LOCUS       sequence-370981-        3449 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 3449)
  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 3449)
  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: JBL6411
FEATURES             Location/Qualifiers
     source          1..3449
                     /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(188..190,191..904)
                     /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             188..901
                     /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(218..246)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     841..861
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             924..953
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             969..986
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1042..1059)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1212..1258
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1411..2070
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1477..1496)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2091..2185
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2592..2609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2726..3271)
                     /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(2806..2825)
                     /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 gatttgaggc tcctagtcct ttgccactct tttttgaggt gcaaagtgtt
      181 aaaaaaaatg agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      241 agatggtgat gttaatgggc acaaattttc tgtccgtgga gagggtgaag gtgatgctac
      301 aaacggaaaa ctcaccctta aatttatttg cactactgga aaactacctg ttccgtggcc
      361 aacacttgtc actactctga cctatggtgt tcaatgcttt tcccgttatc cggatcacat
      421 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
      481 atctttcaaa gatgacggga cctacaagac gcgtgctgaa gtcaagtttg aaggtgatac
      541 ccttgttaat cgtatcgagt taaagggtat tgattttaaa gaagatggaa acattcttgg
      601 acacaaactc gagtacaact ttaactcaca caatgtatac atcacggcag acaaacaaaa
      661 gaatggaatc aaagctaact tcaaaattcg ccacaacgtt gaagatggtt ccgttcaact
      721 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
      781 ccattacctg tcgacacaat ctgtcctttc gaaagatccc aacgaaaagc gtgaccacat
      841 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa
      901 ataaggatct gaagcttggg cccgaacaaa aactcatctc agaagaggat ctgaatagcg
      961 ccgtcgacca tcatcatcat catcattgag tttaaacggt ctccagcttg gctgttttgg
     1021 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     1081 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     1141 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     1201 ctgccaggca tcaaataaaa cgaaaggctc agtagaaaga ctgggccttt cgttttatct
     1261 gttgtttgtc ggtgaactgg atccttactc gagtctagac tgcagttgat cgggcacgta
     1321 agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta
     1381 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     1441 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     1501 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     1561 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     1621 ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     1681 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     1741 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     1801 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     1861 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     1921 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     1981 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2041 tactgcgatg agtggcaggg cggggcgtaa tttgatatcg agctcgcttg gactcctgtt
     2101 gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg
     2161 ccgggcgttt tttattggtg agaatccaag cctccgatca acgtctcatt ttcgccaaaa
     2221 gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg
     2281 atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg ccaacttact
     2341 gatttagtgt atgatggtgt ttttgaggtg ctccagtggc ttctgtttct atcagctgtc
     2401 cctcctgttc agctactgac ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc
     2461 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
     2521 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
     2581 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
     2641 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
     2701 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
     2761 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
     2821 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
     2881 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
     2941 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
     3001 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
     3061 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
     3121 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
     3181 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
     3241 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
     3301 tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     3361 atataagttg taattctcat gtttgacagc ttatcatcga taagcttccg atggcgcgcc
     3421 gagaggcttt acactttatg cttccggct
//
