LOCUS       sequence-237677-        6419 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 6419)
  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 6419)
  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..6419
                     /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(1116..1544)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1226..1247)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1438..1457
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1571..1675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1676..1744,1745..2536)
                     /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(1894..1913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2707..3295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3196..3215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3738..3872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4297..4317)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          4447..4512
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     primer_bind     complement(5044..5063)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5652..5671
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6082..6411)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6096..6231
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6396..6416
                     /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 gagtggccaa agatctctgg cgtaatagcg
     1021 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc taatgggaaa
     1081 ttgtaaacgt taatattttg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1141 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1201 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1261 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     1321 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     1381 agggatgccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     1441 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     1501 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     1561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1621 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1681 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1741 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1801 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1861 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1921 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1981 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2041 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2101 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2161 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2221 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2281 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2341 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2401 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2461 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2521 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2581 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2641 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2701 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2761 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2821 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2881 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2941 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3001 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc aacacagcca gcttggagcg
     3061 aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc
     3121 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3181 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3241 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3301 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3361 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3421 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3481 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gagatctttg
     3541 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3601 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     3661 aactccatca ctaggggttc ctggaggggt ggagtcgtga cgtgaattac gtcatagggt
     3721 tagggaggtc ctggatcgat ccagacatga taagatacat tgatgagttt ggacaaacca
     3781 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3841 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3901 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3961 gtatggctga ttatgatctc tagtcaaggc actatacatc aaatattcct tattaacccc
     4021 tttacaaatt aaaaagctaa aggtacacaa tttttgagca tagttattaa tagcagacac
     4081 tctatgcctg tgtggagtaa gaaaaaacag tatgttatga ttataactgt tatgcctact
     4141 tataaaggtt acagaatatt tttccataat tttcttgtat agcagtgcag ctttttcctt
     4201 tgtggtgtaa atagcaaagc aagcaagagt tctattacta aacacagcat gactcaaaaa
     4261 acttagcaat tctgaaggaa agtccttggg gtcttctacc tttctcttct tttttggagg
     4321 agtagaatgt tgagagtcag cagtagcctc atcatcacta gatggcattt cttctgagca
     4381 aaacaggttt tcctcattaa aggcattcca ccactgctcc cattcatcag ttccataggt
     4441 tggaatctaa aatacacaaa caattagaat cagtagttta acacattata cacttaaaaa
     4501 ttttatattt accttagagc tttaaatctc tgtaggtagt ttgtccaatt atgtcacacc
     4561 acagaagtaa ggttccttca caaagatccg ggaccaaagc ggccatcgtg cctccccact
     4621 cctgcagttc gggggcatgg atgcgcggat agccgctgct ggtttcctgg atgccgacgg
     4681 atttgcactg ccggtagaac tccgcgaggt cgtccagcct caggcagcag ctgaaccaac
     4741 tcgcgagggg atcgagcccg gggtgggcga agaactccag catgagatcc ccgcgctgga
     4801 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     4861 cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc
     4921 ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
     4981 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
     5041 agcaatatca cgggtagcca acgctatgtc ctgatagcgc gccgccacac ccagccggcc
     5101 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc
     5161 gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
     5221 ttcggctggc gcgagcccct gatgctcttg tccagatcat cctgatcgac aagaccggct
     5281 tccatccgag tacgtgctcg ctcgatgcga tgttcgcttg gtggtcgaat gggcaggtag
     5341 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     5401 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     5461 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     5521 acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
     5581 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     5641 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     5701 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
     5761 ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg
     5821 gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata
     5881 aaaccgccca gtctagctat cggcatgtaa gcccactgca agctacctgc tttctctttg
     5941 cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc
     6001 gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt
     6061 gcttgcggca gcgtgaagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     6121 cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag
     6181 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     6241 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     6301 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
     6361 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccaca
//
