LOCUS       sequence-454240-        4236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LS12-responsive riboswitch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4236)
  AUTHORS   Fukunaga K, Teramoto T, Nakashima M, Ohtani T, Katsuki R, Matsuura 
            T, Yokobayashi Y, Kakuta Y
  TITLE     Structural insights into lab-coevolved RNA-RBP pairs and 
            applications of synthetic riboswitches in cell-free system.
  JOURNAL   Nucleic Acids Res. 2025 Mar 20;53(6):gkaf212. doi: 
            10.1093/nar/gkaf212.
  PUBMED    40119732
REFERENCE   2  (bases 1 to 4236)
  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: "Nucleic 
            Acids Res."; date: "2025-03-20"; volume: "53(6):gkaf212. doi"; 
            pages: " 10.1093/nar/gkaf212"
COMMENT     Sequence Label: pIVEX2.3-CS2-3c-ON8-EGFP
FEATURES             Location/Qualifiers
     source          1..4236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     515..534
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        618..636
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             688..696
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             join(729..731,732..734,735..1448)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(729..731,732..734,735..1445)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(774..795)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1035..1054)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1382..1403
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1539..1557)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1553..1600
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1966..1971,1972..1988,1989..1994))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2015..2031)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2015..2031)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2028..2050)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2039..2055
                     /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)."
     promoter        complement(join(2063..2069,2070..2087,2088..2093))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2246..2263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2417..3005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2497..2516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3176..3967,3968..4036))
                     /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     3799..3818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4037..4141)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4209..4227
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc aaggagatgg
      421 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      481 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      541 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      601 tctcgatccc gcgaaattaa tacgactcac tataggcaga gaacgaaagt tccatgacgc
      661 ctctttgatg cgacccggtt atcgaattaa ggaggtaaaa aatgcaagtg aactttcgat
      721 ctcagcccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      781 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      841 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      901 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      961 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1021 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1081 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1141 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1201 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1261 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1321 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1381 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1441 acaagtaata aaagggcgaa ttccagcaca ctggcggccg ttactagtgg atccggctgc
     1501 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     1561 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     1621 cggatatcca caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta
     1681 gcgaagcgag caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtg
     1741 cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc
     1801 tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
     1861 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
     1921 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
     1981 tgataagctg tcaaacatga gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt
     2041 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2101 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2161 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2221 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2281 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2341 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2401 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2461 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2521 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2581 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2641 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2701 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2761 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2821 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2881 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2941 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3001 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3061 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3121 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3181 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3241 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3301 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3361 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3421 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3481 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3541 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3601 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3661 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3721 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3781 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3841 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3901 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3961 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4021 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4081 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4141 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4201 cctataaaaa taggcgtatc acgaggccct ttcgtc
//
