LOCUS       sequence-489587-        4609 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  RNA circularization with CIRC using Azo intron, harbouring an CVB3 
            IRES-EGFP reporter.Plasmid was linearized with BbsI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4609)
  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 4609)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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-Azo-CVB3 IRES-EGFP-BbsI
FEATURES             Location/Qualifiers
     source          1..4609
                     /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      1495..1504
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1501..1503,1504..1506,1507..2220)
                     /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(1501..1503,1504..1506,1507..2217)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1546..1567)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1807..1826)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2154..2175
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2433..2449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2433..2449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2446..2468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2457..2473
                     /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(2481..2487,2488..2505,2506..2511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2526..2547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2664..2681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2835..3423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2915..2934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3594..4409)
                     /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(3594..4409)
                     /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     4317..4336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4410..4514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4582..4600
                     /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 ctataggatt tcgatgtgcc ttgcgccggg aaaccacgca
      481 agggatggtg tcaaattcgg cgaaacctaa gcgcccgccc gggcgtatgg caacgccgag
      541 ccaagcttcg gcgcctgcgc cgatgaaggt gtagagacta gacggcaccc acctaaggca
      601 aacgctatgg tgaaggcata gtccagggag tggcgaaagt cacacaaacc ggaatccgtt
      661 ggtgctgaaa aacaaaaaac aaaaaaaaac aaaaaaagcc gaagagtcag ctcccgacaa
      721 aaaaaaaacc aaaaaaacaa aacacattaa aacagcctgt gggttgatcc cacccacagg
      781 cccattgggc gctagcactc tggtatcacg gtacctttgt gcgcctgttt tataccccct
      841 cccccaactg taacttagaa gtaacacaca ccgatcaaca gtcagcgtgg cacaccagcc
      901 acgttttgat caagcacttc tgttaccccg gactgagtat caatagactg ctcacgcggt
      961 tgaaggagaa agcgttcgtt atccggccaa ctacttcgaa aaacctagta acaccgtgga
     1021 agttgcagag tgtttcgctc agcactaccc cagtgtagat caggtcgatg agtcaccgca
     1081 ttccccacgg gcgaccgtgg cggtggctgc gttggcggcc tgcccatggg gaaacccatg
     1141 ggacgctcta atacagacat ggtgcgaaga gtctattgag ctagttggta gtcctccggc
     1201 ccctgaatgc ggctaatcct aactgcggag cacacaccct caagccagag ggcagtgtgt
     1261 cgtaacgggc aactctgcag cggaaccgac tactttgggt gtccgtgttt cattttattc
     1321 ctatactggc tgcttatggt gacaattgag agatcgttac catatagcta ttggattggc
     1381 catccggtga ctaatagagc tattatatat ccctttgttg ggtttatacc acttagcttg
     1441 aaagaggtta aaacattaca attcattgtt aagttgaata cagcaaaaac tagtgccacc
     1501 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1561 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1621 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1681 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1741 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1801 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1861 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1921 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1981 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2041 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2101 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2161 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     2221 ccggtgctgg agcctcggtg gccatgcttc ttgccccttg ggcctccccc cagcccctcc
     2281 tccccttcct gcacccgtac ccccgtggtc tttgaataaa gtctgacggg agctgactct
     2341 tcggcaaaaa caaaaaacaa aaaaaaacaa aaaaagagca gcggactcat ttgtcttcgt
     2401 ttaaacggcg cgccgagctt ggctcgagca tggtcatagc tgtttcctgt gtgaaattgt
     2461 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2521 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2581 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2641 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2701 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2761 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2821 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2881 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2941 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3001 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3061 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3121 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3181 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3241 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3301 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3361 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3421 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3481 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3541 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttagaaa
     3601 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3661 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3721 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3781 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3841 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3901 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3961 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4021 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4081 aatacctgga atgctgtttt cccagggatc gcagtggtga gtaaccatgc atcatcagga
     4141 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4201 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4261 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4321 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     4381 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
     4441 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4501 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4561 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
