LOCUS       sequence-174668-        3665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the ""Top"" transcript of the Split-Broccoli system 
            upstream of an RNA Toehold switch controlling mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3665)
  AUTHORS   Alam KK, Tawiah KD, Lichte MF, Porciani D, Burke DH
  TITLE     A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In 
            Vivo.
  JOURNAL   ACS Synth Biol. 2017 May 26. doi: 10.1021/acssynbio.7b00059.
  PUBMED    28548488
REFERENCE   2  (bases 1 to 3665)
  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: "ACS Synth 
            Biol. 2017 May 26. doi: 10.1021/acssynbio.7b00059."
COMMENT     Sequence Label: pUC19-Top-Toehold-mCherry
FEATURES             Location/Qualifiers
     source          1..3665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /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     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             479..1189
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(624..642)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     892..911
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1256..1273)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1256..1272)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1265..1287)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1481..1500)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1600..1622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1660..1678)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1746..1850
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1851..1919,1920..2711)
                     /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(2069..2088)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2882..3470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3371..3390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3624..3641
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc
      241 gactctagac gatcccgcga aattgagcta acaccgtgcg tgttgacaat tttacctctg
      301 gcggtgataa tggttgcagg atgatggaga cggtcgggtc caggatcatt catggcaaga
      361 gacggtcggg tccagatgat gcggatccct gtcttatctt atctatctcg tttatccctg
      421 catacagaaa cagaggagat atgcaatgat aaacgagaac ctggcggcag cgcaaaagat
      481 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
      541 tcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
      601 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
      661 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
      721 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
      781 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaagacgg
      841 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
      901 gcagaagaag actatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
      961 gctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1021 ggtcaagacc acctacaagg ccaagaagcc cgtgcaactg cccggcgcgt acaacgtcaa
     1081 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     1141 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaat agcataaccc
     1201 cttggggctc taaacgggtc ttgaggggtt ttgcccgggt accgagctcg aattcactgg
     1261 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1321 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1381 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     1441 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     1501 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     1561 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1621 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     1681 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     1741 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1801 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1861 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     1921 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     1981 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2041 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     2101 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2161 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2221 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2281 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2341 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     2401 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2461 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2521 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2581 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2641 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2701 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2761 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2821 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2881 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2941 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3001 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3061 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3121 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3181 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3241 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3301 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3361 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3421 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3481 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3541 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3601 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     3661 tacgc
//
