LOCUS       sequence-271500-        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the CeNLuc LumiFluor targeted to the nucleus in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6028)
  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 6028)
  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-2xSV40_NLS-CeNLuc
FEATURES             Location/Qualifiers
     source          1..6028
                     /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"
     CDS             1234..1254
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1258..1278
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1285..1287,1288..1998)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIKA
                     NFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1327..1348)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1588..1607)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1935..1956
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2014..2529
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     polyA_signal    2652..2773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2689..2708)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2743..2762
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2780..3235)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2917..2938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3129..3148
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3262..3366
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3368..3725
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3368..3559
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3391..3411)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3576..3711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3760..4554
                     /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             3760..4554
                     /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(3814..3833)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4424..4443
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4742..4761)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4786..4833
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5162..5750
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5651..5670
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(6028,1..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 cgaattcatg gatccaaaaa agaagagaaa ggtagatcca
     1261 aaaaagaaga gaaaggtagg taccgtgagc aagggcgagg agctgttcac cggggtggtg
     1321 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1381 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1441 ctgcccgtgc cctggcccac cctcgtgacc accctgagct ggggcgtgca gtgcttcgcc
     1501 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1561 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1621 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1681 gacggcaaca tcctggggca caagctggag tacaacgcca tccacggcaa cgtctatatc
     1741 accgccgaca agcagaagaa cggcatcaag gccaacttcg gcctcaactg caacatcgag
     1801 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1861 gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac
     1921 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1981 atggacgagc tgtacaagga tatctccgga ggtatggtct tcacactcga agatttcgtt
     2041 ggggactggc gacagacagc cggctacaac ctggaccaag tccttgaaca gggaggtgtg
     2101 tccagtttgt ttcagaatct cggggtgtcc gtaactccga tccaaaggat tgtcctgagc
     2161 ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc cgtatgaagg tctgagcggc
     2221 gaccaaatgg gccagatcga aaaaattttt aaggtggtgt accctgtgga tgatcatcac
     2281 tttaaggtga tcctgcacta tggcacactg gtaatcgacg gggttacgcc gaacatgatc
     2341 gactatttcg gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta
     2401 acagggaccc tgtggaacgg caacaaaatt atcgacgagc gcctgatcaa ccccgacggc
     2461 tccctgctgt tccgagtaac catcaacgga gtgaccggct ggcggctgtg cgaacgcatt
     2521 ctggcgtaag gatcccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     2581 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2641 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2701 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2761 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2821 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2881 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     2941 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3001 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3061 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3121 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3181 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3241 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3301 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3361 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3421 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3481 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3541 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3601 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3661 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3721 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3781 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3841 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3901 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     3961 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4021 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4081 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4141 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4201 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4261 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4321 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4381 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4441 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4501 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4561 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4621 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4681 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4741 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4801 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     4861 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     4921 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     4981 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5041 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5101 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5161 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5221 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5281 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5341 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5401 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5461 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5521 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5581 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5641 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5701 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5761 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5821 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatgagta
     5881 attcatacaa aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc
     5941 actaacgtag aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat
     6001 cactgaggtg gagaagagca tgcgtgag
//
