LOCUS       sequence-472911-        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"
     LTR             77..710
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(902..921)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(971..987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(971..987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(984..1006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    995..1011
                     /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(1019..1025,1026..1043,1044..1049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1064..1085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1325..1342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1496..2084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1576..1595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2255..3046,3047..3115))
                     /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     2878..2897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3116..3220)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3288..3306
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     LTR             3425..3658
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3733..4321
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3839..3850
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4248..4268
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(4329..4991,4992..4994,4995..4997))
                     /codon_start=1
                     /product="humanized monomeric Keima-Red fluorescent 
                     protein"
                     /label=hmKeima-Red
                     /note="mammalian codon-optimized"
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEIAIARHSLLG
                     "
     CDS             complement(5040..5114)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     primer_bind     complement(5174..5198)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(5195..5398)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5224..5244)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5399..5702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    5754..5871
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(6168..6212)
                     /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    6397..6630
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    join(7127..7222,1..30)
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 ttcagcaagc cgagtcctgc gtcgagagag ctcctctggt ttccctttcg ctttcaagtc
       61 cctgttcggg cgccactgct agagattttc cacactgact aaaagggtct gagggatctc
      121 tagttaccag agtcacacaa cagacgggca cacactactt gaagcactca aggcaagctt
      181 tattgaggct taagcagtgg gttccctagt tagccagaga gctcccaggc tcagatctgg
      241 tctaaccaga gagacccagt acaggcaaaa agcagctgct tatatgcagg atctgagggc
      301 tcgccactcc ccagtcccgc ccaggccacg cctccctgga aagtccccag cggaaagtcc
      361 cttgtagcaa gctcgatatc agcagttctt gaagtactcc ggatgcagct ctcgggccac
      421 gtgatgaaat gctaggcggc tgtcaaacct ccactctaac acttctctct ccgggtcatc
      481 catcccatgc aggctcacag ggtgtaacaa gctggtgttc tctcctttat tggcctcttc
      541 taccttatct ggctcaactg gtactagctt gtagcaccat ccaaaggtca gtggatatct
      601 gacccctggc cctggtgtgt agttctgcta atcagggaag tagccttgtg tgtggtagat
      661 ccacagatca aggatatctt gtcttctttg ggagtgaatt agcccttcca actactaagt
      721 ttgtagtaca tatttaacaa atacaatttc tttaaaatga aaataattca gaggaatcac
      781 aggtttagag taaatgaaac cacaggtaat tggcagtggt aatagggtat ggggtgggaa
      841 gtttgggatg attttggtta gcttgagtta tccagttgat ccagacatga taagatacat
      901 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
      961 tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1021 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1081 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccacgggg
     1141 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     1201 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atgagcttgc
     1261 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1321 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1381 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1441 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1501 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1561 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1621 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1681 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1741 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1801 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1861 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1921 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1981 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2041 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2101 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2161 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2221 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2281 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2341 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2401 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2461 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2521 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2581 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2641 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2701 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2761 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2821 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata
     2881 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2941 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3001 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3061 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3121 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3181 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3241 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3301 cgaggccctt tcgtcttcaa gaatgatcta gccctttcct taattaaccc gggcctctca
     3361 ctctctgata ttcatttctt tgcaagttat aaatactgaa taataagatg acatgaacta
     3421 ctactgctag agattttcca cactgactaa aagggtctga gggatctcta gttaccagag
     3481 tcacacaaca gacgggcaca cactacttga agcactcaag gcaagcttta ttgaggctta
     3541 agcagtgggt tccctagtta gccagagagc tcccaggctc agatctggtc taaccagaga
     3601 gacccagtac aagcaaaaag cagatcttgt cttcgttggg agtgaattag cccttccagt
     3661 cccccctttt cttttaaaaa gtggctaaga tctacagctg ccttgtaagt cattggtctt
     3721 aaaggatctc aggcggggag gcggcccaaa gggagatccg actcgtctga gggcgaaggc
     3781 gaagacgcgg aagaggccgc agagccggca gcaggccgcg ggaaggaagg tccgctggat
     3841 tgagggccga agggacgtag cagaaggacg tcccgcgcag aatccaggtg gcaacacagg
     3901 cgagcagcca aggaaaggac gatgatttcc ccgacaacac cacggaattg tcagtgccca
     3961 acagccgagc ccctgtccag cagcgggcaa ggcaggcggc gatgagttcc gccgtggcaa
     4021 tagggagggg gaaagcgaaa gtcccggaaa ggagctgaca ggtggtggca atgccccaac
     4081 cagtgggggt tgcgtcagca aacacagtgc acaccacgcc acgttgcctg acaacgggcc
     4141 acaactcctc ataaagagac agcaaccagg atttatacaa ggaggagaaa atgaaagcca
     4201 tacgggaagc aatagcatga tacaaaggca ttaaagcagc gtatccacat agcgtaaaag
     4261 gagcaacata gttaagaata ccagtcaatc tttcacaaat tttgtaatcc agaggttgat
     4321 taggatcctt agcccagcag ggagtggcgg gcgatggcga tctcgcactg ctccacgctg
     4381 gtgtagtcgc ggttgtggct ggtcacgtcc agcttgcggt cgatctcgtg gcggccgggc
     4441 atcctcacgg gcttcttggc cttgtaggtg ctcttgaact cgcacaggta gtggccgccg
     4501 ccctccagct tcagggccat gtagtcgttg ccgatcagca ttccgtcgcg ggcgaacagg
     4561 cgctcggtgc tgggctccca gccctgggtc ttcttctgca tcacggggcc gttggggggg
     4621 aagttctcgc cgctgatctt cacgttgtag atgaagcagt tgccctggat gctgctgtcg
     4681 ttgctcacgg tgcacacggc gccgtcctcg aagttcatgc tgcgctccca ggtgtagccc
     4741 tcggggaagc tctgcttgaa gtagtcgggg atgtcctcgg ggtacttggt gaaggggatg
     4801 ctgccgtact ggagctgggg gctcaggatg tcccaggcga agggcagggg gccacccttg
     4861 gtcacggtca gcttcacggt ctgctcgccc tcgtagggct tgcccttgcc gtcgccctcc
     4921 acctcgaagt agtggccgtt cacggtgccg ctcatgtaca ccttgtaggt catctgcttg
     4981 gcgatcacgc tcaccatggc ggatcccccg agcttcccct ccggcggcaa cgaatggatc
     5041 ttggcgcgcg gcactgggag ccgccgggcc gagcctgtca agccccgcag cagcagcggc
     5101 gtcaggacgg acatggtggc ggccgccggt gtcttctatg gaggtcaaaa cagcgtggat
     5161 ggcgtctcca ggcgatctga cggttcacta aacgagctct gcttatatag acctcccacc
     5221 gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt
     5281 cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa
     5341 atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca
     5401 tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca
     5461 tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac
     5521 ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc
     5581 attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg
     5641 tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa
     5701 cgcggaactc cactagtcca aactggatct ctgctgtccc tgtaataaac ccgaaaattt
     5761 tgaatttttg taatttgttt ttgtaattct ttagtttgta tgtctgttgc tattatgtct
     5821 actattcttt cccctgcact gtacccccca atcccccctt ttcttttaaa attgtggatg
     5881 aatactgcca tttgtgaatt aattcggcga taccgtcgag atccgttcac taatcgaatg
     5941 gatctgtctc tgtctctctc tccaccttct tcttctattc cttcgggcct gtcgggtccc
     6001 ctcggggttg ggaggtgggt ctgaaacgat aatggtgaat atccctgcct aactctattc
     6061 actatagaaa gtacagcaaa aactattctt aaacctacca agcctcctac tatcattatg
     6121 aataatttta tataccacag ccaatttgtt atgttaaacc aattccacaa acttgcccat
     6181 ttatctaatt ccaataattc ttgttcattc ttttcttgct ggttttgcga ttcttcaatt
     6241 aaggagtgta ttaagcttgt gtaattgtta atttctctgt cccactccat ccaggtcgtg
     6301 tgattccaaa tctgttccag agatttatta ctccaactag cattccaagg cacagcagtg
     6361 gtgcaaatga gttttccaga gcaaccccaa atccccagga gctgttgatc ctttaggtat
     6421 ctttccacag ccaggattct tgcctggagc tgcttgatgc cccagactgt gagttgcaac
     6481 agatgctgtt gcgcctcaat agccctcagc aaattgttct gctgctgcac tataccagac
     6541 aataattgtc tggcctgtac cgtcagcgtc attgacgctg cgcccatagt gcttcctgct
     6601 gctcccaaga acccaaggaa caaagctcct attcccactg ctcttttttc tctctgcacc
     6661 actcttctct ttgccttggt gggtgctact cctaatggtt caatttttac tactttatat
     6721 ttatataatt cacttctcca attgtccctc atatctcctc ctccaggtct gaagatcagc
     6781 ggccggccgc ttgctgtgcg gtggtcttac ttttgttttg ctcttcctct atcttgtcta
     6841 aagcttcctt ggtgtctttt atctctatcc tttgatgcac acaatagagg gttgctactg
     6901 tattatataa tgatctaagt tcttctgatc ctgtctgaag ggatggttgt agctgtccca
     6961 gtatttgtct acagccttct gatgtttcta acaggccagg attaactgcg aatcgttcta
     7021 gctccctgct tgcccatact atatgtttta atttatattt tttctttccc cctggcctta
     7081 accgaatttt ttcccatcgc gatctaattc tcccccgctt aatactgacg ctctcgcacc
     7141 catctctctc cttctagcct ccgctagtca aaatttttgg cgtactcacc agtcgccgcc
     7201 cctcgcctct tgccgtgcgc gc
//
