LOCUS       sequence-271501-        5458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NanoLuciferase targeted to the mitochondira in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5458)
  AUTHORS   Parag-Sharma K, O'Banion CP, Henry EC, Musicant AM, Cleveland JL, 
            Lawrence DS, Amelio AL
  TITLE     Engineered BRET-Based Biologic Light Sources Enable Spatio-Temporal 
            Control Over Diverse Optogenetic Systems.
  JOURNAL   ACS Synth Biol. 2019 Dec 13. doi: 10.1021/acssynbio.9b00277.
  PUBMED    31834783
REFERENCE   2  (bases 1 to 5458)
  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: "ACS Synth 
            Biol. 2019 Dec 13. doi: 10.1021/acssynbio.9b00277."
COMMENT     Sequence Label: pEF1a-T2-NLuc
FEATURES             Location/Qualifiers
     source          1..5458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          231..1173
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1130..1150
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1228..1237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1447..1959
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    2082..2203
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2119..2138)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2173..2192
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2210..2665)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2347..2368)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2559..2578
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2692..2796
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2798..3155
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2798..2989
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2821..2841)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3006..3141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3190..3984
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3190..3984
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3244..3263)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3854..3873
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4172..4191)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4216..4263
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4592..5180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5081..5100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5458,1..1182)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
ORIGIN
        1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
       61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      421 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
      961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgctagcg ctaccggact
     1201 cagatctcga gctcaagctt cgaattcgcc accatggtgg gccggaacag cgctattgcc
     1261 gctggagtgt gcggcgccct gttcatcggc tactgcatct acttcgacag gaagagaagg
     1321 agcgacccca acttcaagaa caggctgagg gagagaagga agaagcagaa gctggccaag
     1381 gagagagccg gcctgagcaa gctgcccgac ctgaaggacg ccgaggccgt gcagaagttc
     1441 ggtaccgtct tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac
     1501 ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc
     1561 gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat
     1621 gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt
     1681 aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg
     1741 gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc
     1801 gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt
     1861 atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga
     1921 gtgaccggct ggcggctgtg cgaacgcatt ctggcgtaag gatcccgcga ctctagatca
     1981 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2041 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2101 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2161 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     2221 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2281 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2341 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2401 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     2461 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     2521 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     2581 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     2641 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     2701 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2761 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     2821 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2881 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2941 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3001 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3061 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3121 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     3181 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3241 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3301 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3361 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3421 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3481 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3541 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3601 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3661 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     3721 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3781 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     3841 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     3901 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     3961 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4021 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4081 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4141 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     4201 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     4261 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     4321 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     4381 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     4441 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4501 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4561 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4621 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4681 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     4741 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4801 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4861 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4921 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4981 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5041 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5101 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5161 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5221 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5281 ttaccgccat gcattagtta ttaatgagta attcatacaa aaggactcgc ccctgccttg
     5341 gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga tcgctgcgtt
     5401 cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca tgcgtgag
//
