LOCUS       sequence-354620-        6121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a bifunctional circular RNA with Squash in one arm and 
            Broccoli in the other arm of the F30 scaffold, in mammalian cells, 
            using the Tornado expression system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6121)
  AUTHORS   Dey SK, Filonov GS, Olarerin-George AO, Jackson BT, Finley LWS, 
            Jaffrey SR
  TITLE     Repurposing an adenine riboswitch into a fluorogenic imaging and 
            sensing tag.
  JOURNAL   Nat Chem Biol. 2021 Dec 22. pii: 10.1038/s41589-021-00925-0. doi: 
            10.1038/s41589-021-00925-0.
  PUBMED    34937909
REFERENCE   2  (bases 1 to 6121)
  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 Chem 
            Biol. 2021 Dec 22. pii: 10.1038/s41589-021-00925-0. doi: 
            10.1038/s41589-021-00925-0."
COMMENT     Sequence Label: pAV-U6+27-Tornado-F30-Squash-Broccoli
FEATURES             Location/Qualifiers
     source          1..6121
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(30..50)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(78..98)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        83..412
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      263..398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             769..1563
                     /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(823..842)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1433..1452
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     intron          1983..2048
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2178..2198
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2623..2757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2855..2872)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3026..3614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3106..3125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3785..4576,4577..4645))
                     /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     4408..4427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4646..4750)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4777..5232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4864..4883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5074..5095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5329..5450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(join(5923..6121,1..50))
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5981..6000)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccg
       61 accttggatc cggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
      121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
      181 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
      241 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
      301 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
      361 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttca
      421 cgctgccgca agcactcagg gcgcaagggc tgctaaagga agcggaacac gtagaaagcc
      481 agtccgcaga aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg
      541 gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta
      601 gactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt
      661 aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg
      721 cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
      781 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
      841 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
      901 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
      961 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1021 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1081 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1141 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1201 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1261 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     1321 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1381 ggattcatcg actgtggccg gctgggtgtg gcggcgcgct atcaggacat agcgttggct
     1441 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1501 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1561 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     1621 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     1681 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccgggc
     1741 tcgatcccct cgcgagttgg ttcagctgct gcctgaggct ggacgacctc gcggagttct
     1801 accggcagtg caaatccgtc ggcatccagg aaaccagcag cggctatccg cgcatccatg
     1861 cccccgaact gcaggagtgg ggaggcacga tggccgcttt ggtccggatc tttgtgaagg
     1921 aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt taaagctcta
     1981 aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta attgtttgtg
     2041 tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat gcctttaatg
     2101 aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct actgctgact
     2161 ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag gactttcctt
     2221 cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt gcttgctttg
     2281 ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg gaaaaatatt
     2341 ctgtaacctt tataagtagg cataacagtt ataatcataa catactgttt tttcttactc
     2401 cacacaggca tagagtgtct gctattaata actatgctca aaaattgtgt acctttagct
     2461 ttttaatttg taaaggggtt aataaggaat atttgatgta tagtgccttg actagagatc
     2521 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2581 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2641 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2701 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcgat
     2761 ccaggacctc cctaacccta tgacgtaatt cacgtcacga ctccacccct ccaggaaccc
     2821 ctagtgatgg agttggccac tccctctctg cgcgctcgct cactgactcg ctgcgctcgg
     2881 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2941 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4081 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4621 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4741 gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa
     4801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5221 cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5281 ggcctcttcg ctataacccg ccatgctact tatctacgta agacatgata agatacattg
     5341 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     5401 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt actagtcctc
     5461 ggctcgagat cgatgcggtc gccatatgga tatcggtaac cgacgtcaag cttgcatgcc
     5521 tgcaggtcct agtaaaaagc gcaccgaagt gcgctctaga gcgtggactg taccctccac
     5581 ttttatccgg gcttgggacc ggcattggca gtgttctaca gtccacgccg actgatggca
     5641 gttgccatga atgatcgagc ccacactcta ctcgacagat acgaatatct ggacccgacc
     5701 gtctcgatca tcagagtatg tggctaccgc ttagagagtt tacggctcta cttcctatcc
     5761 taacccaaac cgtattattg ggctcacctt gtagccacat acacatggca actgccatca
     5821 gtcggcatgg ttaggcggtt tcccgccccc tcccatttta tccgggcttg ggaccggcga
     5881 ctgatggccg tcgactagta tatgtgctgc cgaagcgagc acggtgtttc gtcctttcca
     5941 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     6001 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     6061 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac
     6121 a
//
