LOCUS       sequence-343559-        7307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  labels mitochondria with mEmerald.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7307)
  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 7307)
  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-mEmerald
FEATURES             Location/Qualifiers
     source          1..7307
                     /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             join(643..645,646..648,649..1362)
                     /codon_start=1
                     /product="brightly fluorescent monomeric variant of GFP"
                     /label=mEmerald
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(688..709)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(949..968)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1296..1317
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             1513..1746
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1818..1939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1855..1874)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1909..1928
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1979..2114
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2134..2153)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2135..2153)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2163..2180)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2163..2179)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2172..2194)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2321..2776
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2408..2427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2618..2639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2802..2906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2907..2975,2976..3767)
                     /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(3125..3144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3938..4526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4427..4446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4680..4697
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4814..4835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4850..4855,4856..4873,4874..4880)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4888..4904
                     /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     4893..4915
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4912..4928
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4912..4928
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4947..4967
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4949..4967
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4995..5221
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5222..5402
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5449..5574
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6067..6300
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6485..6529
                     /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        6827..7075
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6827..6847
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6998..7017
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7138..7255
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7304..7307,1..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 ccatggtgag caagggcgag
      661 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      721 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      781 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttgacc
      841 tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      901 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      961 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1021 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1081 aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc
     1141 aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1201 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1261 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1321 gccgccggga tcactctcgg catggacgag ctgtacaagt aacgtacgga tatctgtaca
     1381 agtaacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat
     1441 cggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     1501 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     1561 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     1621 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     1681 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     1741 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     1801 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     1861 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     1921 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     1981 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2041 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2101 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     2161 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2221 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2281 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     2341 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2401 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2461 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2521 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2581 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2641 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2701 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2761 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2821 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2881 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2941 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3001 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3061 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3121 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3181 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3241 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3301 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3361 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3421 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3481 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3541 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3601 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3661 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3721 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3781 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3841 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4021 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4081 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4561 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4621 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4681 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4741 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4801 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     4861 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     4921 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     4981 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     5041 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     5101 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     5161 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     5221 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     5281 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     5341 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     5401 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     5461 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     5521 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     5581 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     5641 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     5701 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     5761 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     5821 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     5881 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     5941 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     6001 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     6061 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     6121 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     6181 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     6241 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     6301 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     6361 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     6421 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     6481 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     6541 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     6601 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     6661 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     6721 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg
     6781 acggtatcga tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc
     6841 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     6901 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     6961 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     7021 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggtcc
     7081 gcaggtatgc acgcgtgaat tctcgacctc gagacaaatg gcagtattca tccacaattt
     7141 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     7201 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     7261 ttattacagg gacagcagag atccactttg gccgcggctc gaggggg
//
