LOCUS       sequence-477504-        6135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian cell expression for subcellular pharmacokinetics with 
            Golgi targeting of iS-methadoneSnFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6135)
  AUTHORS   Muthusamy AK, Kim CH, Virgil SC, Knox HJ, Marvin JS, Nichols AL, 
            Cohen BN, Dougherty DA, Looger LL, Lester HA
  TITLE     Three Mutations Convert the Selectivity of a Protein Sensor from 
            Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and 
            Biofluids.
  JOURNAL   J Am Chem Soc. 2022 May 18;144(19):8480-8486. doi: 
            10.1021/jacs.2c02323. Epub 2022 Apr 21.
  PUBMED    35446570
REFERENCE   2  (bases 1 to 6135)
  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"; doi: 
            "10.1021/jacs.2c02323"; journalName: "J Am Chem Soc"; date: 
            "2022-05-18- 18"; volume: "144"; issue: "19"; pages: "8480-8486"
COMMENT     Sequence Label: pTwist-iS-methadoneSnFR-Golgi
FEATURES             Location/Qualifiers
     source          1..6135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     1..60
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     primer_bind     777..798
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1909..1938
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1994..2115
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2005..2024)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2059..2078
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      2230..2276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2306..2442
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2487..2558
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2784..3372)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2864..2883)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(4118..4186,4187..4978)
                     /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     complement(4336..4355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      4991..5127
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5279..5350
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5355..5401)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    5419..5467
                     /note="synthetic polyadenylation signal"
     enhancer        5548..5851
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5853..6055
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6006..6026
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
       61 gacagatcta ggcttcggga gccgctcctg agcggcagcg ccgcgatgcc aggcgcgtcc
      121 ctacagcggg cctgccgcct gctcgtggcc gtctgcgctc tgcaccttgg cgtcaccctc
      181 gtttactacc tggctggccg cgacctgagc cgcctgcccc aactggtcgg agtctccaca
      241 ccgctgcagg gcggctcgaa cagtgccgcc gccatcgggc agtcctccgg ggagctccgg
      301 accggagggg ccaaggatcc accggtcgcc accgcgaacg acaccgtagt tgtgggctcg
      361 atcgtgttta cagaagggat tatcgtcgca aacatggtgg cagagatgat tgaggcgcat
      421 acagacctta aggtggttcg caaactgaac cttggcgggg agaacgttaa ctttgaagcc
      481 attaaacgcg gaggtgcgaa taatggtatt gacatttacg tggagtacac tgggcacggt
      541 cttgtggata ttctggggtt cccggagccg aacgtctata tcaccgccga caagcagaag
      601 aacggcatca aggcgaactt caagatccgc cacaacatgg aggacggcag cgtgcagctc
      661 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
      721 cactacctga gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg
      781 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
      841 ggcggtaccg gagggagcat gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      901 ctggtcgagc tggacggcgg cgtaaacggc cacaagttca gcgtgcgcgg cgagggcgag
      961 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1021 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1081 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1141 gagcgcacca tcagcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc
     1201 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1261 aacatcctgg ggcacaagct ggagtacaac tttccgccgc ccagctctac tgatccagaa
     1321 ggtgcatacg aaaccgtgaa gaaggagtac aaacgtaaat ggaatattgt atggctcaaa
     1381 ccactgggat tcaacaatac gtatacgctt accgttaaag acgaactggc gaaacagtat
     1441 aaccttaaaa ccttcagtga cttagcgaaa atctcggata agctgattct gggtgcaacg
     1501 atgttctttt tagaagggcc cgatggttac ccaggcctgc aaaaactgta caatttcaaa
     1561 ttcaagcaca ccaaaagcat ggacatgggt attcgctata ccgccattga taataacgaa
     1621 gttcaggtaa ttgatgcctt cgccactgat ggcttgctgg tgagccacaa attaaaaatt
     1681 ctggaggatg ataaagcgtt cttcccgccg tattatgctg cccccatcat ccgtcaggat
     1741 gtcttagata agcatcctga actgaaggac gtgctgaaca aactcgcgaa tcaaatttca
     1801 gcggaagaaa tgcagaaact gaattacaag gtggacggtg agggtcagga cccagcgaaa
     1861 gtagctaagg agtttttgaa agagaaaggt ttaattctgc aggtcgacga acaaaaactc
     1921 atctcagaag aggatctgaa ttaggctagc ttgactgact gagatacagc gtaccttcag
     1981 ctcacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2041 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2101 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     2161 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtattgg cccatctcta
     2221 tcggtatcgt agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgtgcc
     2281 cctcgggccg gattgctatc taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct
     2341 gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc cccaacttgc
     2401 ccacttccat acgtgtcctc cttaccagaa atttatcctt aaggtcgtca gctatcctgc
     2461 aggcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg acaccactag
     2521 gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt accttgggct
     2581 atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg tctgggtttc
     2641 gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg cacccgttct
     2701 ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2761 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2821 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2881 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2941 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3001 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3061 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3121 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3181 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3241 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3301 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3361 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3421 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3481 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3541 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3601 atagttgcat ttaaatttcc gaactctcca aggccctcgt cggaaaatct tcaaaccttt
     3661 cgtccgatcc atcttgcagg ctacctctcg aacgaactat cgcaagtctc ttggccggcc
     3721 ttgcgccttg gctattgctt ggcagcgcct atcgccaggt attactccaa tcccgaatat
     3781 ccgagatcgg gatcacccga gagaagttca acctacatcc tcaatcccga tctatccgag
     3841 atccgaggaa tatcgaaatc ggggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg
     3901 agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc
     3961 aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt
     4021 taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa
     4081 catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac atttccgtgt
     4141 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4201 ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca tcgaactgga
     4261 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc caatgatgag
     4321 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4381 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga
     4441 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4501 tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc
     4561 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4621 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacctt
     4681 gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt tgatagactg
     4741 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4801 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4861 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4921 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaacc
     4981 gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc
     5041 ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg
     5101 tcctccttac cagaaattta tccttaagat cccgaatcgt ttaaactcga ctctggctct
     5161 atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg
     5221 catcgaatct cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca
     5281 tcttggacca ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca
     5341 gctacctctc aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct
     5401 atcaatcgtt gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga
     5461 ttttattatc taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt
     5521 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5581 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5641 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5701 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5761 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5821 catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg
     5881 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     5941 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     6001 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
     6061 agtgaaccgt cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg
     6121 gccgctgcca agctt
//
