LOCUS       sequence-204706-        7791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7791)
  AUTHORS   Quarato G, Guy CS, Grace CR, Llambi F, Nourse A, Rodriguez DA, 
            Wakefield R, Frase S, Moldoveanu T, Green DR
  TITLE     Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding 
            Sites Executes Necroptosis.
  JOURNAL   Mol Cell. 2016 Feb 18;61(4):589-601. doi: 
            10.1016/j.molcel.2016.01.011. Epub 2016 Feb 4.
  PUBMED    26853145
REFERENCE   2  (bases 1 to 7791)
  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"; doi: 
            "10.1016/j.molcel.2016.01.011"; journalName: "Mol Cell"; date: 
            "2016-02-18- 18"; volume: "61"; issue: "4"; pages: "589-601"
COMMENT     Sequence Label: hMLKL NB(1-140)-2xFv-flag
FEATURES             Location/Qualifiers
     source          1..7791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..40
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             724..899
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    962..1161
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1362..1778
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1708..1730
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1746..1762
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1818..2167
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    1826..1844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1862..1880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1898..1916
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1934..1952
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1970..1988
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2006..2024
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2042..2060
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2106..2130
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2612..2932
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2939..3259
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3272..3295
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3332..3355
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3371..3394
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        3408..3907
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3443..3466)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3840..3859
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3928..4527
                     /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(3928..3947)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4424..4444
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4708..5133
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5236..5258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5394..5414)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5399..5728
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5579..5714
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5750..5769
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5850..5867)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6021..6609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6101..6120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6780..7571,7572..7640))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7403..7422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7641..7745)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaatta
       61 gcttggccat tgcatacgtt gtatccatat cataatatgt acatttatat tggctcatgt
      121 ccaacattac cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      661 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 gctcaataaa agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc
      781 gcccgggtac ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg
      841 ctgttccttg ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt
      901 gggggctcgt ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg
      961 taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta
     1021 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1081 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1141 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1201 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1261 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1321 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     1381 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1441 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1501 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1561 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1621 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1681 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1741 cctcccttta tccagccctc actccttctc taggcgccgg aattgaagat ctcgatgagg
     1801 ccctttcgtc ttcactcgag tttactccct atcagtgata gagaacgtat gaagagttta
     1861 ctccctatca gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt
     1921 ataaggagtt tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt
     1981 gatagagaac gtatctacag tttactccct atcagtgata gagaacgtat atccagttta
     2041 ctccctatca gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg cgcctataaa
     2101 agcagagctc gtttagtgaa ccgtcagatc gcctggagca attccacaac acttttgtct
     2161 tatacttgga tccatcgata ccatggaaaa tttgaagcat attatcaccc ttggccaggt
     2221 catccacaaa cggtgtgaag agatgaaata ctgcaagaaa cagtgccggc gcctgggcca
     2281 ccgcgtcctc ggcctgatca agcctctgga gatgctccag gaccaaggaa agaggagcgt
     2341 gccctctgag aagttaacca cagccatgaa ccgcttcaag gctgccctgg aggaggctaa
     2401 tggggagata gaaaagttca gcaatagatc caatatctgc aggtttctaa cagcaagcca
     2461 ggacaaaata ctcttcaagg acgtgaacag gaagctgagt gatgtctgga aggagctctc
     2521 gctgttactt caggttgagc aacgcatgcc tgtttcaccc ataagccaag gagcgtcctg
     2581 ggcacaggaa gatcagcagg atgcggccgc aggcgtccaa gtcgaaacca ttagtcccgg
     2641 cgatggcaga acatttccta aaaggggaca aacatgtgtc gtccattata caggcatgtt
     2701 ggaggacggc aaaaaggtgg acagtagtag agatcgcaat aaacctttca aattcatgtt
     2761 gggaaaacaa gaagtcatta ggggatggga ggagggcgtg gctcaaatgt ccgtcggcca
     2821 acgcgctaag ctcaccatca gccccgacta cgcatacggc gctaccggac atcccggaat
     2881 tattccccct cacgctacct tggtgtttga cgtcgaactg ttgaagctcg agactagagg
     2941 agtgcaggtg gagactatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac
     3001 ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaagttgatt cctcccggga
     3061 cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga
     3121 aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc
     3181 ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt
     3241 ggagcttcta aaactggaag cggccgccag ctggagccac cctcagttcg agaagggagg
     3301 aggaagcggc ggaggcagcg gaggaggaag ctggagccac ccgcagttcg agaaaggagc
     3361 tagcggagag gattataaag atgatgatga taaataacaa ttctaccggg taggggaggc
     3421 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     3481 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     3541 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     3601 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     3661 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     3721 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
     3781 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
     3841 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
     3901 cctttcgacc tgcagcccaa gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     3961 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     4021 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     4081 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     4141 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     4201 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     4261 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     4321 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     4381 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     4441 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     4501 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa
     4561 aggagcgcac gaccccatgc atcggcgtct cgagatatca gtggtccagg ctctagtttt
     4621 gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat
     4681 tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct
     4741 agcttaagta acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt
     4801 cagatcaagg tcaggaacag atggaacagg gtcgacccta gagaaccatc agatgtttcc
     4861 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc
     4921 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct
     4981 cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa
     5041 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag
     5101 tgattgacta cccgtcagcg ggggtctttc atttgggggc tcgtccggga tcgggagacc
     5161 cctgcccagg gaccaccgac ccaccaccgg gaggtaagct ggctgcctcg cgcgtttcgg
     5221 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5281 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5341 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tagatccggc tgtggaatgt
     5401 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5461 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     5521 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5581 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5641 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5701 cttttttgga ggcctaggct tttgcaaaaa gcttactggc ttaactatgc ggcatcagag
     5761 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     5821 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5881 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5941 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6001 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6061 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6121 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6181 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6241 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6301 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6361 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6421 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6481 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6541 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6601 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6661 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6721 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6781 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6841 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6901 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6961 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7021 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7081 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7141 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7201 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7261 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7321 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7381 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7441 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7501 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7561 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7621 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7681 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7741 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat t
//
