LOCUS       sequence-244699-        6801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Efficient expression of tRNA synthetase/tRNA for the incorporation 
            of a photo-cross-linker, 3'-azibutyl-N-carbamoyl-lysine (AbK) into 
            recombinant protein by amber codon (TAG) suppression in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6801)
  AUTHORS   Pan D, Walstein K, Take A, Bier D, Kaiser N, Musacchio A
  TITLE     Mechanism of centromere recruitment of the CENP-A chaperone HJURP 
            and its implications for centromere licensing.
  JOURNAL   Nat Commun. 2019 Sep 6;10(1):4046. doi: 10.1038/s41467-019-12019-6.
  PUBMED    31492860
REFERENCE   2  (bases 1 to 6801)
  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"; journalName: "Nat 
            Commun."; date: "2019-09-6"; volume: "10(1):4046. doi"; pages: " 
            10.1038/s41467-019-12019-6"
COMMENT     Sequence Label: pEVOL-ABK
FEATURES             Location/Qualifiers
     source          1..6801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..306
                     /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     229..248
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1612..1629
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1685..1702)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3939..4598)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4513..4532
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4599..4701)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5093..5110)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5227..5772)
                     /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(5307..5326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5918..6796)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 tcatactccc gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc
       61 actgcgtctt ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca
      121 ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc
      181 acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat
      241 ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt
      301 ctccataccc gtttttttgg gctaacagga ggaattagat ccatggataa aaaaccatta
      361 gatgttttaa tatctgcgac cgggctctgg atgtccagga ctggcacgct ccacaaaatc
      421 aagcaccatg aggtctcaag aagtaaaata tacattgaaa tggcgtgtgg agaccatctt
      481 gttgtgaata attccaggag ttgtagaaca gccagagcat tcagacatca taagtacaga
      541 aaaacctgca aacgatgtag ggtttcggac gaggatatca ataattttct cacaagatca
      601 accgaaagca aaaacagtgt gaaagttagg gtagtttctg ctccaaaggt caaaaaagct
      661 atgccgaaat cagtttcaag ggctccgaag cctctggaaa attctgtttc tgcaaaggca
      721 tcaacgaaca catccagatc tgtaccttcg cctgcaaaat caactccaaa ttcgtctgtt
      781 cccgcatcgg ctcctgctcc ttcacttaca agaagccagc ttgatagggt tgaggctctc
      841 ttaagtccag aggataaaat ttctctaaat atggcaaagc ctttcaggga acttgagcct
      901 gaacttgtga caagaagaaa aaacgatttt cagcggctct ataccaatga tagagaagac
      961 tacctcggta aactcgaacg tgatattacg aaatttttcg tagaccgggg ttttctggag
     1021 ataaagtctc ctatccttat tccggcggaa tacgtggaga gaatgggtat taataatgat
     1081 actgaacttt caaaacagat cttccgggtg gataaaaatc tctgcttgag gccaatgctt
     1141 gccccgactc tgtataacta tatgcgaaaa ctcgatagga ttttaccagg cccaataaaa
     1201 attttcgaag tcggaccttg ttaccggaaa gagtctgacg gcaaagagca cctggaagaa
     1261 tttactatgg tgaacttcgc tcagatgggt tcgggatgta ctcgggaaaa tcttgaagct
     1321 ctcatcaaag agtttctgga ctatctggaa atcgacttcg aaatcgtagg agattcctgt
     1381 atggtctttg gggatactct tgatataatg cacggggacc tggagctttc ttcggcagtc
     1441 gtcgggccag tttctcttga tagagaatgg ggtattgaca aaccatggat aggtgcaggt
     1501 tttggtcttg aacgcttgct caaggttatg cacggcttta aaaacattaa gagggcatca
     1561 aggtccgaat cttactataa tgggatttca accaatctat aataagtcga ccatcatcat
     1621 catcatcatt gagtttaaac ggtctccagc ttggctgttt tggcggatga gagaagattt
     1681 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     1741 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     1801 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     1861 aaacgaaagg ctcagtcgaa agactgggcc ttgtttgtga gctcccggtc atcaatcatc
     1921 cccataatcc ttgttagatt atcaatttta aaaaactaac agttgtcagc ctgtcccgct
     1981 ttaatatcat acgccgttat acgttgttta cgctttgagg aatcccatat ggataaaaaa
     2041 ccattagatg ttttaatatc tgcgaccggg ctctggatgt ccaggactgg cacgctccac
     2101 aaaatcaagc accatgaggt ctcaagaagt aaaatataca ttgaaatggc gtgtggagac
     2161 catcttgttg tgaataattc caggagttgt agaacagcca gagcattcag acatcataag
     2221 tacagaaaaa cctgcaaacg atgtagggtt tcggacgagg atatcaataa ttttctcaca
     2281 agatcaaccg aaagcaaaaa cagtgtgaaa gttagggtag tttctgctcc aaaggtcaaa
     2341 aaagctatgc cgaaatcagt ttcaagggct ccgaagcctc tggaaaattc tgtttctgca
     2401 aaggcatcaa cgaacacatc cagatctgta ccttcgcctg caaaatcaac tccaaattcg
     2461 tctgttcccg catcggctcc tgctccttca cttacaagaa gccagcttga tagggttgag
     2521 gctctcttaa gtccagagga taaaatttct ctaaatatgg caaagccttt cagggaactt
     2581 gagcctgaac ttgtgacaag aagaaaaaac gattttcagc ggctctatac caatgataga
     2641 gaagactacc tcggtaaact cgaacgtgat attacgaaat ttttcgtaga ccggggtttt
     2701 ctggagataa agtctcctat ccttattccg gcggaatacg tggagagaat gggtattaat
     2761 aatgatactg aactttcaaa acagatcttc cgggtggata aaaatctctg cttgaggcca
     2821 atgcttgccc cgactctgta taactatatg cgaaaactcg ataggatttt accaggccca
     2881 ataaaaattt tcgaagtcgg accttgttac cggaaagagt ctgacggcaa agagcacctg
     2941 gaagaattta ctatggtgaa cttcgctcag atgggttcgg gatgtactcg ggaaaatctt
     3001 gaagctctca tcaaagagtt tctggactat ctggaaatcg acttcgaaat cgtaggagat
     3061 tcctgtatgg tctttgggga tactcttgat ataatgcacg gggacctgga gctttcttcg
     3121 gcagtcgtcg ggccagtttc tcttgataga gaatggggta ttgacaaacc atggataggt
     3181 gcaggttttg gtcttgaacg cttgctcaag gttatgcacg gctttaaaaa cattaagagg
     3241 gcatcaaggt ccgaatctta ctataatggg atttcaacca atctataact gcagtttcaa
     3301 acgctaaatt gcctgatgcg ctacgcttat caggcctaca tgatctctgc aatatattga
     3361 gtttgcgtgc ttttgtaggc cggataaggc gttcacgccg catccggcaa gaaacagcaa
     3421 acaatccaaa acgccgcgtt cagcggcgtt ttttctgctt ttcttcgcga attaattccg
     3481 cttcgcaaca tgtgagcacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct
     3541 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga
     3601 tcagtgcacg gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct
     3661 attaagggat tgacgagggc gtatctgcgc agtaagatgc gccccgcatt ggaaacctga
     3721 tcatgtagat cgaacggact ctaaatccgt tcagccgggt tagattcccg gggtttccgc
     3781 caaattcgaa aagcctgctc aacgagcagg cttttttgca tgctcgagca gctcagggtc
     3841 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa
     3901 ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca
     3961 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     4021 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     4081 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     4141 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     4201 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     4261 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     4321 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
     4381 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     4441 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     4501 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     4561 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     4621 aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg
     4681 gaacctctta cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg
     4741 gtatcaacag ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt
     4801 tattcggcgc aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg
     4861 tttttgaggt gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga
     4921 cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg
     4981 gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa
     5041 aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca
     5101 ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg
     5161 gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa
     5221 gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca
     5281 gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct
     5341 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
     5401 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
     5461 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
     5521 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
     5581 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
     5641 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
     5701 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
     5761 aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa
     5821 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
     5881 tgtttgacag cttatcatcg ataagcttgg tacccaatta tgacaacttg acggctacat
     5941 cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt
     6001 taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga
     6061 taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc
     6121 agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca
     6181 agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga
     6241 tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg
     6301 cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc
     6361 gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt
     6421 gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt
     6481 catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg
     6541 gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca ctcggtcggc
     6601 aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa
     6661 tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa
     6721 tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat
     6781 tttgcgcttc agccatactt t
//
