LOCUS       sequence-358316-        6621 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 6621)
  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 6621)
  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_cyto
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        22..401
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        402..605
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     556..576
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     650..669
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        650..668
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1198..1219
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2384..2401)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2390..2614
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2746..3334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2826..2845)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3663..3710
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3735..3754
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3942..4736)
                     /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(4053..4072)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4663..4682
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4835..4854)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        4933..5124
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5081..5101
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5184..5975,5976..6044))
                     /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     5807..5826
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6045..6149)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6176..6604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6263..6282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6473..6494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cttacaattt acgcgcgcgt tgacattgat tattgactag ttattaatag taatcaatta
       61 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      121 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      181 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      241 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      301 atgacggtaa atgacccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      361 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      421 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      481 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      541 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      601 gaactctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      661 actataggga gacccaagct tggtaccgaa ctcggatcca ctagtaacgg ccgccagtgt
      721 gctggaattc ggcttgggga tatccaccat gtctgcgaac gacaccgtag ttgtgggctc
      781 gatcgagttt acagaaggga ttatcgtcgc aaacatggtg gcagagatga ttgaggcgca
      841 tacagacctt aaggtggttc gcaaactgaa ccttggcggg gagaacgtta actttgaagc
      901 cattaaacgc ggaggtgcga ataatggtat tgacatttac gtggagtaca ctgggcacgg
      961 tcttgtggat attctggggt tcccggagcc gaacgtctat atcaccgccg acaagcagaa
     1021 gaacggcatc aaggcgaact tcaagatccg ccacaacatg gaggacggca gcgtgcagct
     1081 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1141 ccactacctg agcacccagt ccgtgctgag caaagacccc aacgagaagc gcgatcacat
     1201 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1261 gggcggtacc ggagggagca tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1321 cctggtcgag ctggacggcg gcgtaaacgg ccacaagttc agcgtgcgcg gcgagggcga
     1381 gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1441 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1501 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1561 ggagcgcacc atcagcttca aggacgacgg cacctacaag acccgcgccg aggtgaagtt
     1621 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1681 caacatcctg gggcacaagc tggagtacaa ctttccgccg cccagctcta ctgatccaga
     1741 aggtgcatac gaaaccgtga agaaggagta caaacgtaaa tggaatattg tatggctcaa
     1801 accactggga ttcaacaata cgtatacgct taccgttaaa gacgaactgg cgaaacagta
     1861 taaccttaaa accttcagtg acttagcgaa aatctcggat aagctgattc tgggtgcaac
     1921 gatgttcttt ttagaagggc ccgatggtta cccaggcctg caaaaactgt acaatttcaa
     1981 attcaagcac accaaaagca tggacatggg tattcgctat accgccattg ataataacga
     2041 agttcaggta attgatgcct gggccactga tggcttgctg gtgagccaca aattaaaaat
     2101 tctggaggat gataaagagt tcttcccgcc gtattatgct gcccccatca tccgtcagga
     2161 tgtcttagat aagcatcctg aactgaagga cgtgctgaac aaactcgcga atcaaatttc
     2221 actggaagaa atgcagaaac tgaattacaa ggtggacggt gagggtcagg acccagcgaa
     2281 agtagctaag gagtttttga aagagaaagg tttaattctg gatattgatg aactggcgct
     2341 gaaatttgcg ggcctggatc tgtaggcggc cgctcgagat cagcctcgac tgtgccttct
     2401 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2461 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2521 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2581 agcaggcgtg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagtggc
     2641 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2701 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg acgtttttcc ataggctccg
     2761 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2821 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2881 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2941 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3001 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3061 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3121 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3181 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3241 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3301 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3361 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3421 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3481 atatgagtaa cctgaggcta tggcagggcc tgccgccccg acgttggctg cgagccctgg
     3541 gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg cgtattggcc
     3601 ccaatggggt ctcggtgggg tatcgacaga gtgccagccc tgggaccgaa ccccgcgttt
     3661 atgaacaaac gacccaacac cgtgcgtttt attctgtctt tttattgccg tcatagcgcg
     3721 ggttccttcc ggtattgtct ccttccgtgt ttcagttagc ctccccctag ggtgggcgaa
     3781 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     3841 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     3901 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     3961 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     4021 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     4081 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     4141 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cggcgagatc ctcgccgtcg
     4201 ggcttgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     4261 tccagatcat cctgatcgac aagaccagct tccatccgag tacgtgctcg ctcgatgcga
     4321 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     4381 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     4441 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     4501 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt
     4561 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     4621 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     4681 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     4741 aacgatcctc atcctgtctc ttgatcgatc tttgcaaaag cctaggcctc caaaaaagcc
     4801 tcctcactac ttctggaata gctcagaggc cgggcctcgg cctctgcata aataaaaaaa
     4861 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
     4921 ggagttaggg gcgggactat ggttgctgac taattgagat gcttgctttg catacttctg
     4981 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcttgctt
     5041 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
     5101 cacagctggt tctttccgcc tcaggactct tcctttttca ataaatcaat ctaaagtata
     5161 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5221 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5281 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5341 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5401 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5461 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5521 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5581 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5641 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5701 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5761 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5821 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5881 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5941 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6001 gcaaaaaagg gaataagggc gacacggaaa tgttgagtac tcatactctt cctttttcaa
     6061 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
     6181 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     6241 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     6301 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     6361 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     6421 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     6481 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     6541 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     6601 aattttaaca aaatattaac g
//
