LOCUS       sequence-409890-        7035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid for the expression of M. mazei pyrrolysine aminoacyl-tRNA 
            synthetase/tRNA pair (PylRS/tRNAPyl) in E. Coli. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7035)
  AUTHORS   Ruskowitz ER, Munoz-Robles BG, Strange AC, Butcher CH, Kurniawan S, 
            Filteau JR, DeForest CA
  TITLE     Spatiotemporal functional assembly of split protein pairs through a 
            light-activated SpyLigation.
  JOURNAL   Nat Chem. 2023 May;15(5):694-704. doi: 10.1038/s41557-023-01152-x. 
            Epub 2023 Apr 17.
  PUBMED    37069270
REFERENCE   2  (bases 1 to 7035)
  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: 
            "10.1038/s41557-023-01152-x"; journalName: "Nat Chem"; date: 
            "2023-05"; volume: "15"; issue: "5"; pages: "694-704"
COMMENT     Sequence Label: pEVOL-NBK-1
FEATURES             Location/Qualifiers
     source          1..7035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..291
                     /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     214..233
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     primer_bind     297..316
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        297..315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             318..340
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1762..1779)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1932..1978
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4158..4817)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4732..4751
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4818..4920)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5312..5329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5446..5991)
                     /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(5526..5545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6137..7015)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPSDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc gtcttttact
       61 ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg taacaaagcg
      121 ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc agaaaagtcc
      181 acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta tccataagat
      241 tagcggatcc tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtaat
      301 acgactcact atagggtttt gtttaacttt aagaaggaga tatacttacc catatggata
      361 aaaagcctct gaacactctg atttctgcga ccggtctgtg gatgtcccgc accggcacca
      421 tccacaaaat caaacaccat gaagttagcc gttccaaaat ctacattgaa atggcttgcg
      481 gcgatcacct ggttgtcaac aactcccgtt cttctcgtac cgctcgcgca ctgcgccacc
      541 acaaatatcg caaaacctgc aaacgttgcc gtgttagcga tgaagatctg aacaaattcc
      601 tgaccaaagc taacgaggat cagacctccg taaaagtgaa ggtagtaagc gctccgaccc
      661 gtactaaaaa ggctatgcca aaaagcgtgg cccgtgcccc gaaacctctg gaaaacaccg
      721 aggcggctca ggctcaacca tccggttcta aattttctcc ggcgatccca gtgtccaccc
      781 aagaatctgt ttccgtacca gcaagcgtgt ctaccagcat tagcagcatt tctaccggtg
      841 ctaccgcttc tgcgctggta aaaggtaaca ctaacccgat tactagcatg tctgcaccgg
      901 tacaggcaag cgccccagct ctgactaaat cccagacgga ccgtctggag gtgctgctga
      961 acccaaagga tgaaatctct ctgaacagcg gcaagccttt ccgtgagctg gaaagcgagc
     1021 tgctgtctcg tcgtaaaaag gatctgcaac agatctacgc tgaggaacgc gagaactatc
     1081 tgggtaagct ggagcgcgaa attactcgct tcttcgtgga tcgcggtttc ctggagatca
     1141 aatctccgat tctgattccg ctggaataca ttgaacgtat gggcatcgat aatgataccg
     1201 aactgtctaa acagatcttc cgtgtggata aaaacttctg tctgcgtccg atgctggccc
     1261 cgaacctgat gaactatgcg cgtaaactgg accgtgccct gccggacccg atcaaaattt
     1321 tcgagatcgg tccttgctac cgtaaagagt ccgacggtaa agagcacctg gaagaattca
     1381 ccatgctgaa cttcgcgcag atgggtagcg gttgcacgcg tgaaaacctg gaatccatta
     1441 tcaccgactt cctgaatcac ctgggtatcg atttcaaaat tgttggtgac agctgtatgg
     1501 tgtttggcga tacgctggat gttatgcacg gcgatctgga gctgtcttcc gcagtagtgg
     1561 gcccaatccc gctggatcgt gagtggggta tcgacaaacc ttggatcggt gcgggttttg
     1621 gtctggagcg tctgctgaaa gtaaaacacg acttcaagaa catcaaacgt gctgcacgtt
     1681 ccgagtccta ttacaatggt atttctacta acctgtaagt ctccagcttg gctgttttgg
     1741 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     1801 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     1861 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     1921 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1981 gttgtttgtg agctcccggt catcaatcat ccccataatc cttgttagat tatcaatttt
     2041 aaaaaactaa cagttgtcag cctgtcccgc tttaatatca tacgccgtta tacgttgttt
     2101 acgctttgag gaatcccaat ggataaaaag cctctgaaca ctctgatttc tgcgaccggt
     2161 ctgtggatgt cccgcaccgg caccatccac aaaatcaaac accatgaagt tagccgttcc
     2221 aaaatctaca ttgaaatggc ttgcggcgat cacctggttg tcaacaactc ccgttcttct
     2281 cgtaccgctc gcgcactgcg ccaccacaaa tatcgcaaaa cctgcaaacg ttgccgtgtt
     2341 agcgatgaag atctgaacaa attcctgacc aaagctaacg aggatcagac ctccgtaaaa
     2401 gtgaaggtag taagcgctcc gacccgtact aaaaaggcta tgccaaaaag cgtggcccgt
     2461 gccccgaaac ctctggaaaa caccgaggcg gctcaggctc aaccatccgg ttctaaattt
     2521 tctccggcga tcccagtgtc cacccaagaa tctgtttccg taccagcaag cgtgtctacc
     2581 agcattagca gcatttctac cggtgctacc gcttctgcgc tggtaaaagg taacactaac
     2641 ccgattacta gcatgtctgc accggtacag gcaagcgccc cagctctgac taaatcccag
     2701 acggaccgtc tggaggtgct gctgaaccca aaggatgaaa tctctctgaa cagcggcaag
     2761 cctttccgtg agctggaaag cgagctgctg tctcgtcgta aaaaggatct gcaacagatc
     2821 tacgctgagg aacgcgagaa ctatctgggt aagctggagc gcgaaattac tcgcttcttc
     2881 gtggatcgcg gtttcctgga gatcaaatct ccgattctga ttccgctgga atacattgaa
     2941 cgtatgggca tcgataatga taccgaactg tctaaacaga tcttccgtgt ggataaaaac
     3001 ttctgtctgc gtccgatgct ggccccgaac ctgatgaact atgcgcgtaa actggaccgt
     3061 gccctgccgg acccgatcaa aattttcgag atcggtcctt gctaccgtaa agagtccgac
     3121 ggtaaagagc acctggaaga attcaccatg ctgaacttcg cgcagatggg tagcggttgc
     3181 acgcgtgaaa acctggaatc cattatcacc gacttcctga atcacctggg tatcgatttc
     3241 aaaattgttg gtgacagctg tatggtgttt ggcgatacgc tggatgttat gcacggcgat
     3301 ctggagctgt cttccgcagt agtgggccca atcccgctgg atcgtgagtg gggtatcgac
     3361 aaaccttgga tcggtgcggg ttttggtctg gagcgtctgc tgaaagtaaa acacgacttc
     3421 aagaacatca aacgtgctgc acgttccgag tcctattaca atggtatttc tactaacctg
     3481 taactgcagt tacaggttag tgtaggcact gcagtttcaa acgctaaatt gcctgatgcg
     3541 ctacgcttat caggcctaca tgatctctgc aatatattga gtttgcgtgc ttttgtaggc
     3601 cggataaggc gttcacgccg catccggcaa gaaacagcaa acaatccaaa acgccgcgtt
     3661 cagcggcgtt ttttctgctt ttcttcgcga attaattccg cttcgcaaca tgtgagcacc
     3721 ggtttattga ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg aggccagttt
     3781 gctcaggctc tccccgtgga ggtaataatt gacgatatga tcagtgcacg gctaactaag
     3841 cggcctgctg actttctcgc cgatcaaaag gcattttgct attaagggat tgacgagggc
     3901 gtatctgcgc agtaagatgc gccccgcatt tggcggaaac cccgggaatc taacccggct
     3961 gaacggattt agagtccatt cgatctacat gatcaggttt ccaattcgaa aagcctgctc
     4021 aacgagcagg cttttttgca tctcgagcag ctcagggtcg aatttgcttt cgaatttctg
     4081 ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta agggcaccaa
     4141 taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     4201 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     4261 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     4321 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     4381 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     4441 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     4501 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     4561 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     4621 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     4681 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     4741 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     4801 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     4861 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     4921 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga
     4981 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg
     5041 ggtgatgctg ccaacttact gatttagtgt atgatggtgt ttttgaggtg ctccagtggc
     5101 ttctgtttct atcagctgtc cctcctgttc agctactgac ggggtggtgc gtaacggcaa
     5161 aagcaccgcc ggacatcagc gctagcggag tgtatactgg cttactatgt tggcactgat
     5221 gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc
     5281 agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
     5341 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
     5401 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
     5461 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
     5521 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
     5581 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
     5641 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
     5701 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
     5761 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
     5821 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
     5881 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
     5941 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
     6001 cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca
     6061 cctgaagtca gccccatacg atataagttg taattctcat gtttgacagc ttatcatcga
     6121 taagcttggt acccaattat gacaacttga cggctacatc attcactttt tcttcacaac
     6181 cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga
     6241 gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca
     6301 aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta
     6361 actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc
     6421 tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta
     6481 cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca
     6541 attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt
     6601 taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga
     6661 accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac
     6721 gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa
     6781 tctctcctgg cgggaacagc aaaatatcac tcggtcggca aacaaattct cgtccctgat
     6841 ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg
     6901 gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca
     6961 gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt
     7021 tcatactccc gccat
//
