LOCUS       sequence-420923-        7068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tornado split nLuc with a mutated 3' ribozyme so that 
            circularization cannot occur..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7068)
  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 7068)
  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: "Cell Chem 
            Biol. 2023 Oct 13:S2451-9456(23)00334-3. doi: 
            10.1016/j.chembiol.2023.09.015."
COMMENT     Sequence Label: pcDNA3.1+-mutTornado-split-nLuc
FEATURES             Location/Qualifiers
     source          1..7068
                     /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
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     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"
     CDS             1126..1155
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2046..2069
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2621..2639)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2622..2639)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2659..2676)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2665..2889
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2935..3363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3022..3041)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3232..3253
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3372..3392)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3377..3706
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3557..3692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3619..3638
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3773..4567
                     /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(3827..3846)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4437..4456
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4741..4862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4778..4797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4832..4851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4911..4927)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4911..4927)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4924..4946)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4935..4951
                     /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(join(4959..4965,4966..4983,4984..4989))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5004..5025
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5142..5159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5313..5901)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5393..5412)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6072..6863,6864..6932))
                     /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     6695..6714
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6933..7037)
                     /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 tagtgaccgg ctaccggctg ttcgaggaga ttctggaaca aaaactcatc
     1141 tcagaagagg atctggaaga gtgagaattc aatcatctac aaggtgagcc caataatacg
     1201 gtttgggtta ggataggaag tagagccgta aactctctaa gcggtagaaa acaaaaaaca
     1261 aaaaaaacaa aaaaaaacca aaaaaacaaa acacattaaa acagcctgtg ggttgatccc
     1321 acccacaggc ccattgggcg ctagcactct ggtatcacgg tacctttgtg cgcctgtttt
     1381 ataccccctc ccccaactgt aacttagaag taacacacac cgatcaacag tcagcgtggc
     1441 acaccagcca cgttttgatc aagcacttct gttaccccgg actgagtatc aatagactgc
     1501 tcacgcggtt gaaggagaaa gcgttcgtta tccggccaac tacttcgaaa aacctagtaa
     1561 caccgtggaa gttgcagagt gtttcgctca gcactacccc agtgtagatc aggtcgatga
     1621 gtcaccgcat tccccacggg cgaccgtggc ggtggctgcg ttggcggtct gcccatgggg
     1681 aaacccatgg gacgctctaa tacagacatg gtgcgaagag tctattgagc tagttggtag
     1741 tcctccggcc cctgaatgcg gctaatccta actgcggagc acacaccctc aagccagagg
     1801 gcagtgtgtc gtaacgggca actctgcagc ggaaccgact actttgggtg tccgtgtttc
     1861 attttattcc tatactggct gcttatggtg acaattgaga gatcgttacc atatagctat
     1921 tggattggcc atccggtgac taatagagct attatatatc cctttgttgg gtttatacca
     1981 cttagcttga aagaggttaa aacattacaa ttcattgtta agttgaatac agcaaaatgc
     2041 gtacggacta caaggacgac gatgacaagg tcttcacact cgaagatttc gttggggact
     2101 gggaacagac agccgcctac aacctggacc aagtccttga acagggaggt gtgtccagtt
     2161 tgctgcagaa tctcgccgtg tccgtaactc cgatccaaag gattgtccgg agcggtgaaa
     2221 atgccctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc gccgaccaaa
     2281 tggcccagat cgaagaggtg tttaaggtgg tgtaccctgt ggatgatcat cactttaagg
     2341 tgatcctgcc ctatggcaca ctggtaatcg acggggttac gccgaacatg ctgaactatt
     2401 tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga
     2461 ccctgtggaa cggcaacaaa attatcgacg agcgcctgat cacccccgac ggctccatgc
     2521 tgttccgagt aaccatcaac agctctagag ccatgtctat gtggccgcgg tcggcgtgga
     2581 ctgtagcggt ccgctcgagc atgcatctag agggccctat tctatagtgt cacctaaatg
     2641 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2701 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2761 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2821 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2881 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2941 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3001 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3061 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3121 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3181 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3241 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3301 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3361 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3421 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3481 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3541 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3601 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3661 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3721 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3781 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3841 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3901 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3961 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4021 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4081 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4141 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4201 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4261 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4321 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4381 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4441 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4501 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4561 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4621 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4681 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4741 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4801 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4861 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4921 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4981 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5041 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5101 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5161 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5221 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5281 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5341 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5401 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5461 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5521 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5581 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5641 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5701 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5761 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5821 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5881 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5941 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6001 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6061 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6121 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6181 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6241 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6301 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6361 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6421 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6481 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6541 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6601 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6661 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6721 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6781 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6841 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6901 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6961 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7021 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
