LOCUS       Exported                7008 bp DNA     circular SYN 01-DEC-2025
DEFINITION  For expression of a circular nLuc mRNA. To express your gene of 
            interest clone into the BsiWI, SacII sites and add the 
            GCCATGTcTATGTGG 5' of the SacII site..
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1+-Tornado-nLuc
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  AUTHORS   Unti MJ, Jaffrey SR
  TITLE     Highly efficient cellular expression of circular mRNA enables 
            prolonged protein expression.
  JOURNAL   Cell Chem Biol. 2023 Oct 13:S2451-9456(23)00334-3. doi: 
            10.1016/j.chembiol.2023.09.015.
  PUBMED    37883972
REFERENCE   2  (bases 1 to 7008)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 1, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Chem 
            Biol. 2023 Oct 13:S2451-9456(23)00334-3. doi: 
            10.1016/j.chembiol.2023.09.015."
COMMENT     Sequence Label: pcDNA3.1+-Tornado-nLuc
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          1067..1081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             901..924
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             937..963
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1007..1060
                     /label=Ribozyme
     misc_feature    1061..1089
                     /label=StemForming
     misc_feature    1092..1890
                     /label=IRES
     CDS             1888..1890
                     /codon_start=1
                     /label=START
                     /translation="M"
     CDS             1897..2409
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     misc_RNA        2410..2428
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat
                     protein"
     misc_feature    2435..2471
                     /label=StemForming
     misc_feature    2472..2525
                     /label=Twister Ribozyme
     promoter        complement(2561..2579)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2562..2579)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2599..2616)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer.
                     Also called BGH reverse"
     polyA_signal    2605..2829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2875..3303
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2962..2981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3172..3193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3312..3332)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3317..3646
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3497..3632
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3559..3578
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3713..4507
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3767..3786)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4377..4396
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4681..4802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4718..4737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4772..4791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4851..4867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4851..4867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4864..4886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4875..4891
                     /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(4899..4929)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4944..4965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(5082..5099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5253..5841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5333..5352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6012..6872)
                     /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     6635..6654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6873..6977)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttaccatg
      901 gactacaagg acgacgatga caagctcgat ggaggatacc cctacgacgt gcccgactac
      961 gccagcggat ccagctaaac tcgatgataa tacgactcac tataggccgc actcgccggt
     1021 cccaagcccg gataaaatgg gagggggcgg gaaaccgcct aaccatgccg agtgcggccg
     1081 catatatgga taaacaaaaa acaaaaaaaa acaaaaaaaa aaccaaaaaa acaaaacaca
     1141 ttaaaagaat tccagcctgt gggttgatcc cacccacagg cccattgggc gctagcactc
     1201 tggtatcacg gtacctttgt gcgcctgttt tataccccct cccccaactg taacttagaa
     1261 gtaacacaca ccgatcaaca gtcagcgtgg cacaccagcc acgttttgat caagcacttc
     1321 tgttaccccg gactgagtat caatagactg ctcacgcggt tgaaggagaa agcgttcgtt
     1381 atccggccaa ctacttcgaa aaacctagta acaccgtgga agttgcagag tgtttcgctc
     1441 agcactaccc cagtgtagat caggtcgatg agtcaccgca ttccccacgg gcgaccgtgg
     1501 cggtggctgc gttggcggcc tgcccatggg gaaacccatg ggacgctcta atacagacat
     1561 ggtgcgaaga gtctattgag ctagttggta gtcctccggc ccctgaatgc ggctaatcct
     1621 aactgcggag cacacaccct caagccagag ggcagtgtgt cgtaacgggc aactctgcag
     1681 cggaaccgac tactttgggt gtccgtgttt cattttattc ctatactggc tgcttatggt
     1741 gacaattgag agatcgttac catatagcta ttggattggc catccggtga ctaatagagc
     1801 tattatatat ccctttgttg ggtttatacc acttagcttg aaagaggtta aaacattaca
     1861 attcattgtt aagttgaata cagcaaaatg cgtacggtct tcacactcga agatttcgtt
     1921 ggggactggc gacagacagc cggctacaac ctggaccaag tccttgaaca gggaggtgtg
     1981 tccagtttgt ttcagaatct cggggtgtcc gtaactccga tccaaaggat tgtcctgagc
     2041 ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc cgtatgaagg tctgagcggc
     2101 gaccaaatgg gccagatcga aaaaattttt aaggtggtgt accctgtgga tgatcatcac
     2161 tttaaggtga tcctgcacta tggcacactg gtaatcgacg gggttacgcc gaacatgatc
     2221 gactatttcg gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta
     2281 acagggaccc tgtggaacgg caacaaaatt atcgacgagc gcctgatcaa ccccgacggc
     2341 tccctgctgt tccgagtaac catcaacgga gtgaccggct ggcggctgtg cgaacgcatt
     2401 ctggcgtaaa catgaggatc acccatgtgg atccgccatg tctatgtggc cgcggtcggc
     2461 gtggactgta gaacactgcc aatgccggtc ccaagcccgg ataaaagtgg agggtacagt
     2521 ccacgccggt ccgctcgagc atgcatctag agggccctat tctatagtgt cacctaaatg
     2581 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2641 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2701 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2761 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2821 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2881 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2941 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3001 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3061 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3121 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3181 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3241 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3301 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3361 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3421 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3481 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3541 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3601 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3661 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3721 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3781 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3841 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3901 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3961 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4021 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4081 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4141 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4201 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4261 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4321 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4381 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4441 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4501 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4561 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4621 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4681 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4741 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4801 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4861 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4921 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4981 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5041 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5101 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5161 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5221 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5281 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5341 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5401 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5461 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5521 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5581 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5641 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5701 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5761 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5821 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5881 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5941 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6001 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6061 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6121 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6181 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6241 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6301 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6361 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6421 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6481 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6541 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6601 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6661 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6721 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6781 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6841 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6901 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6961 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
