LOCUS       sequence-412260-       12847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducibly expresses human MLKL, fused to light-activatable Cry2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12847)
  AUTHORS   Shkarina K, Hasel de Carvalho E, Santos JC, Ramos S, Leptin M, Broz 
            P
  TITLE     Optogenetic activators of apoptosis, necroptosis, and pyroptosis.
  JOURNAL   J Cell Biol. 2022 Jun 6;221(6). pii: 213145. doi: 
            10.1083/jcb.202109038. Epub 2022 Apr 14.
  PUBMED    35420640
REFERENCE   2  (bases 1 to 12847)
  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: "J Cell 
            Biol."; date: "2022-06-6"; volume: "221(6). pii: 213145. doi"; 
            pages: " 10.1083/jcb.202109038. Epub 2022 Apr 14"
COMMENT     Sequence Label: pLVX-opto-hMLKL
FEATURES             Location/Qualifiers
     source          1..12847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    1773..1907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     LTR             2075..2708
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    2755..2880
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3377..3610
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3795..3839
                     /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    4102..4217
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     polyA_signal    4261..4395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4601..5311)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4879..4898)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5148..5166
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      5308..5317
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5336..6826)
                     /codon_start=1
                     /product="N-terminal photolyase homology region of 
                     Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2PHR
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="KMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQF
                     YPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYD
                     PVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIE
                     SVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNE
                     FIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEE
                     SADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLV
                     DAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYI
                     SGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKAS
                     GVELGTNYAKPIVDIDTARELLAKAISRTRGAQIMIGAA"
     promoter        complement(8269..8636)
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    8394..8412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8430..8448
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8466..8484
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8502..8520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8538..8556
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8574..8592
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8610..8628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        8653..9163
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     9082..9100
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             9182..9928
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     promoter        9939..10268
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10119..10254
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             10277..10876
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(10277..10296)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     10773..10793
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10890..11478
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10943..10963)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11361..11372)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11685..12318
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(12446..12462)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12446..12462)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12459..12481)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12470..12486
                     /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(12494..12500,12501..12518,12519..12524))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12539..12560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12677..12694)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtcgacg gatcgggaga tcaacttgtt tattgcagct tataatggtt
     1801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1861 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc
     1921 aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc
     1981 tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt
     2041 gtatttgtta aatatgtact acaaacttag tagttggaag ggctaattca ctcccaaaga
     2101 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattagcag
     2161 aactacacac cagggccagg ggtcagatat ccactgacct ttggatggtg ctacaagcta
     2221 gtaccagttg agccagataa ggtagaagag gccaataaag gagagaacac cagcttgtta
     2281 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtgttaga gtggaggttt
     2341 gacagccgcc tagcatttca tcacgtggcc cgagagctgc atccggagta cttcaagaac
     2401 tgctgatatc gagcttgcta caagggactt tccgctgggg actttccagg gaggcgtggc
     2461 ctgggcggga ctggggagtg gcgagccctc agatcctgca tataagcagc tgctttttgc
     2521 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     2581 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     2641 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     2701 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     2761 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     2821 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     2881 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     2941 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     3001 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     3061 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     3121 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     3181 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac
     3241 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     3301 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     3361 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     3421 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     3481 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     3541 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     3601 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     3661 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     3721 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     3781 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     3841 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     3901 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     3961 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc
     4021 ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga
     4081 acggatctcg acggtatcgc ctttaaaaga aaagggggga ttggggggta cagtgcaggg
     4141 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     4201 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttatcgactt
     4261 aacttgttta ttgcagctta taatggttac aaataaggca atagcatcac aaatttcaca
     4321 aataaggcat ttttttcact gcattctagt tttggtttgt ccaaactcat caatgtatct
     4381 tatcatgtct ggatctcaaa tccctcggaa gctgcgcctg tcttaggttg gagtgataca
     4441 tttttatcac ttttacccgt ctttggatta ggcagtagct ctgacggccc tcctgtctta
     4501 ggttagtgaa aaatgtcact ctcttacccg tcattggctg tccagcttag ctcgcagggg
     4561 aggtggtctg gatccgccgg caccggtgta tacgggaatt ttacttgtac agctcgtcca
     4621 tgccgccggt ggagtggcgg ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct
     4681 cgttgtggga ggtgatgtcc aacttgatgt tgacgttgta ggcgccgggc agctgcacgg
     4741 gcttcttggc cttgtaggtg gtcttgacct cagcgtcgta gtggccgccg tccttcagct
     4801 tcagcctctg cttgatctcg cccttcaggg cgccgtcctc ggggtacatc cgctcggagg
     4861 aggcctccca gcccatggtc ttcttctgca ttacggggcc gtcggagggg aagttggtgc
     4921 cgcgcagctt caccttgtag atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg
     4981 tcaccacgcc gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg
     5041 acagcttcaa gtagtcgggg atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca
     5101 tgaactgagg ggacaggatg tcccaggcga agggcagggg gccacccttg gtcaccttca
     5161 gcttggcggt ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact
     5221 cgtggccgtt cacggagccc tccatgtgca ccttgaagcg catgaactcc ttgatgatgg
     5281 ccatgttatc ctcctcgccc ttgctcacca tggtggcgac cggtggatcc cgggctgctg
     5341 ctccgatcat gatctgtgct ccacgggttc ttgaaatagc tttagctagt agctcacgag
     5401 ctgtgtcgat gtctacaatg ggtttcgcat agtttgttcc gagttccaca ccagaagctt
     5461 tgagtacggt taaaggagcg tcccatggat gatggatcca ttcagttggc aatctcgcaa
     5521 gctcgggaag ccattgcctt atgtactcac cttctgggtc atatttggcg ccttgtaacg
     5581 cgggattgtc caagcgatca agctcgtggc catcggggat actcccagag atatactgcc
     5641 agccaaggat gtcacattcc aaatcagcat ccaaaagtgt atcccagaaa tacttcattc
     5701 cccatttcca tggaaggaga agaaacttca cagcaaagct tgaaacaatc actcttattc
     5761 tgttatgcat ccatccggta gcccaaagct ctctcattcc ggcatccacc aacggataac
     5821 cggtcctgcc ttgtctccag gccttgaact tatcaacatc agcatcccaa gggaaaaacc
     5881 gaagatgact caacaacgat tgctcgtgag taaacgggaa gttgaaacat atataccgag
     5941 aatactctct taaaccgatt cccctaagaa aaagatctgc actttcttct ccttcactgt
     6001 tcttatctct tgcccatata atttgtttca tccgggcaca ctggaaaacg tgtctgacgc
     6061 ttatttcccc gaaatggaga tacggagaaa gtagtgaagt agaattccca acaactttct
     6121 tgctgttctt tgcataatct atcaactgct tctcgatgaa ctcatttagt aacttatcag
     6181 cattgctcca tcctggagac caagctctag ttaacaacgc attgctcggt ttctcggcct
     6241 cattctccag ccctagttct tcaatcgaac acgcccaaat cgcttcagcc gctgcagtta
     6301 ttggcatcaa ccgccaagga ggaggaagca taacggattc aatcgacata tctaagcatt
     6361 tcttccagta agaattgaaa ctcgtaaaag gtttgccctt ttcgcagtat atctcccacg
     6421 gttcatacaa tagatctcca ttgtagcttt gcacagagat cccacgttcc accagcttct
     6481 cctttacggt atggtcccga actaacgaaa caggatcata gaggtggtta aagacgactt
     6541 ttgtagcacc ggtaacgcgg atacaatcca agatcgctga aatcgtgttg tgggttttga
     6601 ttaaagtgag gtcagatcca agagccttca aggattgaga taagtgagca agtgattgtt
     6661 tcatccacca tcttgaagct cttccaggat aaaactgtcc ttcttcttca ggacaccaaa
     6721 tgaagacagg aaaaacagat ccttcgtgag cagctgctgc taatgcagga ttatcctcaa
     6781 tccttaggtc tcttctaaac caaactatag tctttttgtc catcttacca ccgccgcttc
     6841 cgccgcctcc cttagaaaag gtggagagtt tctttaagat ttcatccaca gagggccgca
     6901 cagagggatc atgggcccgg cactcatcaa tgatctcccg cagctctgaa gggcagtctt
     6961 cacccagtgg ctcctgctgc cgcttcacag ccaccagctt gcggatcttc tcagaattac
     7021 agccttgaaa cgggatatct ccagtggcga tttcccagag gacgattcca aagctgtata
     7081 tttcagactt tacatcatat tgataaaata catcttccag ttcctgaggt gagagatatg
     7141 ctgtagattt gactctgtct gtcttttctc tcgtagttcc caaactcatg gaagtctgtg
     7201 ttttcctcaa ctcaaatcct gcaagcttca cttggtagcc ttgagttacc aggaagtttg
     7261 agcttctgat ttttccgtgg agttcaggtg cttctgaatg gtgtagccgg tataggcctc
     7321 gggctgcccc caggactagg accatgcgct tgccaagtgt gaggtctttt tccctatcca
     7381 acagctccct cagggtcccg agttcacagt actccatgac aatggagaat tgaggcggag
     7441 tcactgtttc atcaatgcaa atcccaaata tacgcaggat gttgggagat tcgaatttct
     7501 tcatggtttt gatctcctta ttgaaagtct gcctcactat tgcaatgctg ccagcctgga
     7561 gttttttgaa tacttttatg gccactggag ctctgtggta ttctccttta taaagtgtgc
     7621 tgacttcatt ttcccttagc agaatccacg gggatcctga aagctgctcc ttcttgatct
     7681 ccttgatttg ctcttgcggg atctcctgca tgcattttgg tggtaaatac tgcctcaaag
     7741 tttccttgat ttctttcatg ttgatttcta atcgtctcag tgaagcttct attttttcat
     7801 tatctcttct tagcatctgg aaagctcgcc tgtcttcgtc tgcatcctgc tgatcttcct
     7861 gtgcccagga cgctccttgg cttatgggtg aaacaggcat gcgttgctca acctgaagta
     7921 acagcgagag ctccttccag acatcactca gcttcctgtt cacgtccttg aagagtattt
     7981 tgtcctggct tgctgttaga aacctgcaga tattggatct attgctgaac ttttctatct
     8041 ccccattagc ctcctccagg gcagccttga agcggttcat ggctgtggtt aacttctcag
     8101 agggcacgct cctctttcct tggtcctgga gcatctccag aggcttgatc aggccgagga
     8161 cgcggtggcc caggcgccgg cactgtttct tgcagtattt catctcttca caccgtttgt
     8221 ggatgacctg gccaagggtg ataatatgct tcaaattttc cataattctt tacgagggta
     8281 ggaagtggta cggaaagttg gtataagaca aaagtgttgt ggaattgaag tttactcaaa
     8341 aaatcagcac tcttttatag gcgccctggt ttacataagc aaagcttata cgttctctat
     8401 cactgatagg gagtaaactg gatatacgtt ctctatcact gatagggagt aaactgtaga
     8461 tacgttctct atcactgata gggagtaaac tggtcatacg ttctctatca ctgataggga
     8521 gtaaactcct tatacgttct ctatcactga tagggagtaa agtctgcata cgttctctat
     8581 cactgatagg gagtaaactc ttcatacgtt ctctatcact gatagggagt aaactcgagg
     8641 tgataattcc acggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg
     8701 acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtctcgcac
     8761 attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc
     8821 ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg
     8881 gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg
     8941 agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg
     9001 cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg
     9061 ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc
     9121 agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc atcgaattac
     9181 catgtctaga ctggacaaga gcaaagtcat aaactctgct ctggaattac tcaatggagt
     9241 cggtatcgaa ggcctgacga caaggaaact cgctcaaaag ctgggagttg agcagcctac
     9301 cctgtactgg cacgtgaaga acaagcgggc cctgctcgat gccctgccaa tcgagatgct
     9361 ggacaggcat catacccact cctgccccct ggaaggcgag tcatggcaag actttctgcg
     9421 gaacaacgcc aagtcatacc gctgtgctct cctctcacat cgcgacgggg ctaaagtgca
     9481 tctcggcacc cgcccaacag agaaacagta cgaaaccctg gaaaatcagc tcgcgttcct
     9541 gtgtcagcaa ggcttctccc tggagaacgc actgtacgct ctgtccgccg tgggccactt
     9601 tacactgggc tgcgtattgg aggaacagga gcatcaagta gcaaaagagg aaagagagac
     9661 acctaccacc gattctatgc ccccacttct gaaacaagca attgagctgt tcgaccggca
     9721 gggagccgaa cctgccttcc ttttcggcct ggaactaatc atatgtggcc tggagaaaca
     9781 gctaaagtgc gaaagcggcg ggccgaccga cgcccttgac gattttgact tagacatgct
     9841 cccagccgat gcccttgacg actttgacct tgatatgctg cctgctgacg ctcttgacga
     9901 ttttgacctt gacatgctcc ccgggtaaac gcgcgaatgt gtgtcagtta gggtgtggaa
     9961 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
    10021 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
    10081 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
    10141 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
    10201 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
    10261 tttgcaaaac gcgaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
    10321 cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
    10381 caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
    10441 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
    10501 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
    10561 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
    10621 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
    10681 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
    10741 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
    10801 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
    10861 caagcccggt gcctgaacgc gtctggaaca atcaacctct ggattacaaa atttgtgaaa
    10921 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
    10981 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
    11041 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
    11101 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
    11161 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
    11221 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
    11281 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
    11341 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
    11401 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
    11461 tttgggccgc ctccccgcct ggaattaatt ctgcagtcga gacctagaaa aacatggagc
    11521 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
    11581 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
    11641 ggcagctgta gatcttagcc actttttaaa agaaaagagg ggactggaag ggctaattca
    11701 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
    11761 tgattagcag aactacacac cagggccagg ggtcagatat ccactgacct ttggatggtg
    11821 ctacaagcta gtaccagttg agccagataa ggtagaagag gccaataaag gagagaacac
    11881 cagcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtgttaga
    11941 gtggaggttt gacagccgcc tagcatttca tcacgtggcc cgagagctgc atccggagta
    12001 cttcaagaac tgctgatatc gagcttgcta caagggactt tccgctgggg actttccagg
    12061 gaggcgtggc ctgggcggga ctggggagtg gcgagccctc agatcctgca tataagcagc
    12121 tgctttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
    12181 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
    12241 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
    12301 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
    12361 caaagaaatg aatatcagag agtgagaggc cttgacattg ctagcgttta ccgtcgacct
    12421 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
    12481 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    12541 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
    12601 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
    12661 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
    12721 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
    12781 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
    12841 tggcgtt
//
