LOCUS       sequence-245438-        6831 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Develop a family of genetically encoded fluorescent biosensors for 
            nicotine, termed iNicSnFRs thus enabling optical subcellular 
            pharmacokinetics for nicotine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6831)
  AUTHORS   Shivange AV, Borden PM, Muthusamy AK, Nichols AL, Bera K, Bao H, 
            Bishara I, Jeon J, Mulcahy MJ, Cohen B, O'Riordan SL, Kim C, 
            Dougherty DA, Chapman ER, Marvin JS, Looger LL, Lester HA
  TITLE     Determining the pharmacokinetics of nicotinic drugs in the 
            endoplasmic reticulum using biosensors.
  JOURNAL   J Gen Physiol. 2019 Feb 4. pii: jgp.201812201. doi: 
            10.1085/jgp.201812201.
  PUBMED    30718376
REFERENCE   2  (bases 1 to 6831)
  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 Gen 
            Physiol. 2019 Feb 4. pii: jgp.201812201. doi: 
            10.1085/jgp.201812201."
COMMENT     Sequence Label: pMinDis.X513-iNicSnFR3b-(V7)-PM
FEATURES             Location/Qualifiers
     source          1..6831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..388
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..592
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     543..563
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     637..656
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        637..655
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     736..795
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     primer_bind     1248..1269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2380..2409
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2413..2559
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(2581..2598)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2587..2811
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2943..3531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3023..3042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3860..3907
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3932..3951
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4139..4933)
                     /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
                     GLAPAELFARLKASKPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4250..4269)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4860..4879
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5032..5051)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        5130..5321
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5278..5298
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5381..6172,6173..6241))
                     /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     6004..6023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6242..6346)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6373..6828
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6460..6479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6670..6691
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cgcgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
       61 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      121 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      181 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      241 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      301 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      361 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      421 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      481 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      541 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagaa ctctctggct
      601 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      661 ccaagcttgg taccgaactc ggatccacta gtaacggccg ccagtgtgct ggaattcggc
      721 ttggggatat ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
      781 ccaggttcca ctggtgacag atctgcgaac gacaccgtag ttgtgggctc gatcgagttt
      841 acagaaggga ttatcgtcgc aaacatggtg gcagagatga ttgaggcgca tacagacctt
      901 aaggtggttc gcaaactgaa ccttggcggg gagaacgtta actttgaagc cattaaacgc
      961 ggaggtgcga ataatggtat tgacatttac gtggagtaca ctgggcacgg tcttgtggat
     1021 attctggggt tcccggagcc gaacgtctat atcaccgccg acaagcagaa gaacggcatc
     1081 aaggcgaact tcaagatccg ccacaacatg gaggacggca gcgtgcagct cgccgaccac
     1141 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1201 agcacccagt ccgtgctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1261 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcggtacc
     1321 ggagggagca tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1381 ctggacggcg gcgtaaacgg ccacaagttc agcgtgcgcg gcgagggcga gggcgatgcc
     1441 accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1501 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1561 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1621 atcagcttca aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac
     1681 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1741 gggcacaagc tggagtacaa ctttccgccg cccagctcta ctgatccaga aggtgcatac
     1801 gaaaccgtga agaaggagta caaacgtaaa tggaatattg tatggctcaa accactggga
     1861 ttcaacaata cgtatacgct taccgttaaa gacgaactgg cgaaacagta taaccttaaa
     1921 accttcagtg acttagcgaa aatctcggat aagctgattc tgggtgcaac gatgttcttt
     1981 ttagaagggc ccgatggtta cccaggcctg caaaaactgt acaatttcaa attcaagcac
     2041 accaaaagca tggacatggg tattcgctat accgccattg ataataacga agttcaggta
     2101 attgatgcct gggccactga tggcttgctg gtgagccaca aattaaaaat tctggaggat
     2161 gataaagcgt tcttcccgcc gtattatgct gcccccatca tccgtcagga tgtcttagat
     2221 aagcatcctg aactgaagga cgtgctgaac aaactcgcga atcaaatttc actggaagaa
     2281 atgcagaaac tgaattacaa ggtggacggt gagggtcagg acccagcgaa agtagctaag
     2341 gagtttttga aagagaaagg tttaattctg caggtcgacg aacaaaaact catctcagaa
     2401 gaggatctga atgctgtggg ccaggacacg caggaggtca tcgtggtgcc acactccttg
     2461 ccctttaagg tggtggtgat ctcagccatc ctggccctgg tggtgctcac catcatctcc
     2521 cttatcatcc tcatcatgct ttggcagaag aagccacgtt aggcggccgc tcgagatcag
     2581 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     2641 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     2701 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     2761 aggattggga agacaatagc aggcgtgctg gggatgcggt gggctctatg gcttctgagg
     2821 cggaaagaac cagtggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2881 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2941 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3001 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3061 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3121 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3181 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3241 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3301 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3361 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3421 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3481 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3541 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3601 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3661 aaatcaatct aaagtatata tgagtaacct gaggctatgg cagggcctgc cgccccgacg
     3721 ttggctgcga gccctgggcc ttcacccgaa cttggggggt ggggtgggga aaaggaagaa
     3781 acgcgggcgt attggcccca atggggtctc ggtggggtat cgacagagtg ccagccctgg
     3841 gaccgaaccc cgcgtttatg aacaaacgac ccaacaccgt gcgttttatt ctgtcttttt
     3901 attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc agttagcctc
     3961 cccctagggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     4021 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa
     4081 tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc
     4141 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     4201 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     4261 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     4321 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     4381 cgagatcctc gccgtcgggc ttgctcgcct tgagcctggc gaacagttcg gctggcgcga
     4441 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     4501 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     4561 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     4621 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     4681 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     4741 ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     4801 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     4861 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     4921 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcgatcttt gcaaaagcct
     4981 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccgg gcctcggcct
     5041 ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag
     5101 ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgct
     5161 tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
     5221 aattgagatg cttgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     5281 ctaactgaca cacattccac agctggttct ttccgcctca ggactcttcc tttttcaata
     5341 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5401 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5461 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5521 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5581 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5641 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5701 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5761 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5821 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5881 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5941 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6001 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6061 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6121 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6181 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6241 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6301 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6361 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6421 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     6481 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6541 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     6601 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6661 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     6721 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6781 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttac g
//
