LOCUS       sequence-349062-        6654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Develop a family of genetically encoded fluorescent biosensors for 
            nicotinic agonists, termed iDrugSnFRs, thus enabling optical 
            subcellular pharmacokinetics for nicotinic agonists.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6654)
  AUTHORS   Nichols AL, Blumenfeld Z, Fan C, Luebbert L, Blom AE, Cohen BN, 
            Marvin JS, Borden PM, Kim CH, Muthusamy AK, Shivange AV, Knox HJ, 
            Campello HR, Wang JH, Dougherty DA, Looger LL, Gallagher T, Rees DC,
            Lester HA
  TITLE     Fluorescence activation mechanism and imaging of drug permeation 
            with new sensors for smoking-cessation ligands.
  JOURNAL   eLife. 2022 Jan 4;11. pii: 74648. doi: 10.7554/eLife.74648.
  PUBMED    34982029
REFERENCE   2  (bases 1 to 6654)
  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"; journalName: "eLife."; 
            date: "2022-01-4"; volume: "11. pii: 74648. doi"; pages: " 
            10.7554/eLife.74648"
COMMENT     Sequence Label: pCMV(MinDis)-iDianiSnFR_ER
FEATURES             Location/Qualifiers
     source          1..6654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1228..1249
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2396..2413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2402..2626
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2758..3346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2838..2857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3675..3722
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3747..3766
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3954..4748)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVSGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASKPDGEDLAVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4065..4084)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4675..4694
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4847..4866)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        4945..5136
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5093..5113
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5196..5987,5988..6056))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSTQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6161)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6188..6643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6275..6294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6485..6506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aactctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgaac tcggatccac tagtaacggc cgccagtgtg ctggaattcg gcttggggat
      721 atccaccatg ctgctatccg tgccgctgct gctcggcctc ctcggcctgg ccatcgccag
      781 atctgcgaac gacaccgtag ttgtgggctc gatcgatttt acagaaggga ttatcgtcgc
      841 aaacatggtg gcagagatga ttgaggcgca tacagacctt aaggtggttc gcaaactgaa
      901 ccttggcggg cgtaacgtta actttgaagc cattaaacgc ggaggtgcga ataatggtat
      961 tgacatttac gtggagtaca ctgggtccgg tcttgtggat attctggggt tcccggagcc
     1021 gaacgtctat atcaccgccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg
     1081 ccacaacatg gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1141 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgtgctgag
     1201 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1261 gatcactctc ggcatggacg agctgtacaa gggcggtacc ggagggagca tgagcaaggg
     1321 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg gcgtaaacgg
     1381 ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     1441 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1501 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1561 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcagcttca aggacgacgg
     1621 cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1681 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1741 ctttccgccg cccaactcta ctgatccaga aggtgcatac gaaaccgtga agaaggagta
     1801 caaacgtaaa tggaatattg tatggctcaa accactggga ttcaacaata cgtatacgct
     1861 tggggttaaa gacgaactgg cgaaacagta taaccttaaa accttcagtg acttagcgaa
     1921 aatctcggat aagctgattc tgggtgcaac gatgttcttt ttagaaaatc ccgatggtta
     1981 cccaggcctg caaaaactgt acaatttcaa attcaagcac accaaaagca tggacatggg
     2041 tattcgctat accgccattg ataataacga agttcaggta attgatgcct gggccactga
     2101 tggcttgctg gtgagccaca aattaaaaat tctggaggat gataaagcgt tcttcccgcc
     2161 gtattatgct gcccccatca tccgtcagga tgtcttagat aagcatcctg aactgaagga
     2221 cgtgctgaac aaactcgcga atcaaatttc actggaagaa atgcagaaac tgaattacaa
     2281 ggtggacggt gagggtcagg acccagcgaa agtagctaag gagtttttga aagagaaagg
     2341 tttaattctg cagacagcag aaaaagatga gttgtgagcg gccgctcgag atcagcctcg
     2401 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2461 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2521 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2581 tgggaagaca atggcaggcg tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2641 agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2701 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tgacgttttt
     2761 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2821 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2881 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2941 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3001 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3061 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3121 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3181 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3241 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3301 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3361 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3421 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3481 aatctaaagt atatatgagt aacctgaggc tatggcaggg cctgccgccc cgacgttggc
     3541 tgcgagccct gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg
     3601 ggcgtattgg ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg
     3661 aaccccgcgt ttatgaacaa acgacccaac accgtgcgtt ttattctgtc tttttattgc
     3721 cgtcatagcg cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccct
     3781 agggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc
     3841 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg
     3901 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag
     3961 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
     4021 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
     4081 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
     4141 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacggcgaga
     4201 tcctcgccgt cgggcttgct cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
     4261 tgatgctctt cgtccagatc atcctgatcg acaagaccag cttccatccg agtacgtgct
     4321 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
     4381 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
     4441 aggagatcct gccccgacac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
     4501 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
     4561 tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
     4621 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
     4681 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
     4741 tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa agcctaggcc
     4801 tccaaaaaag cctcctcact acttctggaa tagctcagag gccgggcctc ggcctctgca
     4861 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     4921 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcttgctt
     4981 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
     5041 agatgcttgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
     5101 tgacacacat tccacagctg gttctttccg cctcaggact cttccttttt caataaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgagt actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6181 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     6241 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     6301 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     6361 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     6421 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     6481 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     6541 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     6601 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttacgcgcg cgtt
//
