LOCUS       sequence-225098-        5216 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 5216)
  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 5216)
  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..5216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..301
                     /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     224..243
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             344..367
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             374..388
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             1049..1525
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPW*LPADLAWFKRNTLNKPVIMGLQ
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1556..1585
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1632..1649)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1802..1888
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1980..2007
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2027..2118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2119..2187,2188..2979)
                     /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(2337..2356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3150..3738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3639..3658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3892..3909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3924..4064
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3990..4009)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4150..4172
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4328..5206)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc
       61 gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg
      121 taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc
      181 agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta
      241 tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac tgtttctcca
      301 tacccgtttt ttgggctaac aggaggaatt aaccatggca agctggagcc acccgcagtt
      361 cgaaaagggt gcagatgacg acgacaaggt accgcatatg gataaagact gcgaaatgaa
      421 acgcactacc ctggacagcc cgctgggtaa actggaactg agcggttgcg agcagggcct
      481 gcacgaaatc attttcctgg gcaaaggtac gagcgctgcg gatgcggtcg aagtaccggc
      541 tccggctgcg gttctgggtg gcccggagcc gctgatgcag gctaccgcat ggctgaacgc
      601 atattttcac cagccagagg cgattgaaga gttccctgta ccggccctgc atcacccggt
      661 atttcagcag gaatccttta cccgtcaggt actgtggaaa ctgctgaaag tagtgaaatt
      721 cggcgaggtg atctcctatt cccatctggc ggcgctggct ggcaacccag cggcaactgc
      781 ggcggtgaaa accgctctgt ctggtaatcc ggtgccaatt ctgatcccgt gccaccgtgt
      841 ggttcagggc gacctggacg taggtggcta cgaaggcggc ctggctgtga aagaatggct
      901 gctggcgcac gaaggtcacc gtctgggtaa accaggcctg ggcgaattcc ctcctcctcc
      961 gccaccgcca ccaccgccgc cgccgccacc accacctcct ccgcctcctc caccaccacc
     1021 gccaccgcct ccaccacctg gcggccgcat gatcagtctg attgcggcgt tagcggtaga
     1081 tcgcgttatc ggcatggaaa acgccatgcc gtggtagctg cctgccgatc tcgcctggtt
     1141 taaacgcaac accttaaata aacccgtgat tatgggcctc caaacctggg aatcaatcgg
     1201 tcgtccgttg ccaggacgca aaaatattat cctcagcagt caaccgggta cggacgatcg
     1261 cgtaacgtgg gtgaagtcgg tggatgaagc catcgcggcg tgtggtgacg taccagaaat
     1321 catggtgatt ggcggcggtc gcgtttatga acagttcttg ccaaaagcgc aaaaactgta
     1381 tctgacgcat atcgacgcag aagtggaagg cgacacccat ttcccggatt acgagccgga
     1441 tgactgggaa tcggtattca gcgaattcca cgatgctgat gcgcagaact ctcacagcta
     1501 ttgctttgag attctggagc ggcggggagc tccgggcttc tcctcaattt ccgctcatca
     1561 ccaccatcat caccatcacc accactaact cgagagcttg gctgttttgg cggatgagag
     1621 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     1681 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     1741 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     1801 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     1861 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     1921 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     1981 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actctttttg tttatttttc
     2041 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2101 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2161 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2221 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2281 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2341 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     2401 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2461 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2521 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2581 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2641 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2701 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2761 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2821 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2881 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2941 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3001 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3061 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3121 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3181 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3241 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3301 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3361 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3421 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3481 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3541 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3601 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3661 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3721 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3781 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3841 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3901 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3961 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4021 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4081 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4141 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4201 ggcagcagat caattcgcgc gcgaaggcga agcggcatgc ataatgtgcc tgtcaaatgg
     4261 acgaagcagg gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg
     4321 ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga
     4381 actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt
     4441 caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct
     4501 tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc
     4561 ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat
     4621 caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat
     4681 ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa
     4741 gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt
     4801 gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat
     4861 tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa
     4921 cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg
     4981 gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca
     5041 ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga
     5101 taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat
     5161 taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt ttcata
//
