LOCUS       sequence-216039-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the bacterial expression of the red-shifted LUCID 
            semi-synthetic BRET biosensor for methotrexate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  AUTHORS   Hiblot J, Yu Q, Sabbadini MDB, Reymond L, Xue L, Schena A, Sallin O,
            Hill N, Griss R, Johnsson K
  TITLE     Luciferases with Tunable Emission Wavelengths.
  JOURNAL   Angew Chem Int Ed Engl. 2017 Nov 13;56(46):14556-14560. doi: 
            10.1002/anie.201708277. Epub 2017 Oct 9.
  PUBMED    28941028
REFERENCE   2  (bases 1 to 7759)
  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: 
            "10.1002/anie.201708277"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2017-11-13- 13"; volume: "56"; issue: "46"; pages: 
            "14556-14560"
COMMENT     Sequence Label: pET51b(+)-LUCIDRS-MTX
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(582..1373,1374..1442))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1205..1224
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1443..1535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1616..2204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2105..2124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2358..2375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2390..2532
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2458..2477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2618..2640
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2634..2825)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3397..4479)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4441..4460
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4480..4557)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4763..4782
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4870..4889
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4870..4888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4889..4913
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             4928..4950
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4966..4989
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4996..5010
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             7621..7650
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(7673..7691)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7687..7734
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
      481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     4921 aaataatttt gtttaacttt aagaaggaga tataccatgg caagctggag ccacccgcag
     4981 ttcgaaaagg gtgcagatga cgacgacaag gtaccaatgg acaaggattg tgaaatgaaa
     5041 cgcaccacac tggacagccc tttggggaag ctggagctgt ctggttgtga gcagggtctg
     5101 cacgaaataa tttttctggg caaggggacg tctgcagctg atgccgtgga ggtcccagcc
     5161 cccgctgcgg ttctcggagg tccggagccc ctgatgcagg ctacagcctg gctgaatgcc
     5221 tatttccacc agcccgaggc tatcgaagag ttccccgtgc cggctcttca ccatcccgtt
     5281 ttccagcaag agtcgttcac cagacaggtg ttatggaagc tgctgaaggt tgtgaaattc
     5341 ggagaagtga tttcttactc tcacttagca gccctggcag gcaaccccgc agccacggca
     5401 gcagtgaaga cggcactgag tggcaatcct gtccctatcc tgatcccgtg ccacagagtg
     5461 gtccaggggg atttggatgt gggcggttac gagggtggac tggccgtgaa ggaatggctt
     5521 ctggcccatg aaggccaccg gttggggaag ccaggcttgg gacctcctcc tccgccaccg
     5581 ccaccaccgc cgccgccgcc accaccagga ggatctggcg gatctcctcc tccgcctcct
     5641 ccaccaccac cgccaccgcc tccaccacct cctgccgatc tcgcctggtt taaacgcaac
     5701 accttaaata aacccgtgat tatgggccgc catacctggg aatcaatcgg tcgtccgttg
     5761 ccaggacgca aaaatattat cctcagcagt caaccgggta cggacgatcg cgtaacgtgg
     5821 gtgaagtcgg tggatgaagc catcgcggcg tgtggtgacg taccagaaat catggtgatt
     5881 ggcggcggtc gcgtttatga acagttcttg ccaaaagcgc aaaaactgta tctgacgcat
     5941 atcgacgcag aagtggaagg cgacacccat ttcccggatt acgagccgga tgactgggaa
     6001 tcggtattca gcgaattcca cgatgctgat gcgcagaact ctcacagcta ttgctttgag
     6061 attctggagc ggcggggcgg cggcggcggc atgatcagtc tgattgcggc gttagcggta
     6121 gatcgcgtta tcggcatgga aaacgccatg ccgtggaacg gaatcggtac tggctttcca
     6181 ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg
     6241 cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc
     6301 aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg
     6361 ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat
     6421 gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc
     6481 gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg
     6541 gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc
     6601 caggccttcc gcaccagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg
     6661 taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac
     6721 ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc
     6781 gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag
     6841 cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc
     6901 tggcggctgt gcgaacgcat tctggcgggt ggcaccggtg gctctggcgg taccggtggc
     6961 tctatggtct tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac
     7021 ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc
     7081 gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat
     7141 gtcatcatcc cgtatgaagg tctggacgtc ggccgcaagc tgatcatcga tcagaacgtt
     7201 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     7261 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     7321 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     7381 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     7441 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     7501 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     7561 cgctggctgt ctactctgga gatttccggt ggagctccgg gcttctcctc aatttccgct
     7621 catcaccacc atcatcacca tcaccaccac taattaacct aggctgctgc caccgctgag
     7681 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     7741 ggaggaacta tatccggat
//
