LOCUS       sequence-366983-        3454 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 3454)
  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 3454)
  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: JBL6427
FEATURES             Location/Qualifiers
     source          1..3454
                     /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(193..195,196..909)
                     /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             193..906
                     /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(223..251)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     846..866
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             929..958
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             974..991
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1047..1064)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1217..1263
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1416..2075
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1482..1501)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2096..2190
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2597..2614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2731..3276)
                     /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(2811..2830)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt ccgtggagta
       61 cggggtgcca cgcgtacgcg tgcgctgaga aaatacccgt cgaacctgat ccggataacg
      121 ccggcgaagg gattactctc tccttctcaa gtcctttgcc actctttttt gaggtgcaaa
      181 gtgttaaaaa aaatgagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
      241 gaattagatg gtgatgttaa tgggcacaaa ttttctgtcc gtggagaggg tgaaggtgat
      301 gctacaaacg gaaaactcac ccttaaattt atttgcacta ctggaaaact acctgttccg
      361 tggccaacac ttgtcactac tctgacctat ggtgttcaat gcttttcccg ttatccggat
      421 cacatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc
      481 actatatctt tcaaagatga cgggacctac aagacgcgtg ctgaagtcaa gtttgaaggt
      541 gatacccttg ttaatcgtat cgagttaaag ggtattgatt ttaaagaaga tggaaacatt
      601 cttggacaca aactcgagta caactttaac tcacacaatg tatacatcac ggcagacaaa
      661 caaaagaatg gaatcaaagc taacttcaaa attcgccaca acgttgaaga tggttccgtt
      721 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
      781 gacaaccatt acctgtcgac acaatctgtc ctttcgaaag atcccaacga aaagcgtgac
      841 cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc
      901 tacaaataag gatctgaagc ttgggcccga acaaaaactc atctcagaag aggatctgaa
      961 tagcgccgtc gaccatcatc atcatcatca ttgagtttaa acggtctcca gcttggctgt
     1021 tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt
     1081 ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     1141 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta
     1201 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtag aaagactggg cctttcgttt
     1261 tatctgttgt ttgtcggtga actggatcct tactcgagtc tagactgcag ttgatcgggc
     1321 acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg
     1381 agttatcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     1441 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     1501 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     1561 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     1621 ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     1681 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     1741 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     1801 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     1861 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     1921 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     1981 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     2041 aacagtactg cgatgagtgg cagggcgggg cgtaatttga tatcgagctc gcttggactc
     2101 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     2161 tgccgccggg cgttttttat tggtgagaat ccaagcctcc gatcaacgtc tcattttcgc
     2221 caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg
     2281 aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac
     2341 ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag
     2401 ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca ccgccggaca
     2461 tcagcgctag cggagtgtat actggcttac tatgttggca ctgatgaggg tgtcagtgaa
     2521 gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat atgtgataca
     2581 ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
     2641 cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca
     2701 gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca
     2761 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     2821 ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg
     2881 tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
     2941 aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg
     3001 ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg
     3061 cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc
     3121 taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa
     3181 gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
     3241 gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata
     3301 aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     3361 atacgatata agttgtaatt ctcatgtttg acagcttatc atcgataagc ttccgatggc
     3421 gcgccgagag gctttacact ttatgcttcc ggct
//
