LOCUS       sequence-362638-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets IDH2 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_IDH2
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    10..26
                     /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     15..37
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     34..50
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     34..50
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     69..89
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        71..89
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        117..343
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             344..524
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    571..696
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1189..1422
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1607..1651
                     /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    1832..1949
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2138..2349
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2368..2377
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2374..6477
                     /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             2374..6477
                     /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             6478..6525
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6526..6549
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6559..6615
                     /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             6616..7215
                     /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(6616..6635)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7112..7132
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7231..7819
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7284..7304)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7702..7713)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7975..8223
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7975..7995
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8146..8165
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8245..8320
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8373..8553
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8631..8752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8668..8687)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8722..8741
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8792..8927
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8947..8966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8948..8966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8976..8993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8976..8992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8985..9007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9134..9589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9221..9240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9431..9452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9615..9719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9720..9788,9789..10580)
                     /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(9938..9957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10751..11339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11240..11259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11493..11510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11627..11648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11663..11668,11669..11686,11687..11691,1..2)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
       61 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
      121 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
      181 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
      241 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
      301 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
      361 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      421 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      481 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      541 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
      601 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
      661 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
      721 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
      781 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
      841 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
      901 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
      961 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1021 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1081 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1141 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1201 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1261 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1321 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1381 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1441 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1501 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1561 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     1621 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     1681 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     1741 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagaa
     1801 tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     1861 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     1921 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     1981 tttggcgccg gctcgagggg gcccgggtgc aaagatggat aaagttttaa acagagagga
     2041 atctttgcag ctaatggacc ttctaggtct tgaaaggagt gggaattggc tccgtaggtc
     2101 ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     2161 gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc
     2221 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     2281 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
     2341 agaacacagg accggttcta gagcgctgcc accatggaca agaagtacag catcggcctg
     2401 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     2461 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     2521 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     2581 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     2641 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     2701 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     2761 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     2821 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     2881 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     2941 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     3001 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     3061 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     3121 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     3181 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     3241 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     3301 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     3361 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     3421 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     3481 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     3541 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     3601 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     3661 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     3721 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     3781 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     3841 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     3901 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     3961 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     4021 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     4081 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     4141 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     4201 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     4261 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     4321 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     4381 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     4441 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     4501 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     4561 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     4621 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     4681 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     4741 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     4801 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     4861 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     4921 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     4981 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     5041 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     5101 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     5161 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     5221 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     5281 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     5341 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     5401 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     5461 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     5521 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     5581 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     5641 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     5701 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     5761 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     5821 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     5881 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     5941 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     6001 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     6061 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     6121 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     6181 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     6241 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     6301 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     6361 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     6421 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaag
     6481 cgacctgccg ccacaaagaa ggctggacag gctaagaaga agaaagatta caaagacgat
     6541 gacgataagg gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa
     6601 gagaatcctg gaccgatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     6661 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     6721 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     6781 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc
     6841 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     6901 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     6961 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     7021 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     7081 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     7141 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     7201 aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa
     7261 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     7321 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     7381 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     7441 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     7501 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     7561 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     7621 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     7681 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     7741 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     7801 ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc
     7861 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     7921 aagatttcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cagagagggc
     7981 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     8041 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     8101 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     8161 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     8221 accggggcat gtacaacacc gacggtttta gagctagaaa tagcaagtta aaataaggct
     8281 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttaagc ttggcgtaac
     8341 tagatcttga gacactgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     8401 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     8461 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     8521 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     8581 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     8641 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     8701 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     8761 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     8821 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     8881 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     8941 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     9001 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     9061 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     9121 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     9181 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     9241 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     9301 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     9361 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     9421 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     9481 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     9541 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     9601 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     9661 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     9721 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     9781 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     9841 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     9901 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     9961 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
    10021 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
    10081 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
    10141 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
    10201 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
    10261 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
    10321 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
    10381 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
    10441 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
    10501 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
    10561 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
    10621 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
    10681 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
    10741 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
    10801 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
    10861 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
    10921 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
    10981 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
    11041 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
    11101 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
    11161 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
    11221 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
    11281 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
    11341 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
    11401 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
    11461 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
    11521 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
    11581 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
    11641 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg t
//
