LOCUS       sequence-174674-        3953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Split-Broccoli system and a Trigger/Toehold switch for
            mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3953)
  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 3953)
  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-Split-Broccoli-Toehold-Switch
FEATURES             Location/Qualifiers
     source          1..3953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(59..76)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(59..75)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(68..90)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             497..1207
                     /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(642..660)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     910..929
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1208..1254
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1269..1288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1388..1410
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1448..1466)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1534..1638
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1639..1707,1708..2499)
                     /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(1857..1876)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2670..3258
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3159..3178
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3412..3429
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3546..3567
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3582..3587,3588..3605,3606..3612)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3620..3636
                     /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     3625..3647
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3644..3660
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3644..3660
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      complement(3703..3750)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 caacacgcac ggtgttagct caatttcgcg ggatcgcccg ggtaccgagc tcgaattcac
       61 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
      121 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
      181 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
      241 cgcatctgtg cggtatttca caccgcacga tcccgcgaaa ttgagctaac accgtgcgtg
      301 ttgacaattt tacctctggc ggtgataatg gttgcaggat gatggagacg gtcgggtcca
      361 ggatcattca tggcaagaga cggtcgggtc cagatgatgc ggatccctgt cttatcttat
      421 ctatctcgtt tatccctgca tacagaaaca gaggagatat gcaatgataa acgagaacct
      481 ggcggcagcg caaaagatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
      541 gttcatgcgc ttcaaggttc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
      601 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
      661 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
      721 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
      781 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
      841 ctcctccctg caagacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
      901 ctccgacggc cccgtaatgc agaagaagac tatgggctgg gaggcctcct ccgagcggat
      961 gtaccccgag gacggcgcgc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     1021 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcaactgcc
     1081 cggcgcgtac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     1141 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     1201 caagtaatag cataacccct tggggcctct aaacgggtct tgaggggttt tttgtggtgc
     1261 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     1321 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     1381 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     1441 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     1501 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1561 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1621 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1681 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1741 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1801 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1861 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1921 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1981 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2041 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2101 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2161 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2221 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2281 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2341 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2401 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2461 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2521 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2581 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2641 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2701 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2761 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2821 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2881 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2941 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3001 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3061 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3121 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3181 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3241 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3301 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3361 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3421 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3481 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3541 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3601 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3661 catgattacg ccaagcttgc atgcctgcag gtcgactcta gacaaaaaac ccctcaagac
     3721 ccgtttagag gccccaaggg gttatgctag gatgatggag cccacactct actcgacagg
     3781 atcatcagag tatgtgggtt gacccacata cacatggcaa gagcccacac tctactcgac
     3841 agatgatgcg gatccctatc ttatcttatc tatctcgttt atccctgctt tactgactat
     3901 tgcacagaat agtcagtccc tgcaaccatt atcaccgcca gaggtaaaat tgt
//
