LOCUS       sequence-449395-        5632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of in situ circularizable RNA via IVT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5632)
  AUTHORS   Tong M, Palmer N, Dailamy A, Kumar A, Khaliq H, Han S, Finburgh E, 
            Wing M, Hong C, Xiang Y, Miyasaki K, Portell A, Rainaldi J, Suhardjo
            A, Nourreddine S, Chew WL, Kwon EJ, Mali P
  TITLE     Robust genome and cell engineering via in vitro and in situ 
            circularized RNAs.
  JOURNAL   Nat Biomed Eng. 2024 Aug 26. doi: 10.1038/s41551-024-01245-z.
  PUBMED    39187662
REFERENCE   2  (bases 1 to 5632)
  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 Biomed 
            Eng. 2024 Aug 26. doi: 10.1038/s41551-024-01245-z."
COMMENT     Sequence Label: Circular-ic-CVB3-GFP
FEATURES             Location/Qualifiers
     source          1..5632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   12..141
                     /label=AAV2 ITR
     primer_bind     226..245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        226..244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1094..1096,1097..1099,1100..1813)
                     /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(1094..1096,1097..1099,1100..1810)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1139..1160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1400..1419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1747..1768
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1873..1893)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2693..2822
                     /label=AAV2 ITR
     primer_bind     complement(2841..2858)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2841..2857)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2850..2872)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2999..3454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3086..3105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3296..3317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3480..3584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3585..3653,3654..4445)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3803..3822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4616..5204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5105..5124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5492..5513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5528..5533,5534..5551,5552..5558)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5566..5582
                     /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     5571..5593
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5590..5606
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5590..5606
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt
      181 agccatgctc taggaagatc ggaattcgcc cttaagctag gatgctaata cgactcacta
      241 tagggccatc agtcgccggt cccaagcccg gataaaatgg gagggggcgg gaaaccgcct
      301 aaccatgccg actgatggca gagcggccgc gtcgacgggc ccgcggaatt ccttaaaaca
      361 gcctgtgggt tgatcccacc cacaggccca ttgggcgcta gcactctggt atcacggtac
      421 ctttgtgcgc ctgttttata ccccctcccc caactgtaac ttagaagtaa cacacaccga
      481 tcaacagtca gcgtggcaca ccagccacgt tttgatcaag cacttctgtt accccggact
      541 gagtatcaat agactgctca cgcggttgaa ggagaaagcg ttcgttatcc ggccaactac
      601 ttcgaaaaac ctagtaacac cgtggaagtt gcagagtgtt tcgctcagca ctaccccagt
      661 gtagatcagg tcgatgagtc accgcattcc ccacgggcga ccgtggcggt ggctgcgttg
      721 gcggcctgcc catggggaaa cccatgggac gctctaatac agacatggtg cgaagagtct
      781 attgagctag ttggtagtcc tccggcccct gaatgcggct aatcctaact gcggagcaca
      841 caccctcaag ccagagggca gtgtgtcgta acgggcaact ctgcagcgga accgactact
      901 ttgggtgtcc gtgtttcatt ttattcctat actggctgct tatggtgaca attgagagat
      961 cgttaccata tagctattgg attggccatc cggtgactaa tagagctatt atatatccct
     1021 ttgttgggtt tataccactt agcttgaaag aggttaaaac attacaattc attgttaagt
     1081 tgaatacagc aaaatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1141 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1201 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1261 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1321 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1381 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1441 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1501 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1561 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1621 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1681 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1741 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1801 gctgtacaag taaggatcca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     1861 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     1921 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     1981 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2041 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     2101 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2161 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     2221 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     2281 ctacaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     2341 tcttcgaagt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2401 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2461 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaactg
     2521 ccatcagtcg gcgtggactg tagaacactg ccaatgccgg tcccaagccc ggataaaagt
     2581 ggagggtaca gtccacgctt tttttctcga gttaagggcg aattcccgat aaggatcttc
     2641 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     2701 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     2761 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     2821 agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2881 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2941 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     3001 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3061 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3121 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3181 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3241 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3301 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3361 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3421 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     3481 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3541 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3601 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3661 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3721 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3781 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3841 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3901 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3961 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4021 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4081 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4141 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4201 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4261 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4321 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4381 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4441 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4501 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4741 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5401 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5461 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     5521 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5581 aatttcacac aggaaacagc tatgaccatg attacgccag atttaattaa gg
//
