LOCUS       sequence-490423-        4654 bp    DNA     circular SYN 23-OCT-2025
DEFINITION  RNA circularization with CIRC using Ana intron, harbouring an CVB3 
            IRES-EGFP reporter.Plasmid was linearized with BsaI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4654)
  AUTHORS   Shen Y, Li B, Dong L, Tang W, Ren J, Chen F, Zheng W, Yu Y, Gao L, 
            Wei W
  TITLE     Self-splicing RNA circularization facilitated by intact group I and 
            II introns.
  JOURNAL   Nat Commun. 2025 Aug 10;16(1):7376. doi: 10.1038/s41467-025-62607-y.
  PUBMED    40784880
REFERENCE   2  (bases 1 to 4654)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-08-10"; volume: "16(1):7376. doi"; pages: " 
            10.1038/s41467-025-62607-y"
COMMENT     Sequence Label: CIRC-V1-Ana-CVB3 IRES-EGFP-BsaI
FEATURES             Location/Qualifiers
     source          1..4654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        429..447
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1540..1549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1546..1548,1549..1551,1552..2265)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1546..1548,1549..1551,1552..2262)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1591..1612)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1852..1871)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2199..2220
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2478..2494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2478..2494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2491..2513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2502..2518
                     /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(2526..2532,2533..2550,2551..2556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2571..2592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2709..2726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2880..3468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2960..2979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3639..4454)
                     /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(3639..4454)
                     /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     4362..4381
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4455..4559)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4627..4645
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatcgagc
      421 tcggtaccta atacgactca ctataggaaa taattgagcc ttaaagaaga aattctttaa
      481 gtggatgctc tcaaactcag ggaaacctaa atctagttat agacaaggca atcctgagcc
      541 aagccgaagt agtaattagt aagttaacaa tagatgactt acaactaatc ggaaggtgca
      601 gagactcgac gggagctacc ctaacgtcaa gacgagggta aagagagagt ccaattctca
      661 aagccaatag gcagtagcga aagctgcaag agaatgaaaa tccgttgacc ttaaaaacaa
      721 aaaacaaaaa aaaacaaaaa aagccgaaga gtcagctccc gacaaaaaaa aaaccaaaaa
      781 aacaaaacac attaaaacag cctgtgggtt gatcccaccc acaggcccat tgggcgctag
      841 cactctggta tcacggtacc tttgtgcgcc tgttttatac cccctccccc aactgtaact
      901 tagaagtaac acacaccgat caacagtcag cgtggcacac cagccacgtt ttgatcaagc
      961 acttctgtta ccccggactg agtatcaata gactgctcac gcggttgaag gagaaagcgt
     1021 tcgttatccg gccaactact tcgaaaaacc tagtaacacc gtggaagttg cagagtgttt
     1081 cgctcagcac taccccagtg tagatcaggt cgatgagtca ccgcattccc cacgggcgac
     1141 cgtggcggtg gctgcgttgg cggcctgccc atggggaaac ccatgggacg ctctaataca
     1201 gacatggtgc gaagagtcta ttgagctagt tggtagtcct ccggcccctg aatgcggcta
     1261 atcctaactg cggagcacac accctcaagc cagagggcag tgtgtcgtaa cgggcaactc
     1321 tgcagcggaa ccgactactt tgggtgtccg tgtttcattt tattcctata ctggctgctt
     1381 atggtgacaa ttgagagatc gttaccatat agctattgga ttggccatcc ggtgactaat
     1441 agagctatta tatatccctt tgttgggttt ataccactta gcttgaaaga ggttaaaaca
     1501 ttacaattca ttgttaagtt gaatacagca aaaactagtg ccaccatggt gagcaagggc
     1561 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1621 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1681 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1741 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1801 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1861 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1921 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1981 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     2041 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     2101 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     2161 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     2221 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaccggt gctggagcct
     2281 cggtggccat gcttcttgcc ccttgggcct ccccccagcc cctcctcccc ttcctgcacc
     2341 cgtacccccg tggtctttga ataaagtctg acgggagctg actcttcggc aaaaacaaaa
     2401 aacaaaaaaa aacaaaaaaa tagacgctac ggactttgag accgtttaaa cggcgcgccg
     2461 agcttggctc gagcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2521 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2581 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2641 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3061 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3301 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt agaaaaactc atcgagcatc
     3661 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     3721 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     3781 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     3841 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     3901 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     3961 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     4021 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     4081 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     4141 gttttcccag ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     4201 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     4261 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     4321 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     4381 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     4441 tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4501 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4561 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     4621 tataaaaata ggcgtatcac gaggcccttt cgtc
//
