LOCUS       sequence-449393-        6350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of in vitro circularizable RNA via IVT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6350)
  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 6350)
  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-oc-CVB3-GFP
FEATURES             Location/Qualifiers
     source          1..6350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(142..161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      955..1543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1444..1463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1831..1852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1867..1872,1873..1890,1891..1897)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1905..1921
                     /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     1910..1932
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1929..1945
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1929..1945
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1983..2112
                     /label=AAV2 ITR
     primer_bind     2197..2216
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2197..2215
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(3156..3158,3159..3161,3162..3875)
                     /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(3156..3158,3159..3161,3162..3872)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3201..3222)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3462..3481)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3809..3830
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3935..3955)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   5382..5511
                     /label=AAV2 ITR
     primer_bind     complement(5530..5547)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5530..5546)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5539..5561)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5688..6143
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5775..5794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5985..6006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6169..6273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6274..6342,6343..6350,1..784)
                     /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"
ORIGIN
        1 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
       61 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
      121 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
      181 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
      241 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
      301 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
      361 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
      421 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
      481 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
      541 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
      601 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
      661 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
      721 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
      781 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
      841 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
      901 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      961 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1021 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1081 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     1141 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1201 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1261 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1321 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1381 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1441 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1501 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1561 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     1621 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     1681 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     1741 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     1801 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     1861 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     1921 atttcacaca ggaaacagct atgaccatga ttacgccaga tttaattaag gccttaatta
     1981 ggctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct
     2041 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
     2101 ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct
     2161 ctaggaagat cggaattcgc ccttaagcta gcatgctaat acgactcact atagggccat
     2221 cagtcgccgg tcccaagccc ggataaaatg ggagggggcg ggaaaccgcc taaccatgcc
     2281 gactgatggc agctattatc gagcgaacgc cttatgcgat gaaagtcgca cgtagggtgt
     2341 agaccaagcg aaatcctatg catttaggat agtgaggtat agcaaaggag aagtcgacgg
     2401 gcccgcggaa ttccttaaaa cagcctgtgg gttgatccca cccacaggcc cattgggcgc
     2461 tagcactctg gtatcacggt acctttgtgc gcctgtttta taccccctcc cccaactgta
     2521 acttagaagt aacacacacc gatcaacagt cagcgtggca caccagccac gttttgatca
     2581 agcacttctg ttaccccgga ctgagtatca atagactgct cacgcggttg aaggagaaag
     2641 cgttcgttat ccggccaact acttcgaaaa acctagtaac accgtggaag ttgcagagtg
     2701 tttcgctcag cactacccca gtgtagatca ggtcgatgag tcaccgcatt ccccacgggc
     2761 gaccgtggcg gtggctgcgt tggcggcctg cccatgggga aacccatggg acgctctaat
     2821 acagacatgg tgcgaagagt ctattgagct agttggtagt cctccggccc ctgaatgcgg
     2881 ctaatcctaa ctgcggagca cacaccctca agccagaggg cagtgtgtcg taacgggcaa
     2941 ctctgcagcg gaaccgacta ctttgggtgt ccgtgtttca ttttattcct atactggctg
     3001 cttatggtga caattgagag atcgttacca tatagctatt ggattggcca tccggtgact
     3061 aatagagcta ttatatatcc ctttgttggg tttataccac ttagcttgaa agaggttaaa
     3121 acattacaat tcattgttaa gttgaataca gcaaaatggt gagcaagggc gaggagctgt
     3181 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     3241 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     3301 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     3361 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     3421 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3481 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3541 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     3601 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     3661 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3721 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     3781 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     3841 ggatcactct cggcatggac gagctgtaca agtaaggatc caatcaacct ctggattaca
     3901 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3961 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4021 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     4081 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     4141 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     4201 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     4261 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4321 ttctgcgcgg gacgtccttc tgctacaatc cagcggacct tccttcccgc ggcctgctgc
     4381 cggctctgcg gcctcttccg cgtcttcgaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4441 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4501 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4561 aaaaaaaaaa aaaaaactat agccatacaa taaaagtgcg aaacgttatc ctataagtaa
     4621 gaaagtttta aaattttctt acgaaaagga tagaacttaa aagttctaac tgttctacta
     4681 aagtaataag tgaaaatctt atttaaagca aacaaccaag tagctttaag tctaagtccc
     4741 ctacacaagt tttatactac tatgcaaaac ttgtgaagct aggtaaggtc gtaatccgtg
     4801 aaagtcggat gcggggctcc ttaaaagatt actatggtaa acataagcta atccattaag
     4861 atgcgattta tatgtatttt atactgttaa atatttttgt gcttgtggct tggtataaaa
     4921 cagttaagat gaagtactta actggttttg gaataattgg ttgttaaact aaaacattat
     4981 aaatcgttag tggataccta aggtaatcaa aaatagggat aggtagaatg gaacgtttga
     5041 tgctgtatat gaagaggttt agtagaacct aggacacata tacgggctca gcaggttcat
     5101 agtagctatg atactcagcc ggaagtcaaa ttaattttga aatacttcta tggtaacata
     5161 ggagaaggat aaaactgagt gagccaagga acctagtcgg taatagctgc catcagtcgg
     5221 cgtggactgt agaacactgc caatgccggt cccaagcccg gataaaagtg gagggtacag
     5281 tccacgcttt ttttctcgag ttaagggcga attcccgata aggatcttcc tagagcatgg
     5341 ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga
     5401 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     5461 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gccttaatta
     5521 acctaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5581 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5641 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     5701 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5761 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5821 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5881 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5941 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     6001 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     6061 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     6121 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     6181 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6241 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6301 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
//
