LOCUS       sequence-354622-        6156 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ratiometric Squash-SAM sensor 4-2 as circular RNA in 
            mammalian cells using the Tornado expression system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6156)
  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 6156)
  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-Broccoli-Squash-SAM-sens...
FEATURES             Location/Qualifiers
     source          1..6156
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     122..141
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    707..828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(925..1380)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1062..1083)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1274..1293
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1407..1511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1512..1580,1581..2372)
                     /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(1730..1749)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2543..3131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3032..3051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3285..3302
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3400..3534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3959..3979)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          4109..4174
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4594..5388)
                     /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(4705..4724)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5315..5334
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5745..6074)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5759..5894
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6059..6079
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6107..6156,1..199)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6107..6127
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
       61 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
      121 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
      181 gtggaaagga cgaaacaccg tgctcgcttc ggcagcacat atactagtcg acggccatca
      241 gtcgccggtc ccaagcccgg ataaaatggg agggggcggg aaaccgccta accatgccga
      301 ctgatggcag ttgccatgtg tatgtggcca gatgccttgt aaccgaaagg gacacaaggt
      361 gagcccaata atacggtttg ggttaggata ggaagtagag ccgtaaactc tctaagcggt
      421 gtcgaaagga tggccacata ctctgatgat cgagacggtc gggtccagat attcgtatct
      481 gtcgagtaga gtgtgggctc gatcattcat ggcaactgcc atcagtcggc gtggactgta
      541 gaacactgcc aatgccggtc ccaagcccgg ataaaagtgg agggtacagt ccacgctcta
      601 gagcgcactt cggtgcgctt tttactagga cctgcaggca tgcaagcttg acgtcggtta
      661 ccgatatcca tatggcgacc gcatcgatct cgagccgagg actagtaact tgtttattgc
      721 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
      781 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtcttacg
      841 tagataagta gcatggcggg ttatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      901 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
      961 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1021 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1081 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1141 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1201 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     1261 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     1321 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     1381 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1441 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1501 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1561 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1621 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1681 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1741 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1801 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1861 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1921 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1981 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2041 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2101 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2161 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2221 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2281 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2341 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2401 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2461 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2521 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2581 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2641 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2701 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2761 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2821 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2881 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2941 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3001 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3061 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3241 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3301 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt
     3361 gacgtgaatt acgtcatagg gttagggagg tcctggatcg atccagacat gataagatac
     3421 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3481 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3541 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     3601 aagtaaaacc tctacaaatg tggtatggct gattatgatc tctagtcaag gcactataca
     3661 tcaaatattc cttattaacc cctttacaaa ttaaaaagct aaaggtacac aatttttgag
     3721 catagttatt aatagcagac actctatgcc tgtgtggagt aagaaaaaac agtatgttat
     3781 gattataact gttatgccta cttataaagg ttacagaata tttttccata attttcttgt
     3841 atagcagtgc agctttttcc tttgtggtgt aaatagcaaa gcaagcaaga gttctattac
     3901 taaacacagc atgactcaaa aaacttagca attctgaagg aaagtccttg gggtcttcta
     3961 cctttctctt cttttttgga ggagtagaat gttgagagtc agcagtagcc tcatcatcac
     4021 tagatggcat ttcttctgag caaaacaggt tttcctcatt aaaggcattc caccactgct
     4081 cccattcatc agttccatag gttggaatct aaaatacaca aacaattaga atcagtagtt
     4141 taacacatta tacacttaaa aattttatat ttaccttaga gctttaaatc tctgtaggta
     4201 gtttgtccaa ttatgtcaca ccacagaagt aaggttcctt cacaaagatc cggaccaaag
     4261 cggccatcgt gcctccccac tcctgcagtt cgggggcatg gatgcgcgga tagccgctgc
     4321 tggtttcctg gatgccgacg gatttgcact gccggtagaa ctccgcgagg tcgtccagcc
     4381 tcaggcagca gctgaaccaa ctcgcgaggg gatcgagccc ggggtgggcg aagaactcca
     4441 gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg attccgaagc
     4501 ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg
     4561 cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     4621 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     4681 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     4741 cgccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     4801 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     4861 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     4921 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     4981 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     5041 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     5101 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     5161 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     5221 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     5281 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     5341 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     5401 tcatcctgtc tcttgatcag atcttgatcc cctgcgccat cagatccttg gcggcaagaa
     5461 agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa
     5521 ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact
     5581 gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct
     5641 gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt tccgcttcct
     5701 ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa gctttttgca aaagcctagg
     5761 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     5821 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     5881 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     5941 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     6001 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     6061 cctaactgac acacattcca cagccggatc caaggtcggg caggaagagg gcctatttcc
     6121 catgattcct tcatatttgc atatacgata caaggc
//
