LOCUS       sequence-460774-       13012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout mouse Casp8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13012)
  AUTHORS   Hanggi K, Li J, Gangadharan A, Liu X, Celias DP, Osunmakinde O, 
            Keske A, Davis J, Ahmad F, Giron A, Anadon CM, Gardner A, DeNardo 
            DG, Shaw TI, Beg AA, Yu X, Ruffell B
  TITLE     Interleukin-1alpha release during necrotic-like cell death generates
            myeloid-driven immunosuppression that restricts anti-tumor immunity.
  JOURNAL   Cancer Cell. 2024 Nov 13:S1535-6108(24)00402-1. doi: 
            10.1016/j.ccell.2024.10.014.
  PUBMED    39577420
REFERENCE   2  (bases 1 to 13012)
  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: "Cancer 
            Cell. 2024 Nov 13:S1535-6108(24)00402-1. doi: 
            10.1016/j.ccell.2024.10.014."
COMMENT     Sequence Label: pLenti-multi-CRISPR-sgCasp8_#1-puro
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     463..483
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    582..1170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(635..655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1053..1064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1242..1475
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(1501..1518)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1507..1731
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1777..2205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1864..1883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2074..2095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2214..2234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2219..2548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2399..2534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2461..2480
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2596..2643
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2662..3036
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3166..3287
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3203..3222)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3257..3276
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3336..3352)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3336..3352)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3349..3371)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3360..3376
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3384..3390,3391..3408,3409..3414))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3429..3450
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3567..3584)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3738..4326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3818..3837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4497..5288,5289..5357))
                     /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     5120..5139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5358..5462)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5537..5556)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5728..6107
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6109..6307
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6262..6282
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6325..6505
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6552..6677
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7170..7403
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7588..7632
                     /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    7930..8047
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8098..8346
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8098..8118
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8269..8288
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8367..8442
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8504..8715
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8734..8743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8740..12843
                     /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             8740..12843
                     /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             12844..12891
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12892..12915
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             12925..12981
                     /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             join(12982..13012,1..566)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
ORIGIN
        1 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
       61 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
      121 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
      181 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
      241 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
      301 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
      361 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
      421 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
      481 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
      541 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
      601 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
      661 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
      721 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
      781 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
      841 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
      901 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
      961 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     1021 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     1081 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     1141 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     1201 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     1261 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     1321 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     1381 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     1441 cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag
     1501 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1561 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1621 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1681 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     1741 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     1801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     2221 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2281 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2341 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2401 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2461 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     2521 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     2581 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac
     2641 gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg
     2701 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc
     2761 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc
     2821 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag
     2881 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc
     2941 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc
     3001 aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc
     3061 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3121 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3181 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3241 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3301 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3361 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3421 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3481 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3541 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3601 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3661 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3721 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3781 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3841 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3901 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3961 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4021 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4081 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4141 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4201 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4261 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4321 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4381 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4441 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4501 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4561 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4621 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4681 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4741 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4801 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4861 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4921 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4981 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5041 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5101 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5161 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5221 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5281 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5341 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5401 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5461 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5521 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     5581 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     5641 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     5701 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
     5761 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     5821 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     5881 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     5941 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     6001 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     6061 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     6121 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     6181 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     6241 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     6301 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
     6361 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     6421 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     6481 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     6541 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     6601 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     6661 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     6721 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     6781 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     6841 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     6901 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     6961 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     7021 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     7081 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     7141 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     7201 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     7261 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     7321 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     7381 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     7441 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     7501 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     7561 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     7621 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     7681 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     7741 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     7801 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     7861 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     7921 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     7981 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     8041 aaattttcgg gtttattaca gggacagcag agatccagtt tggttaatta aggtaccgag
     8101 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     8161 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     8221 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     8281 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     8341 aacacctagc ttctgggcat cctcgagttt tagagctaga aatagcaagt taaaataagg
     8401 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttga attcgctagc
     8461 taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     8521 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg
     8581 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     8641 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     8701 gccgccagaa cacaggaccg gttctagagc gctgccacca tggacaagaa gtacagcatc
     8761 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     8821 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     8881 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     8941 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     9001 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     9061 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     9121 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     9181 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     9241 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     9301 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     9361 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     9421 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     9481 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     9541 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     9601 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     9661 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     9721 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     9781 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     9841 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     9901 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     9961 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
    10021 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
    10081 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
    10141 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
    10201 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
    10261 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
    10321 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
    10381 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
    10441 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
    10501 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
    10561 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
    10621 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
    10681 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
    10741 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
    10801 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
    10861 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
    10921 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
    10981 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
    11041 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
    11101 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
    11161 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
    11221 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
    11281 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
    11341 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
    11401 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
    11461 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
    11521 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
    11581 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
    11641 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
    11701 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
    11761 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
    11821 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
    11881 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
    11941 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
    12001 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
    12061 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
    12121 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
    12181 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
    12241 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
    12301 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
    12361 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
    12421 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
    12481 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
    12541 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
    12601 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
    12661 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
    12721 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
    12781 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
    12841 gacaagcgac ctgccgccac aaagaaggct ggacaggcta agaagaagaa agattacaaa
    12901 gacgatgacg ataagggatc cggcgcaaca aacttctctc tgctgaaaca agccggagat
    12961 gtcgaagaga atcctggacc gaccgagtac aagcccacgg tgcgcctcgc ca
//
