LOCUS       sequence-245420-        4444 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 4444)
  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 4444)
  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: pHHM.X513-iNicSnFR3b-(V7)
FEATURES             Location/Qualifiers
     source          1..4444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             103..120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             124..150
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     615..636
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1747..1776
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1845..1863)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1859..1906
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2003..2458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2090..2109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2300..2321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2484..2588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2589..2657,2658..3449)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRFDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2807..2826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3620..4208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4109..4128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4362..4379
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
       61 agaaataatt ttgtttaact ttaagaagga gatatacata tgcatcatca tcatcatcat
      121 ggttatccct atgatgttcc agattatgct ggggcccagc cggccagatc tgcgaacgac
      181 accgtagttg tgggctcgat cgagtttaca gaagggatta tcgtcgcaaa catggtggca
      241 gagatgattg aggcgcatac agaccttaag gtggttcgca aactgaacct tggcggggag
      301 aacgttaact ttgaagccat taaacgcgga ggtgcgaata atggtattga catttacgtg
      361 gagtacactg ggcacggtct tgtggatatt ctggggttcc cggagccgaa cgtctatatc
      421 accgccgaca agcagaagaa cggcatcaag gcgaacttca agatccgcca caacatggag
      481 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      541 gtgctgctgc ccgacaacca ctacctgagc acccagtccg tgctgagcaa agaccccaac
      601 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      661 atggacgagc tgtacaaggg cggtaccgga gggagcatga gcaagggcga ggagctgttc
      721 accggggtgg tgcccatcct ggtcgagctg gacggcggcg taaacggcca caagttcagc
      781 gtgcgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc
      841 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      901 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      961 cccgaaggct acgtccagga gcgcaccatc agcttcaagg acgacggcac ctacaagacc
     1021 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1081 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt tccgccgccc
     1141 agctctactg atccagaagg tgcatacgaa accgtgaaga aggagtacaa acgtaaatgg
     1201 aatattgtat ggctcaaacc actgggattc aacaatacgt atacgcttac cgttaaagac
     1261 gaactggcga aacagtataa ccttaaaacc ttcagtgact tagcgaaaat ctcggataag
     1321 ctgattctgg gtgcaacgat gttcttttta gaagggcccg atggttaccc aggcctgcaa
     1381 aaactgtaca atttcaaatt caagcacacc aaaagcatgg acatgggtat tcgctatacc
     1441 gccattgata ataacgaagt tcaggtaatt gatgcctggg ccactgatgg cttgctggtg
     1501 agccacaaat taaaaattct ggaggatgat aaagcgttct tcccgccgta ttatgctgcc
     1561 cccatcatcc gtcaggatgt cttagataag catcctgaac tgaaggacgt gctgaacaaa
     1621 ctcgcgaatc aaatttcact ggaagaaatg cagaaactga attacaaggt ggacggtgag
     1681 ggtcaggacc cagcgaaagt agctaaggag tttttgaaag agaaaggttt aattctgcag
     1741 gtcgacgaac aaaaactcat ctcagaagag gatctgaatt aagaattcga agcttgatcc
     1801 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1861 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1921 tatatccgga tctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     1981 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2281 gagtccatgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2461 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2521 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2581 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2641 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2701 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2761 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2821 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2881 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2941 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3001 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3061 ctcgctttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3121 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3181 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3241 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3301 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3361 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3421 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3481 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3541 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3601 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3661 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3721 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3781 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3841 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3901 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3961 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4021 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4081 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4141 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4201 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4261 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4321 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4381 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4441 gcag
//
