LOCUS       sequence-246049-        6654 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 6654)
  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 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: "J Gen 
            Physiol. 2019 Feb 4. pii: jgp.201812201. doi: 
            10.1085/jgp.201812201."
COMMENT     Sequence Label: pMinDis.X513-iNicSnFR3a-(CC93)-ER
FEATURES             Location/Qualifiers
     source          1..6654
                     /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"
     primer_bind     1236..1257
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2404..2421)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2410..2634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2766..3354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2846..2865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3683..3730
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3755..3774
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3962..4756)
                     /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(4073..4092)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4683..4702
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4855..4874)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        4953..5144
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5101..5121
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             complement(join(5204..5995,5996..6064))
                     /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     5827..5846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6065..6169)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6196..6651
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6283..6302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6493..6514
                     /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 ccaccatgct gctatccgtg ccgctgctgc tcggcctcct cggcctggcc
      781 atcgccagat ctgcgaacga caccgtagtt gtgggctcga tcaactttac agaagggatt
      841 atcgtcgcaa acatggtggc agagatgatt gaggcgcata cagaccttaa ggtggttcgc
      901 aaactgaacc ttggcgggga gaacgttaac tttgaagcca ttaaacgcgg aggtgcgaat
      961 aatggtattg acatttacgt ggagtacact gggcacggtc ttgtggatat tctggggttc
     1021 ccggagccga acgtctatat caccgccgac aagcagaaga acggcatcaa ggcgaacttc
     1081 aagatccgcc acaacatgga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1141 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1201 gtgctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1261 gccgccggga tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg
     1321 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcggc
     1381 gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg gcgatgccac caacggcaag
     1441 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1501 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1561 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cagcttcaag
     1621 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1681 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1741 gagtacaact ttccgccgcc cagctctact gatccagaag gtgcatacga aaccgtgaag
     1801 aaggagtaca aacgtaaatg gaatattgta tggctcaaac cactgggatt caacaatacg
     1861 tatacgctta ccgttaaaga cgaactggcg aaacagtata accttaaaac cttcagtgac
     1921 ttagcgaaaa tctcggataa gctgattctg ggtgcaacga tgttcttttt agaagggccc
     1981 gatggttacc caggcctgca aaaactgtac aatttcaaat tcaagcacac caaaagcatg
     2041 gacatgggta ttcgctatac cgccattgat aataacgaag ttcaggtaat tgatgcctgg
     2101 gccactgatg gcttgctggt gagccacaaa ttaaaaattc tggaggatga taaagcgttc
     2161 ttcccgccgt attatgctgc ccccatcatc cgtcaggatg tcttagataa gcatcctgaa
     2221 ctgaaggacg tgctgaacaa actcgcgaat caaatttcac tggaagaaat gcagaaactg
     2281 aattacaagg tggacggtga gggtcaggac ccagcgaaag tagctaagga gtttttgaaa
     2341 gagaaaggtt taattctgca gacagcagaa aaagatgagt tgtgagcggc cgctcgagat
     2401 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2461 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2521 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2581 gggaggattg ggaagacaat agcaggcgtg ctggggatgc ggtgggctct atggcttctg
     2641 aggcggaaag aaccagtggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2701 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2761 acgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2821 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2881 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2941 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3001 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3061 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3121 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3181 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3241 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3301 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3361 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3421 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3481 tttaaatcaa tctaaagtat atatgagtaa cctgaggcta tggcagggcc tgccgccccg
     3541 acgttggctg cgagccctgg gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa
     3601 gaaacgcggg cgtattggcc ccaatggggt ctcggtgggg tatcgacaga gtgccagccc
     3661 tgggaccgaa ccccgcgttt atgaacaaac gacccaacac cgtgcgtttt attctgtctt
     3721 tttattgccg tcatagcgcg ggttccttcc ggtattgtct ccttccgtgt ttcagttagc
     3781 ctccccctag ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     3841 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     3901 aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg
     3961 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     4021 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     4081 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     4141 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     4201 cggcgagatc ctcgccgtcg ggcttgctcg ccttgagcct ggcgaacagt tcggctggcg
     4261 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccagct tccatccgag
     4321 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     4381 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     4441 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     4501 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     4561 gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     4621 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     4681 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     4741 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcgatc tttgcaaaag
     4801 cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc cgggcctcgg
     4861 cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg
     4921 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat
     4981 gcttgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg
     5041 actaattgag atgcttgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     5101 accctaactg acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca
     5161 ataaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5221 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5281 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5341 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5401 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5461 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5521 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5581 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5641 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5701 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5761 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5821 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5881 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5941 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6001 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgagtac
     6061 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6121 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6181 gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     6241 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     6301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6541 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6601 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tacg
//
