LOCUS       sequence-204718-        7917 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 7917)
  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 7917)
  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(1-182)-2xFv-flag
FEATURES             Location/Qualifiers
     source          1..7917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..38
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             722..897
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    960..1159
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1360..1776
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1706..1728
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1744..1760
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1816..2165
                     /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    1824..1842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1860..1878
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1896..1914
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1932..1950
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1968..1986
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2004..2022
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2040..2058
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2104..2128
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2736..3056
                     /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             3063..3383
                     /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             3396..3419
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3456..3479
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3495..3518
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        3532..4031
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3567..3590)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3964..3983
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4052..4651
                     /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(4052..4071)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4548..4568
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4832..5257
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5360..5382)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5518..5538)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5523..5852
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5703..5838
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5874..5893
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5974..5991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6145..6733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6225..6244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6904..7695,7696..7764))
                     /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     7527..7546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7765..7869)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattagc
       61 ttggccattg catacgttgt atccatatca taatatgtac atttatattg gctcatgtcc
      121 aacattaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      661 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tcaataaaag agcccacaac ccctcactcg gcgcgccagt cttccgatag actgcgtcgc
      781 ccgggtaccc gtattcccaa taaagcctct tgctgtttgc atccgaatcg tggtctcgct
      841 gttccttggg agggtctcct ctgagtgatt gactacccac gacgggggtc tttcatttgg
      901 gggctcgtcc gggatttgga gacccctgcc cagggaccac cgacccacca ccgggaggta
      961 agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgtt tgatgttatg
     1021 cgcctgcgtc tgtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact
     1081 gacgagttct gaacacccgg ccgcaaccct gggagacgtc ccagggactt tgggggccgt
     1141 ttttgtggcc cgacctgagg aagggagtcg atgtggaatc cgaccccgtc aggatatgtg
     1201 gttctggtag gagacgagaa cctaaaacag ttcccgcctc cgtctgaatt tttgctttcg
     1261 gtttggaacc gaagccgcgc gtcttgtctg ctgcagcgct gcagcatcgt tctgtgttgt
     1321 ctctgtctga ctgtgtttct gtatttgtct gaaaattagg gccagactgt taccactccc
     1381 ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa ccagtcggta
     1441 gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac ctttaacgtc
     1501 ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa gatcaaggtc
     1561 ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac ctgggaagcc
     1621 ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc tccgcctcct
     1681 cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc gcctcgatcc
     1741 tccctttatc cagccctcac tccttctcta ggcgccggaa ttgaagatct cgatgaggcc
     1801 ctttcgtctt cactcgagtt tactccctat cagtgataga gaacgtatga agagtttact
     1861 ccctatcagt gatagagaac gtatgcagac tttactccct atcagtgata gagaacgtat
     1921 aaggagttta ctccctatca gtgatagaga acgtatgacc agtttactcc ctatcagtga
     1981 tagagaacgt atctacagtt tactccctat cagtgataga gaacgtatat ccagtttact
     2041 ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggcg cctataaaag
     2101 cagagctcgt ttagtgaacc gtcagatcgc ctggagcaat tccacaacac ttttgtctta
     2161 tacttggatc catcgatacc atggaaaatt tgaagcatat tatcaccctt ggccaggtca
     2221 tccacaaacg gtgtgaagag atgaaatact gcaagaaaca gtgccggcgc ctgggccacc
     2281 gcgtcctcgg cctgatcaag cctctggaga tgctccagga ccaaggaaag aggagcgtgc
     2341 cctctgagaa gttaaccaca gccatgaacc gcttcaaggc tgccctggag gaggctaatg
     2401 gggagataga aaagttcagc aatagatcca atatctgcag gtttctaaca gcaagccagg
     2461 acaaaatact cttcaaggac gtgaacagga agctgagtga tgtctggaag gagctctcgc
     2521 tgttacttca ggttgagcaa cgcatgcctg tttcacccat aagccaagga gcgtcctggg
     2581 cacaggaaga tcagcaggat gcagacgaag acaggcgagc tttccagatg ctaagaagag
     2641 ataatgaaaa aatagaagct tcactgagac gattagaaat caacatgaaa gaaatcaagg
     2701 aaactttgag gcagtattta ccaccagcgg ccgcaggcgt ccaagtcgaa accattagtc
     2761 ccggcgatgg cagaacattt cctaaaaggg gacaaacatg tgtcgtccat tatacaggca
     2821 tgttggagga cggcaaaaag gtggacagta gtagagatcg caataaacct ttcaaattca
     2881 tgttgggaaa acaagaagtc attaggggat gggaggaggg cgtggctcaa atgtccgtcg
     2941 gccaacgcgc taagctcacc atcagccccg actacgcata cggcgctacc ggacatcccg
     3001 gaattattcc ccctcacgct accttggtgt ttgacgtcga actgttgaag ctcgagacta
     3061 gaggagtgca ggtggagact atctccccag gagacgggcg caccttcccc aagcgcggcc
     3121 agacctgcgt ggtgcactac accgggatgc ttgaagatgg aaagaaagtt gattcctccc
     3181 gggacagaaa caagcccttt aagtttatgc taggcaagca ggaggtgatc cgaggctggg
     3241 aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt
     3301 atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact ctcgtcttcg
     3361 atgtggagct tctaaaactg gaagcggccg ccagctggag ccaccctcag ttcgagaagg
     3421 gaggaggaag cggcggaggc agcggaggag gaagctggag ccacccgcag ttcgagaaag
     3481 gagctagcgg agaggattat aaagatgatg atgataaata acaattctac cgggtagggg
     3541 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     3601 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     3661 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3721 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     3781 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     3841 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     3901 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     3961 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     4021 cgggcctttc gacctgcagc ccaagcttac catgaccgag tacaagccca cggtgcgcct
     4081 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     4141 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     4201 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     4261 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     4321 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     4381 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     4441 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     4501 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     4561 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     4621 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     4681 cgaaaggagc gcacgacccc atgcatcggc gtctcgagat atcagtggtc caggctctag
     4741 ttttgactca acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa
     4801 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     4861 agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga
     4921 agttcagatc aaggtcagga acagatggaa cagggtcgac cctagagaac catcagatgt
     4981 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
     5041 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
     5101 ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc gtgtatccaa
     5161 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     5221 tgagtgattg actacccgtc agcgggggtc tttcatttgg gggctcgtcc gggatcggga
     5281 gacccctgcc cagggaccac cgacccacca ccgggaggta agctggctgc ctcgcgcgtt
     5341 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5401 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5461 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtagatc cggctgtgga
     5521 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5581 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     5641 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     5701 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5761 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5821 gaggcttttt tggaggccta ggcttttgca aaaagcttac tggcttaact atgcggcatc
     5881 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     5941 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6001 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6061 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6121 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6181 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6241 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6301 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6361 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6421 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6481 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6541 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     6601 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6661 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6721 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6781 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6841 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6901 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6961 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7021 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7081 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7141 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7201 caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7261 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7321 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7381 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7441 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7501 ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt
     7561 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7621 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7681 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7741 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7801 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7861 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattat
//
