LOCUS       sequence-460776-       13012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Knockout mouse Mlkl.
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-sgMlkl_#1-puro
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(163..207)
                     /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    392..625
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    1118..1243
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             1290..1470
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(1488..1686)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1513..1533)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1688..2067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2239..2258
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2333..2437
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2438..2506,2507..3298)
                     /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(2656..2675)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3469..4057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3958..3977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4211..4228
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4345..4366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4381..4386,4387..4404,4405..4411)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4419..4435
                     /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     4424..4446
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4443..4459
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4443..4459
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4508..4629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4519..4538)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4573..4592
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4759..5133)
                     /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"
     promoter        complement(5152..5199)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5247..5576)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5261..5396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5315..5334)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5561..5581
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5590..6018)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5700..5721)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5912..5931
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6064..6288
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6277..6294
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             6320..6553
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6625..7213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6731..6742
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7140..7160
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7229..7825)
                     /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     complement(7312..7332)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             complement(7826..7882)
                     /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             complement(7892..7915)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(7916..7963)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(7964..12067)
                     /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             complement(7964..12067)
                     /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"
     regulatory      12064..12073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(12092..12303)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(12365..12440)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(12461..12709)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(12519..12538)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(12689..12709)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    12760..12877
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 ggttgggagg tgggtctgaa acgataatgg tgaatatccc tgcctaactc tattcactat
       61 agaaagtaca gcaaaaacta ttcttaaacc taccaagcct cctactatca ttatgaataa
      121 ttttatatac cacagccaat ttgttatgtt aaaccaattc cacaaacttg cccatttatc
      181 taattccaat aattcttgtt cattcttttc ttgctggttt tgcgattctt caattaagga
      241 gtgtattaag cttgtgtaat tgttaatttc tctgtcccac tccatccagg tcgtgtgatt
      301 ccaaatctgt tccagagatt tattactcca actagcattc caaggcacag cagtggtgca
      361 aatgagtttt ccagagcaac cccaaatccc caggagctgt tgatccttta ggtatctttc
      421 cacagccagg attcttgcct ggagctgctt gatgccccag actgtgagtt gcaacagatg
      481 ctgttgcgcc tcaatagccc tcagcaaatt gttctgctgc tgcactatac cagacaataa
      541 ttgtctggcc tgtaccgtca gcgtcattga cgctgcgccc atagtgcttc ctgctgctcc
      601 caagaaccca aggaacaaag ctcctattcc cactgctctt ttttctctct gcaccactct
      661 tctctttgcc ttggtgggtg ctactcctaa tggttcaatt tttactactt tatatttata
      721 taattcactt ctccaattgt ccctcatatc tcctcctcca ggtctgaaga tcagcggccg
      781 cttgctgtgc ggtggtctta cttttgtttt gctcttcctc tatcttgtct aaagcttcct
      841 tggtgtcttt tatctctatc ctttgatgca cacaatagag ggttgctact gtattatata
      901 atgatctaag ttcttctgat cctgtctgaa gggatggttg tagctgtccc agtatttgtc
      961 tacagccttc tgatgtttct aacaggccag gattaactgc gaatcgttct agctccctgc
     1021 ttgcccatac tatatgtttt aatttatatt ttttctttcc ccctggcctt aaccgaattt
     1081 tttcccatcg cgatctaatt ctcccccgct taatactgac gctctcgcac ccatctctct
     1141 ccttctagcc tccgctagtc aaaatttttg gcgtactcac cagtcgccgc ccctcgcctc
     1201 ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga gagctcctct ggtttccctt
     1261 tcgctttcaa gtccctgttc gggcgccact gctagagatt ttccacactg actaaaaggg
     1321 tctgagggat ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac
     1381 tcaaggcaag ctttattgag gcttaagcag tgggttccct agttagccag agagctccca
     1441 ggctcagatc tggtctaacc agagagaccc agtacaggca aaacgcgctg cttatataga
     1501 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
     1561 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
     1621 gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc
     1681 gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc
     1741 ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag
     1801 ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat
     1861 actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca
     1921 ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa
     1981 gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact
     2041 attaataact agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca
     2101 gcgcaaaacg cctaacccta agcagattct tcatgcaatt gtcggtcaag ccttgccttg
     2161 ttgtagctta aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca
     2221 gatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cataggggat
     2281 cgggagatct cccgatccgt cgacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2341 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2401 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2461 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2521 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2581 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2641 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2701 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2761 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2821 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2881 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2941 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3001 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3061 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3121 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3181 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3241 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3301 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3361 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3421 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3481 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3541 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3601 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3661 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3721 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3781 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3841 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3901 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3961 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4021 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4081 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4141 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4201 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     4261 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4321 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     4381 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     4441 cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca
     4501 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4561 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4621 aaacaagttg gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca
     4681 gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc
     4741 gaaatctcgt agcacgtgtc agtcctgctc ctcggccacg aagtgcacgc agttgccggc
     4801 cgggtcgcgc agggcgaact cccgccccca cggctgctcg ccgatctcgg tcatggccgg
     4861 cccggaggcg tcccggaagt tcgtggacac gacctccgac cactcggcgt acagctcgtc
     4921 caggccgcgc acccacaccc aggccagggt gttgtccggc accacctggt cctggaccgc
     4981 gctgatgaac agggtcacgt cgtcccggac cacaccggcg aagtcgtcct ccacgaagtc
     5041 ccgggagaac ccgagccggt cggtccagaa ctcgaccgct ccggcgacgt cgcgcgcggt
     5101 gagcaccgga acggcactgg tcaacttggc catggtttag ttcctcacct tgtcgtatta
     5161 tactatgccg atatactatg ccgatgatta attgtcaaca cgtgctgatc agatccgaaa
     5221 atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
     5281 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
     5341 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
     5401 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
     5461 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
     5521 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
     5581 cacagaatta attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5641 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5701 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5761 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5821 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5881 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5941 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6001 cgccgctaca gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag
     6061 aagccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct
     6121 tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg
     6181 caatttcctc attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag
     6241 gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc
     6301 agcgggttta aacgggccct gctagagatt ttccacactg actaaaaggg tctgagggat
     6361 ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac tcaaggcaag
     6421 ctttattgag gcttaagcag tgggttccct agttagccag agagctccca ggctcagatc
     6481 tggtctaacc agagagaccc agtacaagca aaaagcagat cttgtcttcg ttgggagtga
     6541 attagccctt ccagtccccc cttttctttt aaaaagtggc taagatctac agctgccttg
     6601 taagtcattg gtcttaaagt cgacgcgggg aggcggccca aagggagatc cgactcgtct
     6661 gagggcgaag gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa
     6721 ggtccgctgg attgagggcc gaagggacgt agcagaagga cgtcccgcgc agaatccagg
     6781 tggcaacaca ggcgagcagc caaggaaagg acgatgattt ccccgacaac accacggaat
     6841 tgtcagtgcc caacagccga gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt
     6901 ccgccgtggc aatagggagg gggaaagcga aagtcccgga aaggagctga caggtggtgg
     6961 caatgcccca accagtgggg gttgcgtcag caaacacagt gcacaccacg ccacgttgcc
     7021 tgacaacggg ccacaactcc tcataaagag acagcaacca ggatttatac aaggaggaga
     7081 aaatgaaagc catacgggaa gcaatagcat gatacaaagg cattaaagca gcgtatccac
     7141 atagcgtaaa aggagcaaca tagttaagaa taccagtcaa tctttcacaa attttgtaat
     7201 ccagaggttg attgtcgact taacgcgttc aggcaccggg cttgcgggtc atgcaccagg
     7261 tgcgcggtcc ttcgggcacc tcgacgtcgg cggtgacggt gaagccgagc cgctcgtaga
     7321 aggggaggtt gcggggcgcg gaggtctcca ggaaggcggg caccccggcg cgctcggccg
     7381 cctccactcc ggggagcacg acggcgctgc ccagaccctt gccctggtgg tcgggcgaga
     7441 ctccgacggt ggccaggaac cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt
     7501 ccatctgttg ctgcgcggcc agccgggaac cgctcaactc ggccatgcgc gggccgatct
     7561 cggcgaacac cgcccccgct tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc
     7621 cgtcgtccgc gacccacacc ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt
     7681 gcagctcggt gacccgctcg atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt
     7741 agtcggcgaa cgcggcggcg agggtgcgta cggccctggg gacgtcgtcg cgggtggcga
     7801 ggcgcaccgt gggcttgtac tcggtcggtc caggattctc ttcgacatct ccggcttgtt
     7861 tcagcagaga gaagtttgtt gcgccggatc ccttatcgtc atcgtctttg taatctttct
     7921 tcttcttagc ctgtccagcc ttctttgtgg cggcaggtcg cttgtcgcct cccagctgag
     7981 acaggtcgat ccgtgtctcg tacaggccgg tgatgctctg gtggatcagg gtggcgtcca
     8041 gcacctcttt ggtgctggtg tacctcttcc ggtcgatggt ggtgtcaaag tacttgaagg
     8101 cggcaggggc tcccagattg gtcagggtaa acaggtggat gatattctcg gcctgctctc
     8161 tgatgggctt atcccggtgc ttgttgtagg cggacagcac tttgtccaga ttagcgtcgg
     8221 ccaggatcac tctcttggag aactcgctga tctgctcgat gatctcgtcc aggtagtgct
     8281 tgtgctgttc cacaaacagc tgtttctgct cattatcctc gggggagccc ttcagcttct
     8341 catagtggct ggccaggtac aggaagttca catatttgga gggcagggcc agttcgtttc
     8401 ccttctgcag ttcgccggca gaggccagca ttctcttccg gccgttttcc agctcgaaca
     8461 gggagtactt aggcagcttg atgatcaggt cctttttcac ttctttgtag cccttggctt
     8521 ccagaaagtc gatgggattc ttctcgaagc tgcttctttc catgatggtg atccccagca
     8581 gctctttcac actcttcagt ttcttggact tgcccttttc cactttggcc accaccagca
     8641 cagaataggc cacggtgggg ctgtcgaagc cgccgtactt cttagggtcc cagtccttct
     8701 ttctggcgat cagcttatcg ctgttcctct tgggcaggat agactctttg ctgaagccgc
     8761 ctgtctgcac ctcggtcttt ttcacgatat tcacttgggg catgctcagc actttccgca
     8821 cggtggcaaa atcccggccc ttatcccaca cgatctcccc ggtttcgccg tttgtctcga
     8881 tcagaggccg cttccggatc tcgccgttgg ccagggtaat ctcggtcttg aaaaagttca
     8941 tgatgttgct gtagaagaag tacttggcgg tagccttgcc gatttcctgc tcgctcttgg
     9001 cgatcatctt ccgcacgtcg tacaccttgt agtcgccgta cacgaactcg ctttccagct
     9061 tagggtactt tttgatcagg gcggttccca cgacggcgtt caggtaggcg tcgtgggcgt
     9121 ggtggtagtt gttgatctcg cgcactttgt aaaactggaa atccttccgg aaatcggaca
     9181 ccagcttgga cttcagggtg atcactttca cttcccggat cagcttgtca ttctcgtcgt
     9241 acttagtgtt catccgggag tccaggatct gtgccacgtg ctttgtgatc tgccgggttt
     9301 ccaccagctg tctcttgatg aagccggcct tatccagttc gctcaggccg cctctctcgg
     9361 ccttggtcag attgtcgaac tttctctggg taatcagctt ggcgttcagc agctgccgcc
     9421 agtagttctt catcttcttc acgacctctt cggagggcac gttgtcgctc ttgccccggt
     9481 tcttgtcgct tctggtcagc accttgttgt cgatggagtc gtccttcaga aagctctgag
     9541 gcacgatatg gtccacatcg tagtcggaca gccggttgat gtccagttcc tggtccacgt
     9601 acatatcccg cccattctgc aggtagtaca ggtacagctt ctcgttctgc agctgggtgt
     9661 tttccacggg gtgttctttc aggatctggc tgcccagctc tttgatgccc tcttcgatcc
     9721 gcttcattct ctcgcggctg ttcttctgtc ccttctgggt ggtctggttc tctctggcca
     9781 tttcgatcac gatgttctcg ggcttgtgcc ggcccatcac tttcacgagc tcgtccacca
     9841 ccttcactgt ctgcaggatg cccttcttaa tggcggggct gccggccaga ttggcaatgt
     9901 gctcgtgcag gctatcgccc tggccggaca cctgggcttt ctggatgtcc tctttaaagg
     9961 tcaggctgtc gtcgtggatc agctgcatga agtttctgtt ggcgaagccg tcggacttca
    10021 ggaaatccag gattgtcttg ccggactgct tgtcccggat gccgttgatc agcttccggc
    10081 tcagcctgcc ccagccggtg tatctccgcc gcttcagctg cttcatcact ttgtcgtcga
    10141 acaggtgggc ataggttttc agccgttcct cgatcatctc tctgtcctca aacagtgtca
    10201 gggtcagcac gatatcttcc agaatgtcct cgttttcctc attgtccagg aagtccttgt
    10261 ccttgataat tttcagcaga tcgtggtatg tgcccaggga ggcgttgaac cgatcttcca
    10321 cgccggagat ttccacggag tcgaagcact cgattttctt gaagtagtcc tctttcagct
    10381 gcttcacggt cactttccgg ttggtcttga acagcaggtc cacgatggcc tttttctgct
    10441 cgccgctcag gaaggcgggc tttctcattc cctcggtcac gtatttcact ttggtcagct
    10501 cgttatacac ggtgaagtac tcgtacagca ggctgtgctt gggcagcacc ttctcgttgg
    10561 gcaggttctt atcgaagttg gtcatccgct cgatgaagct ctgggcggaa gcgcccttgt
    10621 ccaccacttc ctcgaagttc cagggggtga tggtttcctc gctctttctg gtcatccagg
    10681 cgaatctgct gtttcccctg gccagagggc ccacgtagta ggggatgcgg aaggtcagga
    10741 tcttctcgat cttttcccgg ttgtccttca ggaatgggta aaaatcttcc tgccgccgca
    10801 gaatggcgtg cagctctccc aggtggatct ggtgggggat gctgccgttg tcgaaggtcc
    10861 gctgcttccg cagcaggtcc tctctgttca gcttcacgag cagttcctcg gtgccgtcca
    10921 tcttttccag gatgggcttg atgaacttgt agaactcttc ctggctggct ccgccgtcaa
    10981 tgtagccggc gtagccgttc ttgctctggt cgaagaaaat ctctttgtac ttctcaggca
    11041 gctgctgccg cacgagagct ttcagcaggg tcaggtcctg gtggtgctcg tcgtatctct
    11101 tgatcataga ggcgctcagg ggggccttgg tgatctcggt gttcactctc aggatgtcgc
    11161 tcagcaggat ggcgtcggac aggttcttgg cggccagaaa caggtcggcg tactggtcgc
    11221 cgatctgggc cagcaggttg tccaggtcgt cgtcgtaggt gtccttgctc agctgcagtt
    11281 tggcatcctc ggccaggtcg aagttgctct tgaagttggg ggtcaggccc aggctcaggg
    11341 caatcaggtt tccgaacagg ccattcttct tctcgccggg cagctgggcg atcagatttt
    11401 ccagccgtct gctcttgctc agtctggcag acaggatggc cttggcgtcc acgccgctgg
    11461 cgttgatggg gttttcctcg aacagctggt tgtaggtctg caccagctgg atgaacagct
    11521 tgtccacgtc gctgttgtcg gggttcaggt cgccctcgat caggaagtgg ccccggaact
    11581 tgatcatgtg ggccagggcc agatagatca gccgcaggtc ggccttgtcg gtgctgtcca
    11641 ccagtttctt tctcaggtgg tagatggtgg ggtacttctc gtggtaggcc acctcgtcca
    11701 cgatgttgcc gaagatgggg tgccgctcgt gcttcttatc ctcttccacc aggaaggact
    11761 cttccagtct gtggaagaag ctgtcgtcca ccttggccat ctcgttgctg aagatctctt
    11821 gcagatagca gatccggttc ttccgtctgg tgtatcttct tctggcggtt ctcttcagcc
    11881 gggtggcctc ggctgtttcg ccgctgtcga acagcagggc tccgatcagg ttcttcttga
    11941 tgctgtgccg gtcggtgttg cccagcacct tgaatttctt gctgggcacc ttgtactcgt
    12001 cggtgatcac ggcccagccc acagagttgg tgccgatgtc caggccgatg ctgtacttct
    12061 tgtccatggt ggcagcgctc tagaaccggt cctgtgttct ggcggcaaac ccgttgcgaa
    12121 aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa
    12181 ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac
    12241 cggatcaatt gccgacccct ccccccaact tctcggggac tgtgggcgat gtgcgctctg
    12301 cccactgacg ggcaccggag ccaattccca ctcctttcaa gacctagcta gcgaattcaa
    12361 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
    12421 gctatttcta gctctaaaac aacttgatga tctgtcccaa ggtgtttcgt cctttccaca
    12481 agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca
    12541 ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg
    12601 tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag
    12661 ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg gtaccttaat
    12721 taaccaaact ggatctctgc tgtccctgta ataaacccga aaattttgaa tttttgtaat
    12781 ttgtttttgt aattctttag tttgtatgtc tgttgctatt atgtctacta ttctttcccc
    12841 tgcactgtac cccccaatcc ccccttttct tttaaaattg tggatgaata ctgccatttg
    12901 tctgcagaat tggcgcacgc agtgccgatc cgttcactaa tcgaatggat ctgtctctgt
    12961 ctctctctcc accttcttct tctattcctt cgggcctgtc gggtcccctc gg
//
