LOCUS       sequence-229496-        5215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the bacterial expression of the Snifit-MTX with 
            unnatural amino-acid at the position 23 (FRET semi-synthetic 
            biosensor).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5215)
  AUTHORS   Xue L, Prifti E, Johnsson K
  TITLE     A General Strategy for the Semisynthesis of Ratiometric Fluorescent 
            Sensor Proteins with Increased Dynamic Range.
  JOURNAL   J Am Chem Soc. 2016 Apr 27;138(16):5258-61. doi: 
            10.1021/jacs.6b03034. Epub 2016 Apr 15.
  PUBMED    27071001
REFERENCE   2  (bases 1 to 5215)
  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: 
            "10.1021/jacs.6b03034"; journalName: "J Am Chem Soc"; date: 
            "2016-04-27- 27"; volume: "138"; issue: "16"; pages: "5258-61"
COMMENT     Sequence Label: pBAD-MTX-u23_Snifit
FEATURES             Location/Qualifiers
     source          1..5215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             328..351
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             358..372
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             1033..1509
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPW*LPADLAWFKRNTLNKPVIMGLQ
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1540..1569
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1616..1633)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1786..1872
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1964..1991
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2010..2101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2102..2170,2171..2962)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2320..2339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3133..3721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3622..3641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3875..3892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3907..4047
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3973..3992)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4133..4155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4311..5189)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat ggcaagctgg agccacccgc agttcgaaaa gggtgcagat
      361 gacgacgaca aggtaccgca tatggataaa gactgcgaaa tgaaacgcac taccctggac
      421 agcccgctgg gtaaactgga actgagcggt tgcgagcagg gcctgcacga aatcattttc
      481 ctgggcaaag gtacgagcgc tgcggatgcg gtcgaagtac cggctccggc tgcggttctg
      541 ggtggcccgg agccgctgat gcaggctacc gcatggctga acgcatattt tcaccagcca
      601 gaggcgattg aagagttccc tgtaccggcc ctgcatcacc cggtatttca gcaggaatcc
      661 tttacccgtc aggtactgtg gaaactgctg aaagtagtga aattcggcga ggtgatctcc
      721 tattcccatc tggcggcgct ggctggcaac ccagcggcaa ctgcggcggt gaaaaccgct
      781 ctgtctggta atccggtgcc aattctgatc ccgtgccacc gtgtggttca gggcgacctg
      841 gacgtaggtg gctacgaagg cggcctggct gtgaaagaat ggctgctggc gcacgaaggt
      901 caccgtctgg gtaaaccagg cctgggcgaa ttccctcctc ctccgccacc gccaccaccg
      961 ccgccgccgc caccaccacc tcctccgcct cctccaccac caccgccacc gcctccacca
     1021 cctggcggcc gcatgatcag tctgattgcg gcgttagcgg tagatcgcgt tatcggcatg
     1081 gaaaacgcca tgccgtggta gctgcctgcc gatctcgcct ggtttaaacg caacacctta
     1141 aataaacccg tgattatggg cctccaaacc tgggaatcaa tcggtcgtcc gttgccagga
     1201 cgcaaaaata ttatcctcag cagtcaaccg ggtacggacg atcgcgtaac gtgggtgaag
     1261 tcggtggatg aagccatcgc ggcgtgtggt gacgtaccag aaatcatggt gattggcggc
     1321 ggtcgcgttt atgaacagtt cttgccaaaa gcgcaaaaac tgtatctgac gcatatcgac
     1381 gcagaagtgg aaggcgacac ccatttcccg gattacgagc cggatgactg ggaatcggta
     1441 ttcagcgaat tccacgatgc tgatgcgcag aactctcaca gctattgctt tgagattctg
     1501 gagcggcggg gagctccggg cttctcctca atttccgctc atcaccacca tcatcaccat
     1561 caccaccact aactcgagag cttggctgtt ttggcggatg agagaagatt ttcagcctga
     1621 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     1681 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     1741 gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     1801 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     1861 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     1921 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     1981 gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat
     2041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2341 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2641 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3061 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3241 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3901 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     3961 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4021 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4081 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4141 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca gatcaattcg
     4201 cgcgcgaagg cgaagcggca tgcataatgt gcctgtcaaa tggacgaagc agggattctg
     4261 caaaccctat gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac
     4321 ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc
     4381 ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga
     4441 ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt
     4501 tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga
     4561 cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc
     4621 aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc
     4681 gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc
     4741 agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg
     4801 aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc
     4861 cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat
     4921 tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata
     4981 tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg
     5041 gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa
     5101 ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc
     5161 agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt cagag
//
