LOCUS       sequence-491595-        4817 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  RNA circularization with CIRC using Tet 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 4817)
  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 4817)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 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-Tet-CVB3 IRES-EGFP-BbsI
FEATURES             Location/Qualifiers
     source          1..4817
                     /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      1703..1712
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1709..1711,1712..1714,1715..2428)
                     /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(1709..1711,1712..1714,1715..2425)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1754..1775)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2015..2034)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2362..2383
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2641..2657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2641..2657)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2654..2676)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2665..2681
                     /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(2689..2695,2696..2713,2714..2719))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2734..2755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2872..2889)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3043..3631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3123..3142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3802..4617)
                     /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(3802..4617)
                     /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     4525..4544
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4618..4722)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4790..4808
                     /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 tagcaatatt tacctttgga gggaaaagtt
      481 atcaggcatg cacctggtag ctagtcttta aaccaataga ttgcatcggt ttaaaaggca
      541 agaccgtcaa attgcgggaa aggggtcaac agccgttcag taccaagtct caggggaaac
      601 tttgagatgg ccttgcaaag ggtatggtaa taagctgacg gacatggtcc taaccacgca
      661 gccaagtcct aagtcaacag atcttctgtt gatatggatg cagttcacag actaaatgtc
      721 ggtcggggaa gatgtattct tctcataaga tatagtcgga cctctcctta atgggagcta
      781 gcggatgaag tgatgcaaca ctggagccgc tgggaactaa tttgtatgcg aaagtatatt
      841 gattagtttt ggagtactcg taaggtagcc aaatgaaaaa caaaaaacaa aaaaaaacaa
      901 aaaaagccga agagtcagct cccgacaaaa aaaaaaccaa aaaaacaaaa cacattaaaa
      961 cagcctgtgg gttgatccca cccacaggcc cattgggcgc tagcactctg gtatcacggt
     1021 acctttgtgc gcctgtttta taccccctcc cccaactgta acttagaagt aacacacacc
     1081 gatcaacagt cagcgtggca caccagccac gttttgatca agcacttctg ttaccccgga
     1141 ctgagtatca atagactgct cacgcggttg aaggagaaag cgttcgttat ccggccaact
     1201 acttcgaaaa acctagtaac accgtggaag ttgcagagtg tttcgctcag cactacccca
     1261 gtgtagatca ggtcgatgag tcaccgcatt ccccacgggc gaccgtggcg gtggctgcgt
     1321 tggcggcctg cccatgggga aacccatggg acgctctaat acagacatgg tgcgaagagt
     1381 ctattgagct agttggtagt cctccggccc ctgaatgcgg ctaatcctaa ctgcggagca
     1441 cacaccctca agccagaggg cagtgtgtcg taacgggcaa ctctgcagcg gaaccgacta
     1501 ctttgggtgt ccgtgtttca ttttattcct atactggctg cttatggtga caattgagag
     1561 atcgttacca tatagctatt ggattggcca tccggtgact aatagagcta ttatatatcc
     1621 ctttgttggg tttataccac ttagcttgaa agaggttaaa acattacaat tcattgttaa
     1681 gttgaataca gcaaaaacta gtgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     1741 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     1801 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     1861 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1921 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1981 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2041 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2101 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2161 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2221 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     2281 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     2341 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     2401 tctcggcatg gacgagctgt acaagtaacc ggtgctggag cctcggtggc catgcttctt
     2461 gccccttggg cctcccccca gcccctcctc cccttcctgc acccgtaccc ccgtggtctt
     2521 tgaataaagt ctgacgggag ctgactcttc ggcaaaaaca aaaaacaaaa aaaaacaaaa
     2581 aaataactat gactctcttt gtcttcgttt acacggcgcg ccgagcttgg ctcgagcatg
     2641 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     2701 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     2761 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2821 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2881 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2941 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3001 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3061 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3481 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3781 tgagtaaact tggtctgaca gttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3841 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3901 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3961 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     4021 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     4081 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     4141 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     4201 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     4261 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cagggatcgc
     4321 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     4381 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     4441 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     4501 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     4561 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact
     4621 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4681 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4741 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     4801 cacgaggccc tttcgtc
//
