LOCUS       sequence-371634-        5836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid to transcribe mRNA encoding NanoLuc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5836)
  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 5836)
  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: mRNA-CleanCap-NanoLuc
FEATURES             Location/Qualifiers
     source          1..5836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          645..1661
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1585..1607
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1669..1688
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1789..1798
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1804..1869
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1885..2397
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     terminator      2538..2585
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2599..2823
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    2866..2882
                     /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     2871..2893
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2890..2906
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2890..2906
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2955..3076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2966..2985)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3020..3039
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3236..3835)
                     /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(3319..3339)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     3816..3835
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(3846..4174)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3860..3995
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3914..3933)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             complement(4208..5023)
                     /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(4208..5023)
                     /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     4931..4950
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5015..5034
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5248..5836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5737..5756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
       61 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      121 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      181 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      241 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      301 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      361 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      421 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
      481 gggggggggg sgcgccrggg gggggggggg gggggggggg gggggggggg gsgrggsggr
      541 grggkgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      601 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
      661 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
      721 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
      781 gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc
      841 gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg
      901 ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg
      961 ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     1021 gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag
     1081 cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg
     1141 ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg
     1201 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     1261 gggrgggctc gggggrgggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg
     1321 cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt
     1381 cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg
     1441 gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc
     1501 cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc
     1561 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     1621 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     1681 ttattgtgct gtctcatcat tttggcaaag cggccgcggc cagtgaattg taatacgact
     1741 cactataagg aaataagaga gaaaagaaga gtaagaagaa atataagagc caccatggga
     1801 tccgactata aggaccacga cggagactac aaggatcatg atattgatta caaggacgac
     1861 gatgacaagg gaggaggagg aagcgtcttc acactcgaag atttcgttgg ggactggcga
     1921 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
     1981 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
     2041 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
     2101 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     2161 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
     2221 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     2281 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
     2341 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggcgtagtaa
     2401 tgagctggag cctcggtggc catgcttctt gccccttggg cctcccccca gccccccctc
     2461 cccttcctgc acccgtaccc ccgtggtctt tgaataaagt ctgaaaaaaa aaaaaaaaaa
     2521 aaaaaaaaag cggccgccta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2581 ttttggctag cggccggcct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2641 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2701 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2761 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2821 tggcggcgcg ccactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     2881 aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgacg
     2941 gtatacagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3001 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3061 ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga
     3121 tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg
     3181 tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaatcagg
     3241 caccgggctt gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg
     3301 tgacggtgaa gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gtttccagga
     3361 aggcgggcac cccggcgcgc tcggccgcct ccactccggg gagcacgacg gcgctgccca
     3421 gacccttgcc ctggtggtcg ggcgacacgc cgacggtggc caggaaccac gcgggctcct
     3481 tgggccggtg cggcgccagg aggccttcca tctgttgctg cgcggccagc cgggaaccgc
     3541 tcaactcggc catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg
     3601 gcgtggtcca gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga
     3661 gcccgacgcg cgtgaggaag agttcttgca gctcggtgac ccgctcgatg tggcggtccg
     3721 gatcgacggt gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg
     3781 ccctggggac gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg gtcatgcggg
     3841 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     3901 gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
     3961 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     4021 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     4081 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     4141 gagcctgggg actttccaca ccctaactga cacagagctc gtgttacaac caattaacca
     4201 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     4261 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     4321 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     4381 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     4441 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     4501 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     4561 gtgattgcgc ctgagcgagg cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     4621 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     4681 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     4741 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     4801 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     4861 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     4921 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     4981 atcgcggcct ggagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     5041 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     5101 aacatcagag attttgagac acaacgtggc tttgttgaat aaatcgaact tttgctgagt
     5161 tgaaggatca gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5221 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5281 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5341 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5401 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5461 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5521 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5581 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5641 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5701 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5761 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5821 gcggagccta tggaaa
//
