LOCUS       sequence-392918-        6101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Machinery plasmid containing two copies of the Mj pCNFRS and cognate
            tRNA for incorporation of pAzF, pCNF, or pENF at TAG codons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6101)
  AUTHORS   Bednar RM, Jana S, Kuppa S, Franklin R, Beckman J, Antony E, Cooley 
            RB, Mehl RA
  TITLE     Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino
            Acids That Enable Efficient Protein Dual-Labeling in Cells.
  JOURNAL   ACS Chem Biol. 2021 Nov 19;16(11):2612-2622. doi: 
            10.1021/acschembio.1c00649. Epub 2021 Sep 30.
  PUBMED    34590824
REFERENCE   2  (bases 1 to 6101)
  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/acschembio.1c00649"; journalName: "ACS Chem Biol"; date: 
            "2021-11-19- 19"; volume: "16"; issue: "11"; pages: "2612-2622"
COMMENT     Sequence Label: pEVOL-pAzF[TAG]
FEATURES             Location/Qualifiers
     source          1..6101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..299
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     222..241
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     primer_bind     complement(1312..1329)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3232..3891)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3806..3825
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3892..3994)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4386..4403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4520..5065)
                     /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(4600..4619)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5211..6089)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt
       61 cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta
      121 acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag
      181 aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
      241 cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata
      301 cccgtttttt tgggctaaca ggaggaatta gatctatgga cgaatttgaa atgataaaga
      361 gaaacacatc tgaaattatc agcgaggaag agttaagaga ggttttaaaa aaagatgaaa
      421 aatctgctct gataggtttt gaaccaagtg gtaaaataca tttagggcat tatctccaaa
      481 taaaaaagat gattgattta caaaatgctg gatttgatat aattatagtg ttggctgatt
      541 tacatgccta tttaaaccag aaaggagagt tggatgagat tagaaaaata ggagattata
      601 acaaaaaagt ttttgaagca atggggttaa aggcaaaata tgtttatgga agtgaatgga
      661 tgcttgataa ggattataca ctgaatgtct atagattggc tttaaaaact accttaaaaa
      721 gagcaagaag gagtatggaa cttatagcaa gagaggatga aaatccaaag gttgctgaag
      781 ttatctatcc aataatgcag gttaatggtc ctcattatct tggcgttgat gttgcagttg
      841 gagggatgga gcagagaaaa atacacatgt tagcaaggga gcttttacca aaaaaggttg
      901 tttgtattca caaccctgtc ttaacgggtt tggatggaga aggaaagatg agttcttcaa
      961 aagggaattt tatagctgtt gatgactctc cagaagagat tagggctaag ataaagaaag
     1021 catactgccc agctggagtt gttgaaggaa atccaataat ggagatagct aaatacttcc
     1081 ttgaatatcc tttaaccata aaaaggccag aaaaatttgg tggagatttg acagttaata
     1141 gctatgagga gttagagagt ttatttaaaa ataaggaatt gcatccaatg gatttaaaaa
     1201 atgctgtagc tgaagaactt ataaagattt tagagccaat tagaaagaga ttatgatgag
     1261 tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca
     1321 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
     1381 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
     1441 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
     1501 agtcgaaaga ctgggccttg tttgtgagct cccggtcatc aatcatcccc ataatccttg
     1561 ttagattatc aattttaaaa aactaacagt tgtcagcctg tcccgcttta atatcatacg
     1621 ccgttatacg ttgtttacgc tttgaggaat cccatatgga cgaatttgaa atgataaaga
     1681 gaaacacatc tgaaattatc agcgaggaag agttaagaga ggttttaaaa aaagatgaaa
     1741 aatctgctct gataggtttt gaaccaagtg gtaaaataca tttagggcat tatctccaaa
     1801 taaaaaagat gattgattta caaaatgctg gatttgatat aattatagtg ttggctgatt
     1861 tacatgccta tttaaaccag aaaggagagt tggatgagat tagaaaaata ggagattata
     1921 acaaaaaagt ttttgaagca atggggttaa aggcaaaata tgtttatgga agtgaatgga
     1981 tgcttgataa ggattataca ctgaatgtct atagattggc tttaaaaact accttaaaaa
     2041 gagcaagaag gagtatggaa cttatagcaa gagaggatga aaatccaaag gttgctgaag
     2101 ttatctatcc aataatgcag gttaatggtc ctcattatct tggcgttgat gttgcagttg
     2161 gagggatgga gcagagaaaa atacacatgt tagcaaggga gcttttacca aaaaaggttg
     2221 tttgtattca caaccctgtc ttaacgggtt tggatggaga aggaaagatg agttcttcaa
     2281 aagggaattt tatagctgtt gatgactctc cagaagagat tagggctaag ataaagaaag
     2341 catactgccc agctggagtt gttgaaggaa atccaataat ggagatagct aaatacttcc
     2401 ttgaatatcc tttaaccata aaaaggccag aaaaatttgg tggagatttg acagttaata
     2461 gctatgagga gttagagagt ttatttaaaa ataaggaatt gcatccaatg gatttaaaaa
     2521 atgctgtagc tgaagaactt ataaagattt tagagccaat tagaaagaga ttatgactgc
     2581 agtttcaaac gctaaattgc ctgatgcgct acgcttatca ggcctacatg atctctgcaa
     2641 tatattgagt ttgcgtgctt ttgtaggccg gataaggcgt tcacgccgca tccggcaaga
     2701 aacagcaaac aatccaaaac gccgcgttca gcggcgtttt ttctgctttt cttcgcgaat
     2761 taattccgct tcgcaacatg tgagcaccgg tttattgact accggaagca gtgtgaccgt
     2821 gtgcttctca aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga
     2881 cgatatgatc agtgcacggc taactaagcg gcctgctgac tttctcgccg atcaaaaggc
     2941 attttgctat taagggattg acgagggcgt atctgcgcag taagatgcgc cccgcattcc
     3001 ggcggtagtt cagcagggca gaacggcgga ctctaaatcc gcatggcagg ggttcaaatc
     3061 ccctccgccg gaccaaattc gaaaagcctg ctcaacgagc aggctttttt gcatgctcga
     3121 gcagctcagg gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat
     3181 tcaggcgtag caaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc
     3241 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     3301 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     3361 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     3421 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     3481 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     3541 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     3601 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     3661 cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     3721 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     3781 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     3841 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     3901 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     3961 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc
     4021 ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc
     4081 cgtcacaggt atttattcgg cgcaaagtgc gtcgggtgat gctgccaact tactgattta
     4141 gtgtatgatg gtgtttttga ggtgctccag tggcttctgt ttctatcagc tgtccctcct
     4201 gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac cgccggacat cagcgctagc
     4261 ggagtgtata ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt
     4321 ggcaggagaa aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc
     4381 gcttcctcgc tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct
     4441 tacgaacggg gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga
     4501 gggccgcggc aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct
     4561 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4621 ctggcggctc cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc
     4681 tgttatggcc gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc
     4741 tccaagctgg actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt
     4801 aactatcgtc ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact
     4861 ggtaattgat ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag
     4921 gacaagtttt ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc
     4981 tcagagaacc ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat
     5041 tacgcgcaga ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta
     5101 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa
     5161 gttgtaattc tcatgtttga cagcttatca tcgataagct tggtacccaa ttatgacaac
     5221 ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc
     5281 ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga
     5341 ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt
     5401 tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga
     5461 cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc
     5521 aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg tggatggagc
     5581 gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt tatcgccagc
     5641 agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa caggtcgctg
     5701 aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa tattgacggc
     5761 cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg gtgataccat
     5821 tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa cagcaaaata
     5881 tcactcggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg accgcgaatg
     5941 gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa atcgagataa
     6001 ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc
     6061 agcaggggat cattttgcgc ttcagccata cttttcatac t
//
