LOCUS       sequence-392659-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Machinery plasmid expressing constitutive and inducible 7HCou 
            synthetase and cognate amber suppressing tRNA. Incorporates 
            (7-hydroxy-4-coumarin-yl) ethylglycine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6271)
  AUTHORS   Dangerfield TL, Johnson KA
  TITLE     Optimized incorporation of an unnatural fluorescent amino acid 
            affords measurement of conformational dynamics governing 
            high-fidelity DNA replication.
  JOURNAL   J Biol Chem. 2020 Dec 11;295(50):17265-17280. doi: 
            10.1074/jbc.RA120.015557. Epub 2020 Oct 5.
  PUBMED    33020184
REFERENCE   2  (bases 1 to 6271)
  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: 
            "10.1074/jbc.RA120.015557"; journalName: "J Biol Chem"; date: 
            "2020-12-11- 11"; volume: "295"; issue: "50"; pages: "17265-17280"
COMMENT     Sequence Label: pSUP-(2xCouRS-tRNAopt)
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      526..612
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      704..731
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(825..841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(825..841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(942..959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1076..1621)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1156..1175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3735..4394)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4309..4328
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4395..4497)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(5029..5742)
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFNKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     RBS             5831..5839
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 tccaaaggtt gctgaagtta tctatccaat aatgcaggtt aatgggattc attatggtgg
       61 cgttgatgtt gcagttggag ggatggagca gagaaaaata cacatgttag caagggagct
      121 tttaccaaaa aaggttgttt gtattcacaa ccctgtctta acgggtttgg atggagaagg
      181 aaagatgagt tcttcaaaag ggaattttat agctgttgat gactctccag aagagattag
      241 ggctaagata aagaaagcat actgcccagc tggagttgtt gaaggaaatc caataatgga
      301 gatagctaaa tacttccttg aatatccttt aaccataaaa aggccagaaa aatttggtgg
      361 agatttgaca gttaatagct atgaggagtt agagagttta tttaaaaata aggaattgca
      421 tccaatggat ttaaaaaatg ctgtagctga agaacttata aagattttag agccaattag
      481 aaagagatta taactgaatt catgcagtag ggaactgcca ggcatcaaat aaaacgaaag
      541 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
      601 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
      661 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
      721 gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat
      781 gtatccgctc atgagacaat agatctaagc ttggcgtaat catggtcata gctgtttcct
      841 gggatccgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg
      901 gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
      961 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     1021 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     1081 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     1141 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
     1201 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
     1261 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
     1321 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1381 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
     1441 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
     1501 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgtc
     1561 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     1621 agaagatcat cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc
     1681 aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat gtttgacagc
     1741 ttatcatcga taagcttggt accgagctcc cggtcatcaa tcatccccat aatccttgtt
     1801 agattatcaa ttttaaaaaa ctaacagttg tcagcctgtc ccgctttaat atcatacgcc
     1861 gttatacgtt gtttacgctt tgaggaatcc catatggacg aatttgaaat gataaagaga
     1921 aacacatctg aaattatcag cgaggaagag ttaagagagg ttttaaaaaa agatgaaaaa
     1981 tctgctgaga taggttttga accaagtggt aaaatacatt tagggcatta tctccaaata
     2041 aaaaagatga ttgatttaca aaatgctgga tttgatataa ttatacattt gggtgattta
     2101 ggcgcctatt taaaccagaa aggagagttg gatgagatta gaaaaatagg agattataac
     2161 aaaaaagttt ttgaagcaat ggggttaaag gcaaaatatg tttatggaag tgaatatcat
     2221 cttgataagg attatacact gaatgtctat agattggctt taaaaactac cttaaaaaga
     2281 gcaagaagga gtatggaact tatagcaaga gaggatgaaa atccaaaggt tgctgaagtt
     2341 atctatccaa taatgcaggt taatgggatt cattatggtg gcgttgatgt tgcagttgga
     2401 gggatggagc agagaaaaat acacatgtta gcaagggagc ttttaccaaa aaaggttgtt
     2461 tgtattcaca accctgtctt aacgggtttg gatggagaag gaaagatgag ttcttcaaaa
     2521 gggaatttta tagctgttga tgactctcca gaagagatta gggctaagat aaagaaagca
     2581 tactgcccag ctggagttgt tgaaggaaat ccaataatgg agatagctaa atacttcctt
     2641 gaatatcctt taaccataaa aaggccagaa aaatttggtg gagatttgac agttaatagc
     2701 tatgaggagt tagagagttt atttaaaaat aaggaattgc atccaatgga tttaaaaaat
     2761 gctgtagctg aagaacttat aaagatttta gagccaatta gaaagagatt ataactgcag
     2821 tttcaaacgc taaattgcct gatgcgctac gcttatcagg cctacatgat ctctgcaata
     2881 tattgagttt gcgtgctttt gtaggccgga taaggcgttc acgccgcatc cggcaagaaa
     2941 cagcaaacaa tccaaaacgc cgcgttcagc ggcgtttttt ctgcttttct tcgcgaatta
     3001 attccgcttc gcaacatgtg agcaccggtt tattgactac cggaagcagt gtgatgaacc
     3061 acggaacaca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     3121 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     3181 tcctggccag atctaagcgg cctgctgact ttctcgccga tcaaaaggca ttttgctatt
     3241 aagggattga cgagggcgta tctgcgcagt aagatgcgcc ccgcattgtg gtaccaattc
     3301 tttttaagaa ttgatggtat ccggcggtag ttcagcaggg cagaacggcg gactctaaat
     3361 ccgcatggca ggggttcaaa tcccctccgc cggaccaatt ttgaaccccg cttcggcggg
     3421 gttttttgtt ttctgtacat ttcgtcaccc tcccttcgca ataaacgccc gtaatatgac
     3481 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     3541 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     3601 aaacgtctgc gacctgagca acaacatgaa tggtcttgcc gtgtcgacga atttctgcca
     3661 ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg gcaccaataa
     3721 ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta
     3781 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
     3841 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
     3901 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     3961 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     4021 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     4081 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     4141 catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg
     4201 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     4261 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     4321 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     4381 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
     4441 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
     4501 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
     4561 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
     4621 gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc
     4681 tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag
     4741 caccgccgga catcagcgct agcggagtgt ataactagtg tgcaatgtaa catcagagat
     4801 tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg actccccgcg
     4861 cggacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4921 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccataga
     4981 cgtctttgaa ttgttatcag ctatgcgccg accagaacac cttgccgatc agccaaacgt
     5041 ctcttcaggc cactgactag cgataacttt ccccacaacg gaacaactct cattgcatgg
     5101 gatcattggg tactgtgggt ttagtggttg taaaaacacc tgaccgctat ccctgatcag
     5161 tttattgaag gtaaactcat cacccccaag tctggctatg cagaaatcac ctggctcaac
     5221 agcctgctca gggtcaacga gaattaacat tccgtcagga aagcttggct tggagcctgt
     5281 tggtgcggtc atggaattac cttcaacctc aagccagaat gcagaatcac tggctttttt
     5341 ggttgtgctt acccatctct ccgcatcacc tttggtaaag gttctaagct caggtgagaa
     5401 catccctgcc tgaacatgag aaaaaacagg gtactcatac tcacttctaa gtgacggctg
     5461 catactaacc gcttcataca tctcgtagat ttctctggcg attgaagggc taaattcttc
     5521 aacgctaact ttgagaattt ttgtaagcaa tgcggcgtta taagcattta atgcattgat
     5581 gccattaaat aaagcaccaa cgcctgactg ccccatcccc atcttgtctg cgacagattc
     5641 ctgggataag ccaagttcat ttttcttttt ttcataaatt gctttaaggc gacgtgcgtc
     5701 ctcaagctgc tcttgtgtta atggtttctt ttttgtgctc atacgttaaa tctatcaccg
     5761 caagggataa atatctaaca ccgtgcgtgt tgactatttt acctctggcg gtgataatgg
     5821 ttgcatgtac taaggaggtt catatggacg aatttgaaat gataaagaga aacacatctg
     5881 aaattatcag cgaggaagag ttaagagagg ttttaaaaaa agatgaaaaa tctgctgaga
     5941 taggttttga accaagtggt aaaatacatt tagggcatta tctccaaata aaaaagatga
     6001 ttgatttaca aaatgctgga tttgatataa ttatacattt gggtgattta ggcgcctatt
     6061 taaaccagaa aggagagttg gatgagatta gaaaaatagg agattataac aaaaaagttt
     6121 ttgaagcaat ggggttaaag gcaaaatatg tttatggaag tgaatatcat cttgataagg
     6181 attatacact gaatgtctat agattggctt taaaaactac cttaaaaaga gcaagaagga
     6241 gtatggaact tatagcaaga gaggatgaaa a
//
