LOCUS       sequence-229503-        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 51 (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-u51_Snifit
FEATURES             Location/Qualifiers
     source          1..5216
                     /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="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGLQ
                     TWESI*RPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             1540..1569
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(1617..1634)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1787..1873
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1965..1992
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2011..2102
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2103..2171,2172..2963)
                     /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(2321..2340)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3134..3722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3623..3642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3876..3893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3908..4048
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3974..3993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4134..4156
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4312..5190)
                     /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 tgccgtggaa cctgcctgcc gatctcgcct ggtttaaacg caacacctta
     1141 aataaacccg tgattatggg cctccaaacc tgggaatcaa tctagcgtcc 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 tttggcggat gagagaagat tttcagcctg
     1621 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
     1681 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
     1741 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     1801 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     1861 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     1921 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     1981 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
     2041 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2101 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2161 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2221 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2281 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2341 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2401 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2461 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2521 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2581 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2641 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2701 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2761 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2821 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2881 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2941 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3001 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3061 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3241 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3901 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     3961 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4021 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4081 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4141 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc
     4201 gcgcgcgaag gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct
     4261 gcaaacccta tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa
     4321 cttgacggct acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc
     4381 cccggtgcat tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg
     4441 accgacggtg gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg
     4501 ttggtcctcg cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag
     4561 acgcgacggc gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc
     4621 caggtgatcg ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag
     4681 cgactcgtta atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag
     4741 cagctccgaa tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct
     4801 gaaatgcggc tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg
     4861 ccagttaagc cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca
     4921 ttcgcgagcc tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat
     4981 atcacccggt cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat
     5041 ggtgagattg agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata
     5101 accgttggcc tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg
     5161 cagcagggga tcattttgcg cttcagccat acttttcata ctcccgccat tcagag
//
