LOCUS       sequence-477155-        5547 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  A chimeric PylRS for site-specific incorporation of L-lactyl-lysine 
            into target proteins in E.coli cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5547)
  AUTHORS   Shao C, Tang S, Yu S, Liu C, Zhang Y, Wan T, He Z, Yuan Q, Wu S, 
            Zhang H, Wan N, Zhan M, Tan RX, Hao H, Ye H, Wang N
  TITLE     Genetic code expansion reveals site-specific lactylation in living 
            cells reshapes protein functions.
  JOURNAL   Nat Commun. 2025 Jan 8;16(1):227. doi: 10.1038/s41467-024-55165-2.
  PUBMED    39779673
REFERENCE   2  (bases 1 to 5547)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-01-8"; volume: "16(1):227. doi"; pages: " 
            10.1038/s41467-024-55165-2"
COMMENT     Sequence Label: pEvol-chKlacRS-WT
FEATURES             Location/Qualifiers
     source          1..5547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..879)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        906..1190
                     /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     1113..1132
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2493..2510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2566..2583)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3569..4228)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4143..4162
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4229..4331)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4723..4740)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4857..5402)
                     /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(4937..4956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct
       61 cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc
      121 aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg
      181 gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag
      241 atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt
      301 gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg
      361 tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt
      421 tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa
      481 caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa
      541 tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg
      601 gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa
      661 cagcaaaata tcactcggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg
      721 accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa
      781 atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga
      841 gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt
      901 cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg
      961 gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg
     1021 gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca
     1081 cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt
     1141 agcggatcct acctgacgct ttttatcgca actctctact gtttctccat acccgttttt
     1201 ttgggctaac aggaggaatt actagtatgg ataagaagcc gctggatgtt ctgattagcg
     1261 ccaccggtct gtggatgagc cgcacaggta ccctgcataa aattaaacat tatgaagtga
     1321 gccgtagcaa aatttatatt gaaatggcat gtggtgatca tctggttgtt aacaatagtc
     1381 gcagctgtcg taccgcacgt gcatttcgcc atcataaata tcgcaaaacc tgtaaacgtt
     1441 gtcgcgtgag cgatgaagat attaataatt ttctgacccg tagtaccgaa ggtaaaacca
     1501 gcgttaaagt taaagttgtt tccgcaccga aagtgaagaa ggcaatgccg aaaagcgtta
     1561 gccgtgcacc gaaaccgctg gaaaacccgg ttagcgcaaa agcaagcaca gataccagcc
     1621 gtagcgttcc gagccctgca aaaagcaccc cgaatagccc ggttccgaca tcagcaagcg
     1681 ccccagctct gactaaatcc cagacggacc gtctggaggt gctgctgaac ccaaaggatg
     1741 aaatctctct gaacagcggc aagcctttcc gtgagctgga aagcgagctg ctgtctcgtc
     1801 gtaaaaagga tctgcaacag atctacgctg aggaacgcga gaactatctg ggtaagctgg
     1861 agcgcgaaat tactcgcttc ttcgtggatc gcggtttcct ggagatcaaa tctccgattc
     1921 tgattccgct ggaatacatt gaacgtatgg gcatcgataa tgataccgaa ctgtctaaac
     1981 agatcttccg tgtggataaa aacttctgtc tgcgtccgat gatggccccg aacctgctga
     2041 actatgcgcg taaactggac cgtgccctgc cggacccgat caaaattttc gagatcggtc
     2101 cttgctaccg taaagagtcc gacggtaaag agcacctgga agaattcacc atgctgaact
     2161 tctttcagat gggtagcggt tgcacgcgtg aaaacctgga atccattatc accgacttcc
     2221 tgaatcacct gggtatcgat ttcaaaattg ttggtgacag ctgtatggtg tatggcgata
     2281 cgctggatgt tatgcacggc gatctggagc tgtcttccgc agtagtgggc ccaatcccgc
     2341 tggatcgtga gtggggtatc gacaaacctt ggatcggtgc gggttttggt ctggagcgtc
     2401 tgctgaaagt aaaacacgac ttcaagaaca tcaaacgtgc tgcacgttcc gagtcctatt
     2461 acaatggtat ttctactaac ctgtaagtcg accatcatca tcatcatcat tgagtttaaa
     2521 cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     2581 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     2641 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     2701 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     2761 aagactgggc cttgtttgtg agctcccggt catcaatcat ccccataatc cttgttagat
     2821 tatcaatttt aaaaaactaa cagttgtcag cctgtcccgc tttaatatca tacgccgtta
     2881 tacgttgttt acgctttgag gaatcccata tgatacgcgg ccgctttcaa acgctaaatt
     2941 gcctgatgcg ctacgcttat caggcctaca tgatctctgc aatatattga gtttgcgtgc
     3001 ttttgtaggc cggataaggc gttcacgccg catccggcaa gaaacagcaa acaatccaaa
     3061 acgccgcgtt cagcggcgtt ttttctgctt ttcttcgcga attaattccg cttcgcaaca
     3121 tgtgagcacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg
     3181 aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga tcagtgcacg
     3241 gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct attaagggat
     3301 tgacgagggc gtatctgcgc agtaagatgc gccccgcatt ggaaacctga tcatgtagat
     3361 cgaatggact ctaaatccgt tcagccgggt tagattcccg gggtttccgc caaattcgaa
     3421 aagcctgctc aacgagcagg cttttttgca tgctcgagca gctcagggtc gaatttgctt
     3481 tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt
     3541 aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact
     3601 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     3661 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     3721 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     3781 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     3841 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     3901 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     3961 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat
     4021 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     4081 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     4141 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     4201 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     4261 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     4321 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     4381 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc
     4441 aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt
     4501 gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga cggggtggtg
     4561 cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg gcttactatg
     4621 ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc
     4681 ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct
     4741 acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct
     4801 ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc
     4861 cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga
     4921 aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct
     4981 cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat
     5041 tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga
     5101 accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5161 ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag
     5221 tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc
     5281 ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc
     5341 cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc
     5401 aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt
     5461 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgtttgacag
     5521 cttatcatcg ataagcttgg tacccaa
//
