LOCUS       sequence-256279-        7222 bp    DNA     circular SYN 15-MAY-2025
DEFINITION  lentiviral expression of mitochondrial mKeima for generation of 
            stable cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7222)
  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 7222)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 15, 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
FEATURES             Location/Qualifiers
     source          1..7222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    71..188
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        240..543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     698..718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     744..768
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             828..902
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(945..947,948..950,951..1613)
                     /codon_start=1
                     /product="humanized monomeric Keima-Red fluorescent 
                     protein"
                     /label=hmKeima-Red
                     /note="mammalian codon-optimized"
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEIAIARHSLLG
                     "
     misc_feature    1621..2209
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1674..1694)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2092..2103)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2284..2517
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(2636..2654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2722..2826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2827..2895,2896..3687)
                     /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(3045..3064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3858..4446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4347..4366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4600..4617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4857..4878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4893..4898,4899..4916,4917..4923)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4931..4947
                     /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     4936..4958
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4955..4971
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4955..4971
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5021..5040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5232..5865
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5912..6037
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6534..6767
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6952..6996
                     /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"
ORIGIN
        1 ccattcgatt agtgaacgga tctcgacggt atcgccgaat taattcacaa atggcagtat
       61 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
      121 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
      181 aaaattttcg ggtttattac agggacagca gagatccagt ttggactagt ggagttccgc
      241 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      301 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      361 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      421 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      481 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      541 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      601 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      661 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      721 cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc
      781 catccacgct gttttgacct ccatagaaga caccggcggc cgccaccatg tccgtcctga
      841 cgccgctgct gctgcggggc ttgacaggct cggcccggcg gctcccagtg ccgcgcgcca
      901 agatccattc gttgccgccg gaggggaagc tcgggggatc cgccatggtg agcgtgatcg
      961 ccaagcagat gacctacaag gtgtacatga gcggcaccgt gaacggccac tacttcgagg
     1021 tggagggcga cggcaagggc aagccctacg agggcgagca gaccgtgaag ctgaccgtga
     1081 ccaagggtgg ccccctgccc ttcgcctggg acatcctgag cccccagctc cagtacggca
     1141 gcatcccctt caccaagtac cccgaggaca tccccgacta cttcaagcag agcttccccg
     1201 agggctacac ctgggagcgc agcatgaact tcgaggacgg cgccgtgtgc accgtgagca
     1261 acgacagcag catccagggc aactgcttca tctacaacgt gaagatcagc ggcgagaact
     1321 tcccccccaa cggccccgtg atgcagaaga agacccaggg ctgggagccc agcaccgagc
     1381 gcctgttcgc ccgcgacgga atgctgatcg gcaacgacta catggccctg aagctggagg
     1441 gcggcggcca ctacctgtgc gagttcaaga gcacctacaa ggccaagaag cccgtgagga
     1501 tgcccggccg ccacgagatc gaccgcaagc tggacgtgac cagccacaac cgcgactaca
     1561 ccagcgtgga gcagtgcgag atcgccatcg cccgccactc cctgctgggc taaggatcct
     1621 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     1681 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     1741 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     1801 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     1861 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     1921 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     1981 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     2041 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     2101 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     2161 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tgagatcctt
     2221 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     2281 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     2341 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     2401 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     2461 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     2521 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     2581 gtgagaggcc cgggttaatt aaggaaaggg ctagatcatt cttgaagacg aaagggcctc
     2641 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2701 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2761 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2821 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2881 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2941 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3001 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3061 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3121 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3181 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3241 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3301 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3361 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3421 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3481 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3541 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3601 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3661 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3721 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3781 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3841 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3901 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3961 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4021 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4081 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4141 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4201 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4261 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4321 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4381 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4441 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4501 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4561 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4621 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4681 agcaagctca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4741 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4801 tccccgtggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4861 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     4921 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga catgattacg
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt atcatgtctg gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa
     5101 cttcccaccc cataccctat taccactgcc aattacctgt ggtttcattt actctaaacc
     5161 tgtgattcct ctgaattatt ttcattttaa agaaattgta tttgttaaat atgtactaca
     5221 aacttagtag ttggaagggc taattcactc ccaaagaaga caagatatcc ttgatctgtg
     5281 gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt
     5341 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt
     5401 agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat
     5461 ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca
     5521 cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa
     5581 gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg
     5641 agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag
     5701 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5761 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5821 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     5881 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     5941 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     6001 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     6061 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     6121 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     6181 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     6241 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     6301 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     6361 caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     6421 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     6481 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     6541 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     6601 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     6661 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     6721 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     6781 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     6841 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     6901 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     6961 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     7021 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     7081 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     7141 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     7201 ggagagagag acagagacag at
//
