LOCUS       sequence-362654-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets PARP1 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_PARP1
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    25..41
                     /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     30..52
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     49..65
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     49..65
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     84..104
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        86..104
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        132..358
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             359..539
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    586..711
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1204..1437
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1622..1666
                     /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    1847..1964
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2153..2364
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2383..2392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2389..6492
                     /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             2389..6492
                     /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             6493..6540
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6541..6564
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6574..6630
                     /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             6631..7230
                     /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(6631..6650)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7127..7147
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7246..7834
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7299..7319)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7717..7728)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7990..8238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7990..8010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8161..8180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8260..8335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8388..8568
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8646..8767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8683..8702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8737..8756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8807..8942
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8962..8981)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8963..8981)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8991..9008)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8991..9007)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9000..9022)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9149..9604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9236..9255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9446..9467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9630..9734
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9735..9803,9804..10595)
                     /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(9953..9972)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10766..11354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11255..11274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11508..11525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11642..11663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11678..11683,11684..11691,1..10,11..17)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
       61 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
      121 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
      181 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
      241 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
      301 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
      361 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      421 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      481 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      541 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      601 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      661 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      721 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
      781 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
      841 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
      901 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
      961 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1021 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1081 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1141 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1201 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1261 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1321 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1381 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1441 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1501 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1561 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1621 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1681 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1741 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     1801 attatcgttt cagaatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     1861 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     1921 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     1981 acagcagaga tccactttgg cgccggctcg agggggcccg ggtgcaaaga tggataaagt
     2041 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa
     2101 ttggctccgt aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag
     2161 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc
     2221 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2281 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2341 aacgggtttg ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag
     2401 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     2461 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     2521 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     2581 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     2641 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     2701 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     2761 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     2821 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     2881 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     2941 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     3001 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     3061 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     3121 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     3181 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     3241 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     3301 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     3361 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     3421 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     3481 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     3541 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     3601 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     3661 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     3721 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     3781 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     3841 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     3901 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     3961 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     4021 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     4081 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     4141 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     4201 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     4261 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     4321 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     4381 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     4441 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     4501 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     4561 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     4621 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     4681 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     4741 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     4801 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     4861 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     4921 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     4981 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     5041 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     5101 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     5161 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     5221 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     5281 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     5341 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     5401 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     5461 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     5521 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     5581 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     5641 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     5701 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     5761 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     5821 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     5881 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     5941 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     6001 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     6061 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     6121 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     6181 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     6241 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     6301 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     6361 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     6421 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     6481 ctgggaggcg acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa
     6541 gattacaaag acgatgacga taagggatcc ggcgcaacaa acttctctct gctgaaacaa
     6601 gccggagatg tcgaagagaa tcctggaccg atgaccgagt acaagcccac ggtgcgcctc
     6661 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6721 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     6781 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     6841 gccgccgtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     6901 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     6961 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     7021 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     7081 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     7141 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     7201 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     7261 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     7321 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     7381 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     7441 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     7501 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     7561 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     7621 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     7681 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     7741 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     7801 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
     7861 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     7921 actcccaacg aagacaagat ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     7981 ggacccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     8041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     8101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     8161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     8221 tggaaaggac gaaacaccgg cagaaagtca agaagacagg ttttagagct agaaatagca
     8281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     8341 taagcttggc gtaactagat cttgagacac tgctttttgc ttgtactggg tctctctggt
     8401 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     8461 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     8521 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     8581 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     8641 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     8701 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     8761 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     8821 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     8881 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     8941 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     9001 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     9061 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     9121 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     9181 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     9241 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     9301 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     9361 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     9421 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     9481 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     9541 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     9601 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     9661 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     9721 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     9781 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     9841 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     9901 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     9961 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
    10021 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
    10081 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
    10141 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
    10201 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
    10261 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
    10321 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
    10381 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
    10441 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
    10501 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
    10561 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
    10621 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
    10681 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
    10741 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
    10801 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
    10861 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
    10921 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
    10981 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
    11041 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
    11101 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
    11161 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
    11221 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
    11281 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
    11341 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
    11401 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
    11461 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
    11521 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
    11581 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
    11641 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg c
//
