281 cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     8341 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     8401 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     8461 ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt
     8521 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     8581 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     8641 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     8701 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     8761 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     8821 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     8881 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     8941 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     9001 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     9061 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     9121 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     9181 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     9241 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     9301 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     9361 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     9421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     9481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     9541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     9601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     9661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     9721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     9781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     9841 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     9901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     9961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
    10021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
    10081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
    10141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
    10201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
    10261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
    10321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
    10381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
    10441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
    10501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
    10561 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
    10621 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    10681 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    10741 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    10801 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
    10861 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    10921 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    10981 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    11041 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    11101 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    11161 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    11221 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    11281 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
    11341 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
    11401 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
    11461 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
    11521 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
    11581 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
    11641 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa g
//
