LOCUS       sequence-349859-        5910 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 5910)
  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 5910)
  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: pAAV9-hSyn iCyt_BrEt_SnFR_ER
FEATURES             Location/Qualifiers
     source          1..5910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   76..205
                     /label=AAV2 ITR
     promoter        287..734
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     primer_bind     1261..1282
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1385..1406)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_feature    2423..3011
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2476..2496)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2894..2905)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3014..3030)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3015..3031)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3060..3181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3071..3090)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3125..3144
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3343..3472
                     /label=AAV2 ITR
     rep_origin      complement(3709..4297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3789..3808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4468..5259,5260..5328))
                     /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     5091..5110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5329..5433)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5459..5910,1..4))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5596..5617)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5808..5827
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
       61 tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
      121 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
      181 gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct
      241 acgtagccat gctctaggaa gatctctgca gagggccctg cgtatgagtg caagtgggtt
      301 ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc gacccactgg
      361 acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg gggaggggaa
      421 acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca gtgccttcgc
      481 ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg acgtcactcg
      541 ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc gccgccggcc
      601 cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac catctgcgct
      661 gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt
      721 gcctgagagc gcagtcgaat tcggcttggg gatatccacc atgctgctat ccgtgccgct
      781 gctgctcggc ctcctcggcc tggccatcgc cagatctgcg aacgacaccg tagttgtggg
      841 ctcgatcatc tttacagaag ggattatcgt cgcaaacatg gtggcagaga tgattgaggc
      901 gcatacagac cttaaggtgg ttcgcaaact gaaccttggc gggcagggtg ttaactttga
      961 agccattaaa cgcggaggtg cgaataatgg tattgacatt tacgtggagt acactgggca
     1021 cggtcttgtg gatattctgg ggttcccgga gccgaacgtc tatatcaccg ccgacaagca
     1081 gaagaacggc atcaaggcga acttcaagat ccgccacaac gtggaggacg gcagcgtgca
     1141 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1201 caaccactac ctgagcaccc agtccgtgct gagcaaagac cccaacgaga agcgcgatca
     1261 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1321 caagggcggt accggaggga gcatgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1381 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgc gcggcgaggg
     1441 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1501 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1561 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1621 ccaggagcgc accatcagct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa
     1681 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1741 cggcaacatc ctggggcaca agctggagta caactttccg ccgcccgcga ctactgatcc
     1801 agaaggtgca tacgaaaccg tgaagaagga gtacaaacgt aaatggaata ttgtatggct
     1861 caaaccactg ggattcaaca atacgtatac gcttaccgtt aaagacgaac tggcgaaaca
     1921 gtataacctt aaaaccttca gtgacttagc gaaaatctcg gataagctga ttctgggtgc
     1981 aacgatgtgg tttttagaag ggcccgatgg ttacccaggc ctgcaaaaac tgtacaattt
     2041 caaattcaag cacaccaaaa gcatggacat gggtattcgc tataccgcca ttgataataa
     2101 cgaagttcag gtaattgatg cctgggccac tgatggcttg ctggtgagcc acaaattaaa
     2161 aattctggag gatgataaag cgttcttccc gccgtattat gctgccccca tcatccgtca
     2221 ggatgtctta gataagcatc ctgaactgaa ggacgtgctg aacaaactcg cgaatcaaat
     2281 ttcactggaa gaaatgcaga aactgaatta caaggtggac ggtgagggtc aggacccagc
     2341 gaaagtagct aaggagtttt tgaaagagaa aggtttaatt ctgcagacag cagaaaaaga
     2401 tgagttgtga aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     2461 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     2521 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     2581 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     2641 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     2701 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     2761 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     2821 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     2881 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     2941 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3001 cgcctccccg catcgatacc gtcgacctcg acccgggcgg ccgcttcgag cagacatgat
     3061 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3121 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3181 taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt
     3241 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc ctagagcatg
     3301 gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg
     3361 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3421 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta
     3481 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3541 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3601 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3661 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3721 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3781 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3841 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3901 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3961 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4021 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4081 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4141 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4201 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4261 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4321 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4381 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4441 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4501 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4561 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4621 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4681 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4741 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4801 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4861 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4921 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4981 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5041 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5101 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5161 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5221 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5281 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5341 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5401 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5461 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5521 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5581 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5641 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5701 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5761 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5821 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5881 cccgccgcgc ttaatgcgcc gctacagggc
//
