LOCUS       sequence-338581-        6790 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid for pylBCD variant in pBAD backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6790)
  AUTHORS   Ho JML, Miller CA, Smith KA, Mattia JR, Bennett MR
  TITLE     Improved pyrrolysine biosynthesis through phage assisted 
            non-continuous directed evolution of the complete pathway.
  JOURNAL   Nat Commun. 2021 Jun 24;12(1):3914. doi: 10.1038/s41467-021-24183-9.
  PUBMED    34168131
REFERENCE   2  (bases 1 to 6790)
  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: "2021-06-24"; volume: "12(1):3914. doi"; pages: " 
            10.1038/s41467-021-24183-9"
COMMENT     Sequence Label: pJH643
FEATURES             Location/Qualifiers
     source          1..6790
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(266..925)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     840..859
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(926..1028)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1143..1170)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1262..1348)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1501..1518
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(1595..1612)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(2370..2654)
                     /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     complement(2428..2447)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             2681..3559
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     complement(3921..3938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4055..4600)
                     /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(4135..4154)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(4712..4717,4718..4734,4735..4740)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
       61 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
      121 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtctg agattatcaa
      181 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
      241 tatatgagta aacttggtct gacagttacg ccccgccctg ccactcatcg cagtactgtt
      301 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
      361 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
      421 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
      481 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
      541 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
      601 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
      661 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
      721 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
      781 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
      841 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
      901 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
      961 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     1021 gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga
     1081 caccaggatt tatttattct gctgtattta gaaaaataaa caaaagagtt tgtagaaacg
     1141 caaaaaggcc atccgtcagg atggccttct gcttaatttg atgcctggca gtttatggcg
     1201 ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg
     1261 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
     1321 ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga
     1381 gaccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt
     1441 gggaccaccg cgctactgcc gccaggcaaa ttctgtttta tcagaccgct tctgcgttct
     1501 gatttaatct gtatcaggct gaaaatcttc tctcatccgc caaaacagcc aagcttcgaa
     1561 ttcccatatg gtaccagctg cagatctcga gtcaatgatg atgatgatga tgtgagcctt
     1621 tatacagttc gtccataccg tgcgtgatgc cggccgcggt cacgaattcc agcaggacca
     1681 tgtggtcacg cttttcgttc ggatctttac tcagcacaga ctgcgtgctc aggtaatgat
     1741 tgtccggcag caggaccggg ccatcaccaa tcggggtgtt ttgctgatag tggtcggcca
     1801 gttgaacgct gccatcttcc acattatggc ggatcttgaa gttagccttg ataccgttct
     1861 tctgcttatc tgcggtgatc tacacgttgt gcgaattgaa gttatattcc agtttatggc
     1921 ccagaatcta accgtcttcc ttaaaatcga tgcctttcag ttcaatacgg ttaaccagcg
     1981 tatcaccttc gaatttcact tcggcgcgcg ttttgtaggt gccgtcatct ttaaatgaga
     2041 tggtacgttc ttggacataa ccttccggca tcgcggactt gaaaaagtcg tggcgtttca
     2101 tgtgatccgg gtaacgactg aaacactgaa caccatacgt cagcgtggtg accagggtcg
     2161 gccacggaac cggcagttta cccgtggtgc aaataaactt cagcgtcagt ttaccctagg
     2221 ttgcatcgcc ttcaccttcg ccgcgcacgc tgaatttgtg accattgaca tcaccatcca
     2281 gttccaccag aatcggcacc acgcccgtaa acagttcttc gcccttgctc atcctaggtt
     2341 aattcctcct gttagcccaa aaaacgggta tggagaaaca gtagagagtt gcgataaaaa
     2401 gcgtcaggta ggatccgcta atcttatgga taaaaatgct atggcatagc aaagtgtgac
     2461 gccgtgcaaa taatcaatgt ggacttttct gccgtgatta tagacacttt tgttacgcgt
     2521 ttttgtcatg gctttggtcc cgctttgtta cagaatgctt ttaataagcg gggttaccgg
     2581 tttggttagc gagaagagcc agtaaaagac gcagtgacgg caatgtctga tgcaatatgg
     2641 acaattggtt tcttctctga atggcgggag tatgaaaagt atggctgaag cgcaaaatga
     2701 tcccctgctg ccgggatact cgtttaatgc ccatctggtg gcgggtttaa cgccgattga
     2761 ggccaacggt tatctcgatt tttttatcga ccgaccgctg ggaatgaaag gttatattct
     2821 caatctcacc attcgcggtc agggggtggt gaaaaatcag ggacgagaat ttgtttgccg
     2881 accgggtgat attttgctgt tcccgccagg agagattcat cactacggtc gtcatccgga
     2941 ggctcgcgaa tggtatcacc agtgggttta ctttcgtccg cgcgcctact ggcatgaatg
     3001 gcttaactgg ccgtcaatat ttgccaatac ggggttcttt cgcccggatg aagcgcacca
     3061 gccgcatttc agcgacctgt ttgggcaaat cattaacgcc gggcaagggg aagggcgcta
     3121 ttcggagctg ctggcgataa atctgcttga gcaattgtta ctgcggcgca tggaagcgat
     3181 taacgagtcg ctccatccac cgatggataa tcgggtacgc gaggcttgtc agtacatcag
     3241 cgatcacctg gcagacagca attttgatat cgccagcgtc gcacagcatg tttgcttgtc
     3301 gccgtcgcgt ctgtcacatc ttttccgcca gcagttaggg attagcgtct taagctggcg
     3361 cgaggaccaa cgtatcagcc aggcgaagct gcttttgagc accacccgga tgcctatcgc
     3421 caccgtcggt cgcaatgttg gttttgacga tcaactctat ttctcgcggg tatttaaaaa
     3481 atgcaccggg gccagcccga gcgagttccg tgccggttgt gaagaaaaag tgaatgatgt
     3541 agccgtcaag ttgtcataat tggtaacgaa tcagacaatt gacggcttga cggagtagca
     3601 tagggtttgc agaatccctg cttcgtccat ttgacaggca cattatgcat gccgcttcgc
     3661 cttcgcgcgc gaattgatct gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3721 acacatgcag ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg
     3781 gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag
     3841 tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga
     3901 tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     3961 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     4021 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     4081 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     4141 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     4201 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     4261 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     4321 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     4381 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     4441 aggctaaact gaaaggacaa gttttggtgg ctgcgctcct ccaagccagt tacctcggtt
     4501 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     4561 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
     4621 gataaaatat ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
     4681 ccccatacga tataagttgt aattctcatg tttgacagct tatcatcgat aagctttaat
     4741 gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg aaatctaaca
     4801 atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata ggcttggtta
     4861 tgccggtact gccgggcctc ttgcgggatc tggcgaaagg gggatgtgct gcaaggcgat
     4921 taagttgggt aacgccatgc aaaaaagcct gctcgttgag caggcttttc gaatttggcg
     4981 gaaaccccgg gaatctaacc cggctgaacg gatttagagt ccattcgatc tacatgatca
     5041 ggtttccaat gcggggcgca tcttactgcg cagatacgcc ctcgtcaatc ccttaatagc
     5101 aaaatgcctt ttgatcggcg agaaagtcag caggccgctt agttagccag ggtggttttt
     5161 cttttcacca gcgagacggg caacagaacg ccatgagcgg cctcatttct tattatgagt
     5221 tacaacagtc cgcaccgctg ccggtagctc cttcctgtgg gcgcgggcca tagatctcag
     5281 cggtggcagc agcctaggtt acaggttggt agaaatcccg ttatagtaag actcggacct
     5341 tgcagctctc ttgatatttt taaagtcgtg tttaaccttt agaaggcgtt cgagcccgaa
     5401 acctgcccct atccagggtt tatcaatacc ccattcccgg tcaagcggta tgggtccgac
     5461 tactgcagag gaaagttcca ggtctccgtg cattacatca agggtatccc catagaccat
     5521 gcaggaatcg cctacgatct tgaaatcaat tcccaggtgg ttcaggaagt ccgtaattat
     5581 gctttcaaga ttttcccgtg tgcatcccga tcccatctgg cagaagttca gcatggtaaa
     5641 ctcttcgagg tgttctttgc cgtcggactc ttttctgtag catgggccta tttcaaaaat
     5701 ttttattgga tcaggcaggg ccctgtcaag cttgcgcagg tagttgtaaa ggtttggagc
     5761 aagcatgggt ctcaggcaga agttcttgtc aaccctgaag atctgttttg aaagttcggt
     5821 atcattatca atgcccatcc tttcgatata ctcaagaggg atcaggatcg gggattttat
     5881 ttccagaaaa cccctgtcca caaagaacct ggtaatttca cgctcgagtt tccccagata
     5941 attctccctt tcttccgcgt agatctgctg caggtctttt tttctgcgag agagcaattc
     6001 ggactcaagc tccctgaaag gcttgccgga attcagggaa atctcatctt ttgggtttaa
     6061 caggacttca agcctgtcag tctggctctt cgtaagtgcg ggggcacttg cagaggtcgg
     6121 aaccggagag ttcggggtag atttcgccgg agacggaaca gaacgagagg tgtcggtaga
     6181 cgctttcgca gaaaccggat tttccagcgg tttcggcgca cgagaaacag atttcggcat
     6241 cgcttttttc actttcggcg cagaaacaac tttaacttta acagaggttt tgccttcggt
     6301 agaacgggtc aggaagttgt tgatatcttc gtcagaaaca cgacaacgtt tgcaggtttt
     6361 acggtattta tgatgacgga atgcacgtgc ggtacgacaa gaacgagagt tgttcacaac
     6421 cagatggtca ccacacgcca tttcgatgta gattttagaa cgagaaacct catagtgctt
     6481 gatcttgtgc agcgtgccgg tacgggacat ccacagaccg gtcgcagaga tcagaacatc
     6541 cagcggcttc ttatccatac tagttaccct ctagattgag ttaatctcca tgtagcgtta
     6601 caagtattac acaaagtttt ttatgttgag aatatttttt tgatgggaag gcacttattt
     6661 ttgatcgttc gctcaaagaa gcatcgaacg ctgggttgcg gccgcccggg tcgaatttgc
     6721 tttcgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt
     6781 ttaagggcac
//
