LOCUS       sequence-130022-        4597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control cobalamin riboswitch reporter vector for use in E. 
            coli; deletion of regulatory kissing loop interaction by conversion 
            of L5 to GAAA tetra loop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4597)
  AUTHORS   Johnson JE Jr, Reyes FE, Polaski JT, Batey RT
  TITLE     B12 cofactors directly stabilize an mRNA regulatory switch.
  JOURNAL   Nature. 2012 Dec 6;492(7427):133-7. doi: 10.1038/nature11607. Epub 
            2012 Oct 14.
  PUBMED    23064232
REFERENCE   2  (bases 1 to 4597)
  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: 
            "10.1038/nature11607"; journalName: "Nature"; date: "2012-12-6- 6"; 
            volume: "492"; issue: "7427"; pages: "133-7"
COMMENT     Sequence Label: pENV8(GAAA)-GFPuv
FEATURES             Location/Qualifiers
     source          1..4597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      128..214
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      306..333
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             627..1343
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(657..685)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1280..1300
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1684..1702)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1770..1874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1875..1943,1944..2735)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2093..2112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2906..3494
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3395..3414
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3648..3665
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3680..3820
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3922..4113)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ctcgggttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
       61 gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc
      121 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
      181 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
      241 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat
      301 taagcagaag gccatcctga cggatggcct ttttgcgcta gccacagcta acaccacgtc
      361 gtccctatct gctgccctag gtctatgagt ggttgctgga taactttacg ggcatgcata
      421 aggctcgtat aatatattca tataataatg gcctaaaagc gtagtgggaa agtgacgtga
      481 aattcgtcca gattgaaaga tacggttata ctccgaatgc cacctaggcc atacaacgag
      541 caaggagact caacatgctg atgattacgc caagcttgca tgcctgcagg tcgactctag
      601 aggatccccg ggtaccggta gaaaaaatga gtaaaggaga agaacttttc actggagttg
      661 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
      721 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
      781 aactacctgt tccatggcca acacttgtca ctactttctc ttatggtgtt caatgctttt
      841 cccgttatcc ggatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt
      901 atgtacagga acgcactata tctttcaaag atgacgggaa ctacaagacg cgtgctgaag
      961 tcaagtttga aggtgatacc cttgttaatc gtatcgagtt aaaaggtatt gattttaaag
     1021 aagatggaaa cattctcgga cacaaactcg agtacaacta taactcacac aatgtataca
     1081 tcacggcaga caaacaaaag aatggaatca aagctaactt caaaattcgc cacaacattg
     1141 aagatggatc cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc
     1201 ctgtcctttt accagacaac cattacctgt cgacacaatc tgccctttcg aaagatccca
     1261 acgaaaagcg tgaccacatg gtccttcttg agtttgtaac tgctgctggg attacacatg
     1321 gcatggatga gctctacaaa taatgaattc caactgagcg ccggtcgcta ccattaccaa
     1381 cttgtctggt gtcaaaaata ataggcctac tagtcggccg tacgggccct ttcgtctcgc
     1441 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     1501 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1561 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     1621 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcggcctta
     1681 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1741 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1801 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1861 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1921 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1981 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2041 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2101 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2161 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2221 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2281 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2341 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2401 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2461 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2521 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2581 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2641 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2701 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2761 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2821 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2881 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2941 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3001 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3061 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3121 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3181 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3241 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3301 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3361 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3421 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3481 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3541 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3601 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3661 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3721 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3781 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3841 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3901 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3961 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4021 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4081 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4141 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4201 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4261 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4321 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4381 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4441 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4501 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4561 cacgatcatg cgcacccgtg gccaggaccc aacgctg
//
