LOCUS       sequence-325939-        5016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sMHT-67.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5016)
  AUTHORS   Fulk EM, Huh D, Atkinson JT, Lie M, Masiello CA, Silberg JJ
  TITLE     A Split Methyl Halide Transferase AND Gate That Reports by 
            Synthesizing an Indicator Gas.
  JOURNAL   ACS Synth Biol. 2020 Nov 20;9(11):3104-3113. doi: 
            10.1021/acssynbio.0c00355. Epub 2020 Oct 26.
  PUBMED    33104325
REFERENCE   2  (bases 1 to 5016)
  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/acssynbio.0c00355"; journalName: "ACS Synth Biol"; date: 
            "2020-11-20- 20"; volume: "9"; issue: "11"; pages: "3104-3113"
COMMENT     Sequence Label: pEMF035
FEATURES             Location/Qualifiers
     source          1..5016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(572..666)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(687..1346)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1261..1280
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1515..1533
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1554..1572,1573..1578,1579..1595,1596..1601,
                     1602..1627)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1554..1572
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1579..1597
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2044..2115
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2131..2158
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2276..2989)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             2996..3007
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        join(3091..3109,3110..3115,3116..3132,3133..3138,
                     3139..3164)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    3091..3109
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3116..3134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        3960..3994
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             4029..4655
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4105..4124)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      4661..4747
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(join(4912..5016,1..484))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4992..5011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
       61 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      121 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      181 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      241 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      301 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
      361 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      421 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      481 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      541 ttaagggatt ttggtcatga ctagtgcttg gattctcacc aataaaaaac gcccggcggc
      601 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
      661 ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt
      721 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
      781 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
      841 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
      901 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
      961 tcaccataac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     1021 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     1081 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga
     1141 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     1201 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     1261 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     1321 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     1381 tcaaaaaata cgaccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     1441 ggccggccaa cgtctcattt tcgccagata tcgacgtcta agaaaccatt attatcatga
     1501 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcacctc gagtccctat
     1561 cagtgataga gattgacatc cctatcagtg atagagatac tgagcacatc agcaggacgc
     1621 actgaccgat aggacccata caaaggaagt tttgatgagc accgtggcga acattgcgcc
     1681 ggtgtttacc ggcgattgca aaaccattcc gaccccggaa gaatgcgcga cctttctgta
     1741 taaagtggtg aacagcggcg gctgggaaaa atgctgggtg gaagaagtga ttccgtggga
     1801 tctgggcgtg ccgaccccgc tggtgctgca tctggtgaaa aacaacgcgc tgccggcggc
     1861 cgcgggtggc ggtggctctg gaggtggtgg gtccatgaac gaaaaagaag aactgaaatc
     1921 taaaaaagcg gaactgcgca accgcatcga acagctgaaa cagaaacgcg aacagctgaa
     1981 acagaaaatc gcgaacctgc gcaaagaaat cgaagcgtat aaataataat ctagaccagg
     2041 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     2101 tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt
     2161 tatagtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt
     2221 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt catcatttgt
     2281 acagttcatc cataccatgc gtgatgcccg ctgcggttac gaactccagc agaaccatat
     2341 gatcgcgttt ctcgttcgga tctttagaca gaacgctttg cgtgctcaga tagtgattgt
     2401 ctggcagcag aacaggacca tcaccgattg gagtgttttg ctggtagtga tcagccagct
     2461 gcacgctgcc atcctccacg ttgtggcgaa ttttaaaatt cgctttaatg ccattttttt
     2521 gtttatcggc ggtgatgtaa acattgtggc tgttaaaatt gtattccagc ttatggccca
     2581 ggatattgcc gtcttcttta aagtcaatgc ctttcagctc aatgcggttt accagggtat
     2641 cgccttcaaa tttcacttcc gcacgcgttt tgtacgtgcc gtcatcctta aaggaaatcg
     2701 tgcgttcctg cacatagcct tccggcatgg cggacttgaa gaagtcatgc tgcttcatat
     2761 ggtccggata acgagcaaag cactgaacac cataagtcag cgtcgttacc agagtcggcc
     2821 aaggtaccgg cagtttacca gtagtacaga tgaacttcag cgtcagttta ccattagttg
     2881 cgtcaccttc accctcgcca cgcacggaaa acttatgacc gttgacatca ccatccagtt
     2941 ccaccagaat agggacgaca ccagtgaaca gctcttcgcc tttacgcatc tagtatttct
     3001 cctctttttc ttaagcatta tctctgatga ggcgggtaat tcaaagggag taagaatgat
     3061 tggctatata ggggaagaga ctctaccggt tccctatcag tgatagagat tgacatccct
     3121 atcagtgata gagatactga gcacatcagc aggacgcact gaccaactaa cgaaaacgtc
     3181 acacggagca ggttattaaa tgtctatcgc ggccaccctg gaaaacgact tagcacgcct
     3241 tgagaatgaa aatgctcgtc tggagaaaga tatcgcgaat ctcgagcgtg acctggcaaa
     3301 attagagcgt gaggaagctt atttcggtgg cggtggctct ggaggtggtg ggtccgcggc
     3361 cgcaaacggc aaaggcctgg tgccgggctg cggcggcggc tatgatgtgg tggcgatggc
     3421 gaacccggaa cgctttatgg tgggcctgga tattagcgaa aacgcgctga aaaaagcgcg
     3481 cgaaaccttt agcaccatgc cgaacagcag ctgctttagc tttgtgaaag aagatgtgtt
     3541 tacctggcgc ccggaacagc cgtttgattt tatttttgat tatgtgtttt tttgcgcgat
     3601 tgatccgaaa atgcgcccgg cgtggggcaa agcgatgtat gaactgctga aaccggatgg
     3661 cgaactgatt accctgatgt atccgattac caaccatgaa ggcggcccgc cgtttagcgt
     3721 gagcgaaagc gaatatgaaa aagtgctggt gccgctgggc tttaaacagc tgagcctgga
     3781 agattatagc gatctggcgg tggaaccgcg caaaggcaaa gaaaaactgg cgcgctggaa
     3841 aaaaatgaac aactaataat aatctaacta aaaaggcctc ccaaatcggg gggccttttt
     3901 tattgataac aaaaggtacc cgtggatcct ctagagtcga cctgcaggca tgcaagcttt
     3961 ttacggctag ctcagtccta ggtatagtgc tagcccagcc agagaaacac tctttaacag
     4021 ggggtagtat gatgtctcgt ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     4081 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag
     4141 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca
     4201 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     4261 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     4321 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     4381 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgcag
     4441 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     4501 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     4561 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat
     4621 tagaaaaaca acttaaatgt gaaagtgggt cttaaggcat caaataaaac gaaaggctca
     4681 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     4741 gacaaatccg ccgccctaga cctagggcgt tcggctgcgg cgagcggtat cagctcactc
     4801 aaaggcggta atacggttat ccacagaatc aggggataac gcatgcaaga acatgtgagc
     4861 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4921 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4981 gacaggacta taaagatacc aggcgtttcc ccctgg
//
