LOCUS       sequence-477495-        4444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of iS-methadoneSnFR for purification and in 
            vitro assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4444)
  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 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"; 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: pHHM.X513.iS-methadoneSnFR
FEATURES             Location/Qualifiers
     source          1..4444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      414..1002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     903..922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1156..1173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1258..1277
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1258..1276
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1308..1330
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1341..1358
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1362..1388
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     1853..1874
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2985..3014
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3083..3101)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3097..3144
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3241..3696
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3328..3347)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3538..3559
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3722..3826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3827..3895,3896..4444,1..243)
                     /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(4045..4064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
       61 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
      121 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
      181 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
      241 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
      301 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
      361 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      421 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      481 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      541 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
      601 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      661 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      721 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      781 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      841 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      901 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      961 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     1021 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1081 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     1141 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     1201 ccgcctctcc ccgcgcgttg gccgattcat taatgcagga tctcgatccc gcgaaattaa
     1261 tacgactcac tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt
     1321 aagaaggaga tatacatatg catcatcatc atcatcatgg ttatccctat gatgttccag
     1381 attatgctgg ggcccagccg gccagatctg cgaacgacac cgtagttgtg ggctcgatcg
     1441 tgtttacaga agggattatc gtcgcaaaca tggtggcaga gatgattgag gcgcatacag
     1501 accttaaggt ggttcgcaaa ctgaaccttg gcggggagaa cgttaacttt gaagccatta
     1561 aacgcggagg tgcgaataat ggtattgaca tttacgtgga gtacactggg cacggtcttg
     1621 tggatattct ggggttcccg gagccgaacg tctatatcac cgccgacaag cagaagaacg
     1681 gcatcaaggc gaacttcaag atccgccaca acatggagga cggcagcgtg cagctcgccg
     1741 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1801 acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1861 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg
     1921 gtaccggagg gagcatgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1981 tcgagctgga cggcggcgta aacggccaca agttcagcgt gcgcggcgag ggcgagggcg
     2041 atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2101 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2161 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2221 gcaccatcag cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg
     2281 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2341 tcctggggca caagctggag tacaactttc cgccgcccag ctctactgat ccagaaggtg
     2401 catacgaaac cgtgaagaag gagtacaaac gtaaatggaa tattgtatgg ctcaaaccac
     2461 tgggattcaa caatacgtat acgcttaccg ttaaagacga actggcgaaa cagtataacc
     2521 ttaaaacctt cagtgactta gcgaaaatct cggataagct gattctgggt gcaacgatgt
     2581 tctttttaga agggcccgat ggttacccag gcctgcaaaa actgtacaat ttcaaattca
     2641 agcacaccaa aagcatggac atgggtattc gctataccgc cattgataat aacgaagttc
     2701 aggtaattga tgccttcgcc actgatggct tgctggtgag ccacaaatta aaaattctgg
     2761 aggatgataa agcgttcttc ccgccgtatt atgctgcccc catcatccgt caggatgtct
     2821 tagataagca tcctgaactg aaggacgtgc tgaacaaact cgcgaatcaa atttcagcgg
     2881 aagaaatgca gaaactgaat tacaaggtgg acggtgaggg tcaggaccca gcgaaagtag
     2941 ctaaggagtt tttgaaagag aaaggtttaa ttctgcaggt cgacgaacaa aaactcatct
     3001 cagaagagga tctgaattaa gaattcgaag cttgatccgg ctgctaacaa agcccgaaag
     3061 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3121 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatc tggcgtaata
     3181 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     3241 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3301 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3361 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3421 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3481 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccatgttc tttaatagtg
     3541 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3601 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3661 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     3721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3781 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgctttgatc gttgggaacc
     4321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4441 aata
//
