LOCUS       sequence-460789-       13012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout mouse Ripk1.
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-sgRipk1_#1-puro
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      202..630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(289..308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     499..520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(639..659)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        644..973
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      824..959
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     886..905
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        1021..1068
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1087..1461
                     /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    1591..1712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1628..1647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1682..1701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1761..1777)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1761..1777)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1774..1796)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1785..1801
                     /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(1809..1815,1816..1833,1834..1839))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1854..1875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1992..2009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2163..2751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2243..2262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2922..3713,3714..3782))
                     /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     3545..3564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3783..3887)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3962..3981)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4153..4532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4534..4732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4687..4707
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4750..4930
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4977..5102
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5595..5828
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6013..6057
                     /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    6355..6472
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6523..6771
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6523..6543
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6694..6713
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6792..6867
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6929..7140
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      7159..7168
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7165..11268
                     /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             7165..11268
                     /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             11269..11316
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11317..11340
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11350..11406
                     /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             11407..12003
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     11900..11920
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    12019..12607
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12072..12092)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12490..12501)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12679..12912
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12938..12955)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(12944..13012,1..156)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
       61 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
      121 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
      181 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
      241 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      301 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      361 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      421 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
      481 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
      541 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      601 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
      661 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      721 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
      781 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
      841 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
      901 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
      961 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
     1021 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     1081 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
     1141 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
     1201 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     1261 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     1321 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     1381 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     1441 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
     1501 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     1561 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     1621 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1681 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     1741 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1801 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1861 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1921 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1981 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2041 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2101 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2161 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2221 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2281 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2341 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2401 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2461 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2521 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2581 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2641 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2701 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2761 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2821 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2881 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2941 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3001 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3061 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3121 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3181 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3241 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3301 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3361 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3421 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3481 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3541 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3601 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3661 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3721 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3781 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3841 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3901 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     3961 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     4021 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     4081 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     4141 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     4201 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     4261 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4321 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4381 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     4441 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     4501 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     4561 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4621 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     4681 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     4741 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     4801 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     4861 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     4921 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     4981 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     5041 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     5101 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     5161 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     5221 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     5281 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     5341 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     5401 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     5461 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     5521 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     5581 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     5641 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     5701 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     5761 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     5821 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     5881 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     5941 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     6001 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     6061 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     6121 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     6181 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     6241 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     6301 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     6361 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     6421 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     6481 ttacagggac agcagagatc cagtttggtt aattaaggta ccgagggcct atttcccatg
     6541 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     6601 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     6661 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     6721 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgatggcatc
     6781 cagtgacctg cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     6841 cttgaaaaag tggcaccgag tcggtgcttt tttgaattcg ctagctaggt cttgaaagga
     6901 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     6961 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac
     7021 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     7081 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     7141 gaccggttct agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc
     7201 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     7261 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     7321 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     7381 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     7441 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     7501 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     7561 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     7621 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     7681 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     7741 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     7801 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     7861 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     7921 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     7981 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     8041 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     8101 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     8161 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     8221 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     8281 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     8341 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     8401 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     8461 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     8521 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     8581 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     8641 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     8701 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     8761 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     8821 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     8881 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     8941 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     9001 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     9061 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     9121 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     9181 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     9241 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     9301 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     9361 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     9421 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     9481 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     9541 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     9601 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     9661 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     9721 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     9781 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     9841 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     9901 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     9961 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
    10021 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
    10081 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
    10141 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
    10201 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
    10261 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
    10321 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
    10381 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
    10441 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
    10501 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
    10561 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
    10621 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
    10681 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
    10741 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
    10801 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
    10861 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
    10921 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
    10981 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
    11041 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
    11101 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
    11161 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
    11221 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc
    11281 gccacaaaga aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag
    11341 ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct
    11401 ggaccgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
    11461 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
    11521 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
    11581 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
    11641 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
    11701 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
    11761 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
    11821 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
    11881 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
    11941 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
    12001 tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
    12061 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
    12121 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
    12181 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
    12241 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
    12301 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
    12361 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
    12421 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
    12481 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
    12541 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
    12601 tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
    12661 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
    12721 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
    12781 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
    12841 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
    12901 aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
    12961 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tg
//
