LOCUS       sequence-270330-        7783 bp    DNA     circular SYN 14-APR-2025
DEFINITION  stably express both mKeima and FRB-Fis1 for CID induced mitophagy in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7783)
  AUTHORS   Vargas JNS, Wang C, Bunker E, Hao L, Maric D, Schiavo G, Randow F, 
            Youle RJ
  TITLE     Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during 
            Selective Autophagy.
  JOURNAL   Mol Cell. 2019 Apr 18;74(2):347-362.e6. doi: 
            10.1016/j.molcel.2019.02.010. Epub 2019 Mar 7.
  PUBMED    30853401
REFERENCE   2  (bases 1 to 7783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2019.02.010"; journalName: "Mol Cell"; date: 
            "2019-04-18- 18"; volume: "74"; issue: "2"; pages: "347-362.e6"
COMMENT     Sequence Label: pHAGE-mt-mKeima-P2A-FRB-Fis1
FEATURES             Location/Qualifiers
     source          1..7783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2062..2179
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2231..2534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2535..2738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2689..2709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2735..2759
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2819..2893
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(2936..2938,2939..2941,2942..3601)
                     /codon_start=1
                     /product="humanized monomeric Keima-Red fluorescent 
                     protein"
                     /label=hmKeima-Red
                     /note="mammalian codon-optimized"
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEIAIARHSLLG
                     "
     CDS             3617..3673
                     /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             3692..3970
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     misc_feature    4173..4761
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4226..4246)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4644..4655)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4836..5069
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5188..5206)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5274..5378
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5379..5447,5448..6239)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5597..5616)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6410..6998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6899..6918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7152..7169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7409..7430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7445..7450,7451..7468,7469..7475)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7483..7499
                     /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     7488..7510
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7507..7523
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7507..7523
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7573..7592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttaattcaca
     2041 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2101 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2161 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggactag
     2221 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     2281 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     2341 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2401 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2461 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2521 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     2581 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     2641 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     2701 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
     2761 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccggcgg ccgccaccat
     2821 gtccgtcctg acgccgctgc tgctgcgggg cttgacaggc tcggcccggc ggctcccagt
     2881 gccgcgcgcc aagatccatt cgttgccgcc ggaggggaag ctcgggggat ccgccatggt
     2941 gagcgtgatc gccaagcaga tgacctacaa ggtgtacatg agcggcaccg tgaacggcca
     3001 ctacttcgag gtggagggcg acggcaaggg caagccctac gagggcgagc agaccgtgaa
     3061 gctgaccgtg accaagggtg gccccctgcc cttcgcctgg gacatcctga gcccccagct
     3121 ccagtacggc agcatcccct tcaccaagta ccccgaggac atccccgact acttcaagca
     3181 gagcttcccc gagggctaca cctgggagcg cagcatgaac ttcgaggacg gcgccgtgtg
     3241 caccgtgagc aacgacagca gcatccaggg caactgcttc atctacaacg tgaagatcag
     3301 cggcgagaac ttccccccca acggccccgt gatgcagaag aagacccagg gctgggagcc
     3361 cagcaccgag cgcctgttcg cccgcgacgg aatgctgatc ggcaacgact acatggccct
     3421 gaagctggag ggcggcggcc actacctgtg cgagttcaag agcacctaca aggccaagaa
     3481 gcccgtgagg atgcccggcc gccacgagat cgaccgcaag ctggacgtga ccagccacaa
     3541 ccgcgactac accagcgtgg agcagtgcga gatcgccatc gcccgccact ccctgctggg
     3601 cgctagcgga agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     3661 gaaccctgga cctctcgaga tggcttctag aatcctctgg catgagatgt ggcatgaagg
     3721 cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt
     3781 gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt
     3841 taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa
     3901 atcagggaat gtcaaggacc tcctccaagc ctgggacctc tattatcatg tgttccgacg
     3961 aatctcaaag actagttacg tccgcgggtt gctgcagaca gagccccaga acaaccaggc
     4021 caaggaactg gagcggctca ttgacaaggc catgaagaaa gatggactcg tgggcatggc
     4081 catcgtggga ggcatggccc tgggtgtggc gggactggcc ggactcatcg gacttgctgt
     4141 gtccaagtcc aaatcctgaa cgcgtggatc ctaatcaacc tctggattac aaaatttgtg
     4201 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     4261 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     4321 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     4381 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     4441 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     4501 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     4561 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     4621 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     4681 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     4741 ccctttgggc cgcctccccg cctgagatcc tttaagacca atgacttaca aggcagctgt
     4801 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     4861 aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg
     4921 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     4981 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     5041 cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta
     5101 tttataactt gcaaagaaat gaatatcaga gagtgagagg cccgggttaa ttaaggaaag
     5161 ggctagatca ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5221 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5281 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5341 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5401 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5461 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5521 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5581 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     5641 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5701 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5761 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5821 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5881 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5941 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6001 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6061 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6121 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6181 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6241 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6301 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6361 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6421 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6481 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6541 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6601 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6661 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6721 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6781 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6841 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6901 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6961 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7021 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     7081 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     7141 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     7201 ctctccccgc gcgttggccg attcattaat gcagcaagct catggctgac taattttttt
     7261 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     7321 cttttttgga ggcctaggct tttgcaaaaa gctccccgtg gcacgacagg tttcccgact
     7381 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     7441 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     7501 ttcacacagg aaacagctat gacatgatta cgaatttcac aaataaagca tttttttcac
     7561 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact
     7621 ggataactca agctaaccaa aatcatccca aacttcccac cccataccct attaccactg
     7681 ccaattacct gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt
     7741 aaagaaattg tatttgttaa atatgtacta caaacttagt agt
//
