LOCUS       sequence-248078-        6236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of bifunctional circular RNA (Broccoli and empty).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6236)
  AUTHORS   Litke JL, Jaffrey SR
  TITLE     Highly efficient expression of circular RNA aptamers in cells using 
            autocatalytic transcripts.
  JOURNAL   Nat Biotechnol. 2019 Apr 8. pii: 10.1038/s41587-019-0090-6. doi: 
            10.1038/s41587-019-0090-6.
  PUBMED    30962542
REFERENCE   2  (bases 1 to 6236)
  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 
            Biotechnol. 2019 Apr 8. pii: 10.1038/s41587-019-0090-6. doi: 
            10.1038/s41587-019-0090-6."
COMMENT     Sequence Label: pAV-U6+27-Tornado-F30-Broccoli-empty
FEATURES             Location/Qualifiers
     source          1..6236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        25..273
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     25..45
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     196..215
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    686..807
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   856..985
                     /label=AAV2 ITR
     rep_origin      complement(1079..1534)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1216..1237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1428..1447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1561..1665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1666..1734,1735..2526)
                     /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(1884..1903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2697..3285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3186..3205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3554..3688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4113..4133)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          4263..4328
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4748..5542)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVAARYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4859..4878)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5469..5488
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5899..6228)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5913..6048
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6213..6233
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gccggatcca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
       61 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
      121 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      181 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      241 gctttatata tcttgtggaa aggacgaaac accgtgctcg cttcggcagc acatatacta
      301 gtcgacgggc cgcactcgcc ggtcccaagc ccggataaaa tgggaggggg cgggaaaccg
      361 cctaaccatg ccgagtgcgg ccgcttgcca tgtgtatcgg tccggagacg gtcgggtcca
      421 gatattcgta tctgtcgagt agagtgtggg ctccggtccg atactctgat gatgggtccc
      481 atcattcatg gcaagtggcc gcggtcggcg tggactgtag aacactgcca atgccggtcc
      541 caagcccgga taaaagtgga gggtacagtc cacgctctag agcgcacttc ggtgcgcttt
      601 ttactaggac ctgcaggcat gcaagcttga cgtcggttac cgatatccat atggcgaccg
      661 catcgatctc gagccgagga ctagtaactt gtttattgca gcttataatg gttacaaata
      721 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
      781 tttgtccaaa ctcatcaatg tatcttatca tgtcttacgt agataagtag catggcgggt
      841 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
      901 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      961 cctcagtgag cgagcgagcg cgcagagagg gacagatctg cagatctgct ggcgtaatag
     1021 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa
     1081 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1141 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     1201 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1261 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     1321 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     1381 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     1441 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     1501 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     1561 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1621 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1681 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1741 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     1801 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     1861 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     1921 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     1981 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2041 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2101 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2161 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2221 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2281 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2341 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2401 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2461 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2521 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2581 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2641 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2701 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     2761 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     2821 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     2881 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     2941 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3001 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3061 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3121 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3181 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3241 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3301 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3361 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3421 agccgaacga ccgagcgcag cgagtcagtg agcgagcgcg cagagaggga gtggccaact
     3481 ccatcactag gggttcctgg aggggtggag tcgtgacgtg aattacgtca tagggttagg
     3541 gaggtcctgg atcgatccag acatgataag atacattgat gagtttggac aaaccacaac
     3601 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3661 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3721 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     3781 ggctgattat gatctctagt caaggcacta tacatcaaat attccttatt aaccccttta
     3841 caaattaaaa agctaaaggt acacaatttt tgagcatagt tattaatagc agacactcta
     3901 tgcctgtgtg gagtaagaaa aaacagtatg ttatgattat aactgttatg cctacttata
     3961 aaggttacag aatatttttc cataattttc ttgtatagca gtgcagcttt ttcctttgtg
     4021 gtgtaaatag caaagcaagc aagagttcta ttactaaaca cagcatgact caaaaaactt
     4081 agcaattctg aaggaaagtc cttggggtct tctacctttc tcttcttttt tggaggagta
     4141 gaatgttgag agtcagcagt agcctcatca tcactagatg gcatttcttc tgagcaaaac
     4201 aggttttcct cattaaaggc attccaccac tgctcccatt catcagttcc ataggttgga
     4261 atctaaaata cacaaacaat tagaatcagt agtttaacac attatacact taaaaatttt
     4321 atatttacct tagagcttta aatctctgta ggtagtttgt ccaattatgt cacaccacag
     4381 aagtaaggtt ccttcacaaa gatccggacc aaagcggcca tcgtgcctcc ccactcctgc
     4441 agttcggggg catggatgcg cggatagccg ctgctggttt cctggatgcc gacggatttg
     4501 cactgccggt agaactccgc gaggtcgtcc agcctcaggc agcagctgaa ccaactcgcg
     4561 aggggatcga gcccggggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
     4621 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
     4681 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga
     4741 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     4801 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     4861 tatcacgggt agccaacgct atgtcctgat agcgcgccgc cacacccagc cggccacagt
     4921 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     4981 gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
     5041 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     5101 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     5161 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     5221 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     5281 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     5341 atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     5401 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     5461 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     5521 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagatcttg
     5581 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     5641 tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa
     5701 ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc
     5761 ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt
     5821 tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc cctgagtgct
     5881 tgcggcagcg tgaagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt
     5941 ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca
     6001 gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag
     6061 gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     6121 ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact
     6181 tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat tccaca
//
