LOCUS       sequence-454242-        4239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LS4-responsive riboswitch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4239)
  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 4239)
  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-CS1M2b-3c-ON8-EGFP
FEATURES             Location/Qualifiers
     source          1..4239
                     /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             691..699
                     /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(732..734,735..737,738..1451)
                     /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(732..734,735..737,738..1448)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(777..798)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1038..1057)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1385..1406
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1542..1560)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1556..1603
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1969..1974,1975..1991,1992..1997))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2018..2034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2018..2034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2031..2053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2042..2058
                     /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(2066..2072,2073..2090,2091..2096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2111..2132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2249..2266)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2420..3008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2500..2519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3179..3970,3971..4039))
                     /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     3802..3821
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4040..4144)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4212..4230
                     /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 tataggcaaa gaaaggcgaa agccttgtga
      661 ggcctctttg atgcgacccg gttatcgaat taaggaggta aaaaatgcaa gtcaaggctt
      721 tggcctctct catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      781 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      841 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      901 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
      961 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1021 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1081 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1141 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1201 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1261 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1321 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1381 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1441 tgtacaagta ataaaagggc gaattccagc acactggcgg ccgttactag tggatccggc
     1501 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     1561 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1621 atccggatat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa
     1681 gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct ccgagaacgg
     1741 gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag tgactggcga
     1801 tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata accaagccta
     1861 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     1921 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
     1981 cgatgataag ctgtcaaaca tgagaattcg taatcatgtc atagctgttt cctgtgtgaa
     2041 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2101 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2161 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2221 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2281 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2341 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2821 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3001 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3061 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3121 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3181 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3241 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3301 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3361 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3421 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3481 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3541 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3601 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3661 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3721 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3781 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3841 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3901 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3961 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4021 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4081 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4141 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4201 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
