LOCUS       sequence-449390-        5485 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 5485)
  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 5485)
  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-EMCV-GFP
FEATURES             Location/Qualifiers
     source          1..5485
                     /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"
     misc_feature    join(361..934,935..937,938..946)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(528..545)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     755..774
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(947..949,950..952,953..1666)
                     /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(947..949,950..952,953..1663)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(992..1013)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1253..1272)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1600..1621
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1726..1746)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2546..2675
                     /label=AAV2 ITR
     primer_bind     complement(2694..2711)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2694..2710)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2703..2725)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2852..3307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2939..2958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3149..3170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3333..3437
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3438..3506,3507..4298)
                     /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(3656..3675)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4469..5057
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4958..4977
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5345..5366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5381..5386,5387..5404,5405..5411)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5419..5435
                     /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     5424..5446
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5443..5459
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5443..5459
                     /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 ccgccccccc
      361 cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg
      421 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg
      481 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg
      541 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca
      601 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct
      661 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca
      721 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa
      781 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg
      841 cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg
      901 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaaccatgg tgagcaaggg
      961 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1021 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1081 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1141 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1201 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1261 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1321 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1381 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1441 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1501 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1561 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1621 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaggat ccaatcaacc
     1681 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     1741 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     1801 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     1861 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     1921 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     1981 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     2041 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     2101 tgccacctgg attctgcgcg ggacgtcctt ctgctacaat ccagcggacc ttccttcccg
     2161 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcga agtaaaaaaa aaaaaaaaaa
     2221 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2281 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     2341 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa ctgccatcag tcggcgtgga ctgtagaaca
     2401 ctgccaatgc cggtcccaag cccggataaa agtggagggt acagtccacg ctttttttct
     2461 cgagttaagg gcgaattccc gataaggatc ttcctagagc atggctacgt agataagtag
     2521 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     2581 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     2641 gcccgggcgg cctcagtgag cgagcgagcg cgcagcctta attaacctaa ttcactggcc
     2701 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2761 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2821 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     2881 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2941 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3001 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3061 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3121 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3181 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3241 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3301 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3361 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3421 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3481 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3541 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3601 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3661 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     3721 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3781 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3841 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3901 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3961 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4021 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4081 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4141 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4201 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4261 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4321 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4381 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4441 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4501 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4561 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4621 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4681 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4741 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4801 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4861 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4921 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4981 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5041 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5101 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5161 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5221 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5281 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     5341 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     5401 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     5461 atgattacgc cagatttaat taagg
//
