LOCUS       sequence-436932-        5363 bp    DNA     circular SYN 15-APR-2025
DEFINITION  encodes EcTyrRS-pAAFRS-9 and EcTyr-tRNA-TAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5363)
  AUTHORS   Ficaretta ED, Singha Roy SJ, Voss L, Chatterjee A
  TITLE     Native Aminoacyl-tRNA Synthetase/tRNA Pair Drives Highly Efficient 
            Noncanonical Amino Acid Incorporation in Escherichia coli.
  JOURNAL   ACS Chem Biol. 2024 Jun 24. doi: 10.1021/acschembio.4c00221.
  PUBMED    38913984
REFERENCE   2  (bases 1 to 5363)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Chem 
            Biol. 2024 Jun 24. doi: 10.1021/acschembio.4c00221."
COMMENT     Sequence Label: pEVOL-tac-EcTyrRS-pAAFRS-9-lpp-tRNA_CUA^EcTyr
FEATURES             Location/Qualifiers
     source          1..5363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(290..949)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKEGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     864..883
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(950..1052)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1444..1461)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1578..2123)
                     /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(1658..1677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2440..3522)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3484..3503
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3523..3600)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(3648..3653,3654..3669,3670..3676)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3684..3700
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(5059..5076)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 aaaaaaatat tctcaacata aaaaactttg tgtaatactt gtaacgctgg tggggttccc
       61 gagcggccaa agggagcaga ctctaaatct gccgtcatcg acttcgaagg ttcgaatcct
      121 tcccccacca ccaaattcga aaagcctgct caacgagcag gcttttttgc atgctcgagc
      181 agctcagggt cgaatttgct ttcgaatttc tgccattcat ccgcttatta tcacttattc
      241 aggcgtagca accaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc
      301 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
      361 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
      421 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
      481 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
      541 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
      601 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
      661 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccttctttca
      721 ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
      781 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
      841 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
      901 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
      961 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     1021 ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc
     1081 agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg
     1141 tcacaggtat ttgttcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt
     1201 gtatgatggt gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt
     1261 tcagctactg acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg
     1321 agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg
     1381 caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc
     1441 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
     1501 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
     1561 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
     1621 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1681 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
     1741 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
     1801 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
     1861 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
     1921 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
     1981 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
     2041 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
     2101 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga
     2161 tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt
     2221 tgtaattctc atgtttgaca gcttatcatc gataagcttg caatttatct cttcaaatgt
     2281 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     2341 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     2401 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2521 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     2581 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     2641 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     2701 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     2761 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     2821 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     2881 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     2941 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     3001 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     3061 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     3121 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     3181 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     3241 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     3301 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     3361 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     3421 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     3481 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     3541 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     3601 cgggatctcg acgctctccc ttatgcgact cctgcattag ggagctgttg acaattaatc
     3661 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacaaag gaggtgcggc
     3721 cgcatggcaa gcagtaactt gattaaacaa ttgcaagagc gggggctggt agcccaggtg
     3781 acggacgagg aagcgttagc agagcgactg gcgcaaggcc cgatcgcgct cgggtgcggc
     3841 ttcgatccta ccgctgacag cttgcatttg gggcatcttg ttccattgtt atgcctgaaa
     3901 cgcttccagc aggcgggcca caagccggtt gcggttgtag gcggcgcgac gggtctgatt
     3961 ggcgacccga gcttcaaagc tgccgagcgt aagctgaaca ccgaagaaac tgttcaggag
     4021 tgggtggaca aaatccgtaa gcaggttgcc ccgttcctcg atttcgactg tggagaaaac
     4081 tctgctatcg cggcgaacaa ctatgactgg ttcggcaata tgaatgtgct gaccttcctg
     4141 cgcgatattg gcaaacactt ctccgttaac cagatgatca acaaagaagc ggttaagcag
     4201 cgtctcaacc gtgaagatca ggggatttcg ttcactgagt tttcctacaa cctgttgcag
     4261 ggttattgtt atgcctgttg taacaaacag tacggtgtgg tgctgcaaat tggtggttct
     4321 gaccagtggg gtaacatcac ttctggtatc gacctgaccc gtcgtctgca tcagaatcag
     4381 gtgtttggcc tgaccgttcc gctgatcact aaagcagatg gcaccaaatt tggtaaaact
     4441 gaaggcggcg cagtctggtt agatccgaag aaaaccagcc cgtacaaatt ctaccagttc
     4501 tggatcaaca ctgcggatgc cgacgtttac cgcttcctga agttcttcac ctttatgagc
     4561 attgaagaga tcaacgccct ggaagaagaa gataaaaaca gcggtaaagc accgcgcgcc
     4621 cagtatgtac tggcggagca ggtgactcgt ctggttcacg gtgaagaagg tttacaggcg
     4681 gcaaaacgta ttaccgaatg cctgttcagc ggttctttga gtgcgctgag tgaagcggac
     4741 ttcgaacagc tggcgcagga cggcgtaccg atggttgaga tggaaaaggg cgcagacctg
     4801 atgcaggcac tggtcgattc tgaactgcaa ccttcccgtg gtcaggcacg taaaactatc
     4861 gcctccaatg ccatcaccat taacggtgaa aaacagtccg atcctgaata cttctttaaa
     4921 gaagaagatc gtctgtttgg tcgttttacc ttactgcgtc gcggtaaaaa gaattactgt
     4981 ctgatttgct ggaaataagc ggccgcgttt aaacggtctc cagcttggct gttttggcgg
     5041 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     5101 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     5161 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     5221 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggccttgttt gtgagctccc
     5281 ggtcatcaat catccccata atccttgtta gcctgcagcg ccgcttcttt gagcgaacga
     5341 tcaaaaataa gtggcgcccc atc
//
