LOCUS       sequence-429011-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid to transcribe circular RNA encoding NanoLuc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  AUTHORS   Chen R, Wang SK, Belk JA, Amaya L, Li Z, Cardenas A, Abe BT, Chen 
            CK, Wender PA, Chang HY
  TITLE     Engineering circular RNA for enhanced protein production.
  JOURNAL   Nat Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi: 
            10.1038/s41587-022-01393-0.
  PUBMED    35851375
REFERENCE   2  (bases 1 to 7106)
  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: "Nat 
            Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi: 
            10.1038/s41587-022-01393-0."
COMMENT     Sequence Label: circRNA-synIRES-R25-NanoLuc
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        125..401
                     /label=chicken beta-actin promoter
     intron          402..1418
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1342..1364
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1426..1445
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1488..1507
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1488..1506
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2572..2637
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2653..3165
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     terminator      3562..3609
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3623..3847
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3890..3906
                     /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     3895..3917
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3914..3930
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3914..3930
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3979..4100
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3990..4009)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4044..4063
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4260..4859)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4343..4363)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4840..4859
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4870..5198)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4884..5019
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4938..4957)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             complement(5232..6047)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5232..6047)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5955..5974
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6039..6058
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6272..6860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6761..6780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(6926..7106,1..123)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
       61 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      121 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
      181 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg
      241 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
      301 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      361 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      421 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      481 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      541 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      601 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      661 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      721 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      781 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
      841 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
      901 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
      961 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     1021 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     1081 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     1141 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     1201 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1261 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1321 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     1381 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1441 ttgtgctgtc tcatcatttt ggcaaagcgg ccgcggccag tgaattgtaa tacgactcac
     1501 tataggggtt aacaaaaaaa aaaaaaaaaa aaaaaaaatc accgaccgat ctatctgaac
     1561 cggtgggaat tctagagaaa atttcgtctg gattagttac ttatcgtgta aaatctgata
     1621 aatggaattg gttctacata aatgcctaac gactatccct ttggggagta gggtcaagtg
     1681 actcgaaacg atagacaact tgctttaaca agttggagat atagtctgct ctgcatggtg
     1741 acatgcagct ggatataatt ccggggtaag attaacgacc ttatctgaac ataacgctac
     1801 cgtttaatat tgcgtcatat tcggcgacca tttgtgtggt aaaaaaaaaa aaccaaaaaa
     1861 aaaaaacaaa aaaaaaaaat aattgactaa gatatcttaa aacagcggat gggtacccca
     1921 ccatccgacc cactgggtgt agtactctgg tacttcgtac ctttgtacgc ctgttcttcc
     1981 cattgtaccc ttcctgaact tccaacccaa gtaacgttag aagctcaaca tttagtacaa
     2041 caggaagcac cacatccagt ggtgtttagt acaagcactt ctgtttcccc ggagcgaggt
     2101 ataggctgta cccactgcca aaaaccttta accgttatcc gccaaccaac tacgtaaaag
     2161 ctagtagtat tatgttttta actaggcgtt cgatcaggtg gatttcccct ccactagttt
     2221 ggtcgatgag gctaggaatt ccccacgggt gaccgtgtcc tagcctgcgt ggcggccaac
     2281 ccagcccact cactatttgt tttcgcgccc agttgcaaaa agtgtcgggg ctgggacgcc
     2341 tttttataga catggtgtga agactcgcat gtgcttggtt gtgattcctc cggcccctga
     2401 atgcggctaa ccttaaccct ggagccttgt gtcacaaacc agtgatgata aggtcgtaat
     2461 gagcaattcc gggacgggac cgactacttt gggtgtccgt gtttcttatt tttcttatta
     2521 ttgtcttatg gtcacagcat atatataaca tatactgtga tcatgggatc cgactataag
     2581 gaccacgacg gagactacaa ggatcatgat attgattaca aggacgacga tgacaaggga
     2641 ggaggaggaa gcgtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc
     2701 tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg
     2761 gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac
     2821 atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa
     2881 atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc
     2941 acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa
     3001 ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctgtg gaacggcaac
     3061 aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc
     3121 aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgtagtaaca attggctgga
     3181 gcctcggtgg ccatgcttct tgccccttgg gcctcccccc agcccctcct ccccttcctg
     3241 cacccgtacc cccgtggtct ttgaataaag tctgaaccac acaaatggtc gccgactcag
     3301 tagatgtttt cttgggttaa ttgaggcctg agtataaggt gacttatact tgtaatctat
     3361 ctaaacgggg aacctctcta gtagacaatc ccgtgctaaa ttgtaggact gccctttaat
     3421 aaatacttct atatttaaag aggtatttat gaaaagcgga atttatcaga ttaaaaatac
     3481 tttctctaga gtcgacctgc agacgcgttc agatagatcg gtcggtgaaa aaaaaaaaaa
     3541 aaaaaaaaaa aaagcggccg cctagcataa ccccttgggg cctctaaacg ggtcttgagg
     3601 ggttttttgg ctagcggccg gcctgtgcct tctagttgcc agccatctgt tgtttgcccc
     3661 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3721 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3781 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3841 tctatggcgg cgcgccactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3901 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc
     3961 gacggtatac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     4021 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4081 taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg
     4141 aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg
     4201 gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaat
     4261 caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc cttcgggcac ctcgacgtcg
     4321 gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt tgcggggcgc ggaggtttcc
     4381 aggaaggcgg gcaccccggc gcgctcggcc gcctccactc cggggagcac gacggcgctg
     4441 cccagaccct tgccctggtg gtcgggcgac acgccgacgg tggccaggaa ccacgcgggc
     4501 tccttgggcc ggtgcggcgc caggaggcct tccatctgtt gctgcgcggc cagccgggaa
     4561 ccgctcaact cggccatgcg cgggccgatc tcggcgaaca ccgcccccgc ttcgacgctc
     4621 tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg cgacccacac cttgccgatg
     4681 tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg tgacccgctc gatgtggcgg
     4741 tccggatcga cggtgtggcg cgtggcgggg tagtcggcga acgcggcggc gagggtgcgt
     4801 acggccctgg ggacgtcgtc gcgggtggcg aggcgcaccg tgggcttgta ctcggtcatg
     4861 cgggagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag
     4921 ctcagaggca gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg
     4981 cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac
     5041 tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     5101 ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     5161 tggggagcct ggggactttc cacaccctaa ctgacacaga gctcgtgtta caaccaatta
     5221 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     5281 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     5341 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     5401 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     5461 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     5521 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     5581 attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa
     5641 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     5701 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     5761 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     5821 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     5881 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     5941 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     6001 tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta
     6061 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     6121 atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct
     6181 gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6241 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6301 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6361 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6421 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6481 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6541 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6601 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6661 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6721 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6781 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6841 gggggcggag cctatggaaa cctgcaggtt attaatagta atcaattacg gggtcattag
     6901 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     6961 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     7021 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     7081 cagtacatca agtgtatcat atgcca
//
