LOCUS       sequence-366968-        3444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sfGFP reporter under the control of Mutant E. coli thiB TPP 
            Riboswitch under the control of a Constitutive promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3444)
  AUTHORS   Berman KE, Steans R, Hertz LM, Lucks JB
  TITLE     A transient intermediate RNA structure underlies the regulatory 
            function of the E. coli thiB TPP translational riboswitch.
  JOURNAL   RNA. 2023 Nov;29(11):1658-1672. doi: 10.1261/rna.079427.122. Epub 
            2023 Jul 7.
  PUBMED    37419663
REFERENCE   2  (bases 1 to 3444)
  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"; doi: 
            "10.1261/rna.079427.122"; journalName: "RNA"; date: "2023-11"; 
            volume: "29"; issue: "11"; pages: "1658-1672"
COMMENT     Sequence Label: JBL6412
FEATURES             Location/Qualifiers
     source          1..3444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             join(183..185,186..899)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             183..896
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(213..241)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     836..856
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             919..948
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             964..981
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1037..1054)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1207..1253
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1406..2065
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1472..1491)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2086..2180
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2587..2604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2721..3266)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2801..2820)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt ccgttctcaa
       61 cggggtgcca cgcgtacgcg tgcgctgaga aaatacccgt cgaacctgat ccggataacg
      121 ccggcgaagg gatttgagga gtcctttgcc actctttttt gaggtgcaaa gtgttaaaaa
      181 aaatgagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg
      241 gtgatgttaa tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat gctacaaacg
      301 gaaaactcac ccttaaattt atttgcacta ctggaaaact acctgttccg tggccaacac
      361 ttgtcactac tctgacctat ggtgttcaat gcttttcccg ttatccggat cacatgaaac
      421 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt
      481 tcaaagatga cgggacctac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg
      541 ttaatcgtat cgagttaaag ggtattgatt ttaaagaaga tggaaacatt cttggacaca
      601 aactcgagta caactttaac tcacacaatg tatacatcac ggcagacaaa caaaagaatg
      661 gaatcaaagc taacttcaaa attcgccaca acgttgaaga tggttccgtt caactagcag
      721 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
      781 acctgtcgac acaatctgtc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc
      841 ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaataag
      901 gatctgaagc ttgggcccga acaaaaactc atctcagaag aggatctgaa tagcgccgtc
      961 gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt tttggcggat
     1021 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     1081 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     1141 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     1201 aggcatcaaa taaaacgaaa ggctcagtag aaagactggg cctttcgttt tatctgttgt
     1261 ttgtcggtga actggatcct tactcgagtc tagactgcag ttgatcgggc acgtaagagg
     1321 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttatcgag
     1381 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     1441 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     1501 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     1561 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     1621 atttcgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     1681 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     1741 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     1801 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     1861 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     1921 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     1981 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     2041 cgatgagtgg cagggcgggg cgtaatttga tatcgagctc gcttggactc ctgttgatag
     2101 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     2161 cgttttttat tggtgagaat ccaagcctcc gatcaacgtc tcattttcgc caaaagttgg
     2221 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt
     2281 ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac ttactgattt
     2341 agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag ctgtccctcc
     2401 tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag
     2461 cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg
     2521 tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
     2581 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc
     2641 ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag
     2701 agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc
     2761 tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2821 cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
     2881 ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg
     2941 ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg
     3001 taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac
     3061 tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa
     3121 ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
     3181 ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga
     3241 ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct
     3301 agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata
     3361 agttgtaatt ctcatgtttg acagcttatc atcgataagc ttccgatggc gcgccgagag
     3421 gctttacact ttatgcttcc ggct
//
