LOCUS       sequence-225097-        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 36 (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-u36_Snifit
FEATURES             Location/Qualifiers
     source          1..5216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      34..61
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        81..172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(173..241,242..1033)
                     /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(391..410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1204..1792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1693..1712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1946..1963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1978..2118
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2044..2063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2204..2226
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2382..3260)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3287..3571
                     /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     3494..3513
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3614..3637
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3644..3658
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             4319..4795
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNT*NKPVIMGLQ
                     TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             4826..4855
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(4902..4919)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5072..5158
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
       61 ttgcgtttct acaaactctt tttgtttatt tttctaaata cattcaaata tgtatccgct
      121 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      181 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      241 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      301 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      361 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
      421 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
      481 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
      541 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
      601 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
      661 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
      721 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
      781 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
      841 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
      901 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
      961 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1021 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1081 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1321 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1561 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1621 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1681 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1741 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1801 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     1921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     1981 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     2041 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     2101 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     2161 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     2221 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag
     2281 gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta
     2341 tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct
     2401 acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat
     2461 tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg
     2521 gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
     2581 cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc
     2641 gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
     2701 ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta
     2761 atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa
     2821 tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc
     2881 tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc
     2941 cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
     3001 tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt
     3061 cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg
     3121 agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
     3181 tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga
     3241 tcattttgcg cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt
     3301 ccatattgca tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa
     3361 ccggtaaccc cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa
     3421 acgcgtaaca aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc
     3481 gtcacacttt gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc
     3541 tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac aggaggaatt
     3601 aaccatggca agctggagcc acccgcagtt cgaaaagggt gcagatgacg acgacaaggt
     3661 accgcatatg gataaagact gcgaaatgaa acgcactacc ctggacagcc cgctgggtaa
     3721 actggaactg agcggttgcg agcagggcct gcacgaaatc attttcctgg gcaaaggtac
     3781 gagcgctgcg gatgcggtcg aagtaccggc tccggctgcg gttctgggtg gcccggagcc
     3841 gctgatgcag gctaccgcat ggctgaacgc atattttcac cagccagagg cgattgaaga
     3901 gttccctgta ccggccctgc atcacccggt atttcagcag gaatccttta cccgtcaggt
     3961 actgtggaaa ctgctgaaag tagtgaaatt cggcgaggtg atctcctatt cccatctggc
     4021 ggcgctggct ggcaacccag cggcaactgc ggcggtgaaa accgctctgt ctggtaatcc
     4081 ggtgccaatt ctgatcccgt gccaccgtgt ggttcagggc gacctggacg taggtggcta
     4141 cgaaggcggc ctggctgtga aagaatggct gctggcgcac gaaggtcacc gtctgggtaa
     4201 accaggcctg ggcgaattcc ctcctcctcc gccaccgcca ccaccgccgc cgccgccacc
     4261 accacctcct ccgcctcctc caccaccacc gccaccgcct ccaccacctg gcggccgcat
     4321 gatcagtctg attgcggcgt tagcggtaga tcgcgttatc ggcatggaaa acgccatgcc
     4381 gtggaacctg cctgccgatc tcgcctggtt taaacgcaac acctagaata aacccgtgat
     4441 tatgggcctc caaacctggg aatcaatcgg tcgtccgttg ccaggacgca aaaatattat
     4501 cctcagcagt caaccgggta cggacgatcg cgtaacgtgg gtgaagtcgg tggatgaagc
     4561 catcgcggcg tgtggtgacg taccagaaat catggtgatt ggcggcggtc gcgtttatga
     4621 acagttcttg ccaaaagcgc aaaaactgta tctgacgcat atcgacgcag aagtggaagg
     4681 cgacacccat ttcccggatt acgagccgga tgactgggaa tcggtattca gcgaattcca
     4741 cgatgctgat gcgcagaact ctcacagcta ttgctttgag attctggagc ggcggggagc
     4801 tccgggcttc tcctcaattt ccgctcatca ccaccatcat caccatcacc accactaact
     4861 cgagagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca
     4921 gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca
     4981 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct
     5041 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     5101 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     5161 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggac
//
