LOCUS       sequence-358312-        6633 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 6633)
  AUTHORS   Nichols AL, Blumenfeld Z, Luebbert L, Knox HJ, Muthusamy AK, Marvin 
            JS, Kim CH, Grant SN, Walton DP, Cohen BN, Hammar R, Looger L, 
            Artursson P, Dougherty DA, Lester HA
  TITLE     Selective Serotonin Reuptake Inhibitors within Cells: Temporal 
            Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane.
  JOURNAL   J Neurosci. 2023 Mar 2:JN-RM-1519-22. doi: 
            10.1523/JNEUROSCI.1519-22.2022.
  PUBMED    36868853
REFERENCE   2  (bases 1 to 6633)
  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: "J Neurosci.
            2023 Mar 2:JN-RM-1519-22. doi: 10.1523/JNEUROSCI.1519-22.2022."
COMMENT     Sequence Label: pCMV(MinDis)-iEscSnFR_ER
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     678..697
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        678..696
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1277..1298
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2445..2462)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2451..2675
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2807..3395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2887..2906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3724..3771
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3796..3815
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4003..4797)
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASKPDGEDLAVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4114..4133)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4724..4743
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4896..4915)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        4973..5164
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5121..5141
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5224..6015,6016..6084))
                     /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     5847..5866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6085..6189)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6216..6633,1..38)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6303..6322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6513..6534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 taacgcgaat tttaacaaaa tattaacgct tacaatttac gcgcgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga actctctggc taactagaga acccactgct
      661 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgaact
      721 cggatccact agtaacggcc gccagtgtgc tggaattcgg cttggggata tccaccatgc
      781 tgctatccgt gccgctgctg ctcggcctcc tcggcctggc catcgccaga tctgcgaacg
      841 acaccgtagt tgtgggctcg atcgagttta cagaagggat tatcgtcgca aacatggtgg
      901 cagagatgat tgaggcgcat acagacctta aggtggttcg caaactgaac cttggcgggg
      961 agaacgttaa ctttgaagcc attaaacgcg gaggtgcgaa taatggtatt gacatttacg
     1021 tggagtacac tgggcacggt cttgtggata ttctggggtt cccggagccg aacgtctata
     1081 tcaccgccga caagcagaag aacggcatca aggcgaactt caagatccgc cacaacatgg
     1141 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1201 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgtgctgagc aaagacccca
     1261 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1321 gcatggacga gctgtacaag ggcggtaccg gagggagcat gagcaagggc gaggagctgt
     1381 tcaccggggt ggtgcccatc ctggtcgagc tggacggcgg cgtaaacggc cacaagttca
     1441 gcgtgcgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct
     1501 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1561 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1621 tgcccgaagg ctacgtccag gagcgcacca tcagcttcaa ggacgacggc acctacaaga
     1681 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1741 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tttccgccgc
     1801 ccagctctac tgatccagaa ggtgcatacg aaaccgtgaa gaaggagtac aaacgtaaat
     1861 ggaatattgt atggctcaaa ccactgggat tcaacaatac gtatacgctt accgttaaag
     1921 acgaactggc gaaacagtat aaccttaaaa ccttcagtga cttagcgaaa atctcggata
     1981 agctgattct gggtgcaacg atgttctttt tagaagggcc cgatggttac ccaggcctgc
     2041 aaaaactgta caatttcaaa ttcaagcaca ccaaaagcat ggacatgggt attcgctata
     2101 ccgccattga taataacgaa gttcaggtaa ttgatgcctg ggccactgat ggcttgctgg
     2161 tgagccacaa attaaaaatt ctggaggatg ataaagagtt cttcccgccg tattatgctg
     2221 cccccatcat ccgtcaggat gtcttagata agcatcctga actgaaggac gtgctgaaca
     2281 aactcgcgaa tcaaatttca ctggaagaaa tgcagaaact gaattacaag gtggacggtg
     2341 agggtcagga cccagcgaaa gtagctaagg agtttttgaa agagaaaggt ttaattctgc
     2401 agacagcaga aaaagatgag ttgtgagcgg ccgctcgaga tcagcctcga ctgtgccttc
     2461 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2521 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2581 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2641 tagcaggcgt gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagtgg
     2701 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2761 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct gacgtttttc cataggctcc
     2821 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2881 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2941 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3001 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3061 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3121 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3181 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3241 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3301 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3361 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3421 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3541 tatatgagta acctgaggct atggcagggc ctgccgcccc gacgttggct gcgagccctg
     3601 ggccttcacc cgaacttggg gggtggggtg gggaaaagga agaaacgcgg gcgtattggc
     3661 cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga accccgcgtt
     3721 tatgaacaaa cgacccaaca ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc
     3781 gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccta gggtgggcga
     3841 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     3901 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     3961 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     4021 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     4081 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     4141 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     4201 ttccaccatg atattcggca agcaggcatc gccatgggtc acggcgagat cctcgccgtc
     4261 gggcttgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     4321 gtccagatca tcctgatcga caagaccagc ttccatccga gtacgtgctc gctcgatgcg
     4381 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     4441 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     4501 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     4561 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag
     4621 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     4681 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     4741 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     4801 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
     4861 ctcctcacta cttctggaat agctcagagg ccgggcctcg gcctctgcat aaataaaaaa
     4921 aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggactat
     4981 ggttgctgac taattgagat gcttgctttg catacttctg cctgctgggg agcctgggga
     5041 ctttccacac ctggttgctg actaattgag atgcttgctt tgcatacttc tgcctgctgg
     5101 ggagcctggg gactttccac accctaactg acacacattc cacagctggt tctttccgcc
     5161 tcaggactct tcctttttca ataaatcaat ctaaagtata tatgagtaaa cttggtctga
     5221 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5281 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5341 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5401 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5461 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5521 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5581 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5641 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5701 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5761 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5821 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5881 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5941 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6001 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6061 gacacggaaa tgttgagtac tcatactctt cctttttcaa tattattgaa gcatttatca
     6121 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6181 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
     6241 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     6301 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     6361 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     6421 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     6481 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     6541 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     6601 ttggttaaaa aatgagctga tttaacaaaa att
//
