LOCUS       sequence-449384-        6203 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 6203)
  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 6203)
  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-EMCV-GFP
FEATURES             Location/Qualifiers
     source          1..6203
                     /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"
     misc_feature    join(2423..2996,2997..2999,3000..3008)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2590..2607)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2817..2836
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3009..3011,3012..3014,3015..3728)
                     /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(3009..3011,3012..3014,3015..3725)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3054..3075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3315..3334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3662..3683
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3788..3808)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   5235..5364
                     /label=AAV2 ITR
     primer_bind     complement(5383..5400)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5383..5399)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5392..5414)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5541..5996
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5628..5647)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5838..5859
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6022..6126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6127..6195,6196..6203,1..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 ttccgccccc cccccctctc cctccccccc ccctaacgtt actggccgaa
     2461 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     2521 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     2581 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     2641 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     2701 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     2761 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     2821 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     2881 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg
     2941 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     3001 ccacaaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     3061 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     3121 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     3181 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3241 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     3301 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     3361 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3421 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     3481 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     3541 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3601 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     3661 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3721 acaagtaagg atccaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3781 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3841 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3901 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3961 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4021 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     4081 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     4141 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctaca
     4201 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     4261 gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4321 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4381 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tatagccata
     4441 caataaaagt gcgaaacgtt atcctataag taagaaagtt ttaaaatttt cttacgaaaa
     4501 ggatagaact taaaagttct aactgttcta ctaaagtaat aagtgaaaat cttatttaaa
     4561 gcaaacaacc aagtagcttt aagtctaagt cccctacaca agttttatac tactatgcaa
     4621 aacttgtgaa gctaggtaag gtcgtaatcc gtgaaagtcg gatgcggggc tccttaaaag
     4681 attactatgg taaacataag ctaatccatt aagatgcgat ttatatgtat tttatactgt
     4741 taaatatttt tgtgcttgtg gcttggtata aaacagttaa gatgaagtac ttaactggtt
     4801 ttggaataat tggttgttaa actaaaacat tataaatcgt tagtggatac ctaaggtaat
     4861 caaaaatagg gataggtaga atggaacgtt tgatgctgta tatgaagagg tttagtagaa
     4921 cctaggacac atatacgggc tcagcaggtt catagtagct atgatactca gccggaagtc
     4981 aaattaattt tgaaatactt ctatggtaac ataggagaag gataaaactg agtgagccaa
     5041 ggaacctagt cggtaatagc tgccatcagt cggcgtggac tgtagaacac tgccaatgcc
     5101 ggtcccaagc ccggataaaa gtggagggta cagtccacgc tttttttctc gagttaaggg
     5161 cgaattcccg ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt
     5221 aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     5281 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     5341 ctcagtgagc gagcgagcgc gcagccttaa ttaacctaat tcactggccg tcgttttaca
     5401 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     5461 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     5521 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5581 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5641 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5701 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5761 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5821 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5881 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5941 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     6001 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6061 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6121 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6181 cttcctgttt ttgctcaccc aga
//
