LOCUS       sequence-343560-        7322 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  labels mitochondria with TagRFP-T.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7322)
  AUTHORS   Kidwell CU, Casalini JR, Pradeep S, Scherer SD, Greiner D, Bayik D, 
            Watson DC, Olson GS, Lathia JD, Johnson JS, Rutter J, Welm AL, 
            Zangle TA, Roh-Johnson M
  TITLE     Transferred mitochondria accumulate reactive oxygen species, 
            promoting proliferation.
  JOURNAL   eLife. 2023 Mar 6;12:e85494. doi: 10.7554/eLife.85494.
  PUBMED    36876914
REFERENCE   2  (bases 1 to 7322)
  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"; journalName: "eLife."; 
            date: "2023-03-6"; volume: "12:e85494. doi"; pages: " 
            10.7554/eLife.85494"
COMMENT     Sequence Label: pLKO.1 mito-TagRFP-T
FEATURES             Location/Qualifiers
     source          1..7322
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             535..609
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      637..646
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             643..1374
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTETLYPADGGLEGRTD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     LTR             1528..1761
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1833..1954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1870..1889)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1924..1943
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1994..2129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2149..2168)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2150..2168)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2178..2195)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2178..2194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2187..2209)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2336..2791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2423..2442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2633..2654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2817..2921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2922..2990,2991..3782)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3140..3159)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3953..4541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4442..4461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4695..4712
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4829..4850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4865..4870,4871..4888,4889..4895)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4903..4919
                     /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     4908..4930
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4927..4943
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4927..4943
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4962..4982
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4964..4982
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5010..5236
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5237..5417
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5464..5589
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6082..6315
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6500..6544
                     /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"
     promoter        6842..7090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6842..6862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7013..7032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7153..7270
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7319..7322,1..507)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatccgacgt caccggagct taccatgtcc
      541 gtcctgacgc cgctgctgct gcggggcttg acaggctcgg cccggcggct cccagtgccg
      601 cgcgccaaga tccattcgtt gggggatcca ccggtcgcca ccatggtgtc taagggcgaa
      661 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac
      721 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
      781 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc
      841 atgtacggca gcagaacctt catcaaccac acccagggca tccccgattt ctttaagcag
      901 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
      961 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     1021 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1081 aacaccgaga ccctgtaccc cgctgacggc ggcctggaag gcagaaccga catggccctg
     1141 aagctcgtgg gcgggggcca cctgatctgc aacttcaaga ccacatacag atccaagaaa
     1201 cccgctaaga acctcaagat gcccggcgtc tactatgtgg accacagact ggaaagaatc
     1261 aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc
     1321 gacctcccta gcaaactggg gcacaaactt aatggcatgg acgagctgta caagtaacgt
     1381 acggatatct gtacaagtaa cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     1441 cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct gtagatctta
     1501 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     1561 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     1621 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     1681 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     1741 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
     1801 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     1861 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1921 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     1981 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2041 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     2101 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     2161 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2221 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2281 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     2341 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2401 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2461 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2521 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2581 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2641 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2701 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2761 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     2821 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2881 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     2941 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3001 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3061 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3121 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3181 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3241 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3301 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3361 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3421 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3481 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3541 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3601 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3661 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3721 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3781 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3841 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3901 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3961 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4021 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4081 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4141 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4201 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4261 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4321 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4381 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4441 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4501 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4561 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4621 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4681 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     4741 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     4801 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     4861 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     4921 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     4981 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
     5041 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
     5101 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
     5161 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
     5221 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     5281 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     5341 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     5401 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     5461 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     5521 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     5581 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     5641 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     5701 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     5761 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     5821 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     5881 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     5941 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     6001 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     6061 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     6121 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     6181 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     6241 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     6301 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     6361 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     6421 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     6481 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     6541 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     6601 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     6661 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     6721 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     6781 agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg cccccttcac
     6841 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     6901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     6961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     7021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     7081 acgaaacacc ggtccgcagg tatgcacgcg tgaattctcg acctcgagac aaatggcagt
     7141 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     7201 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     7261 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg
     7321 gg
//
