LOCUS       sequence-339046-        6884 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid for pylBCD variant in pCDF backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6884)
  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 6884)
  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: pJH291
FEATURES             Location/Qualifiers
     source          1..6884
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(54..140)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     293..310
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(387..404)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(1162..1446)
                     /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(1220..1239)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1473..2351
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     complement(2713..2730)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2847..3392)
                     /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(2927..2946)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(3504..3509,3510..3526,3527..3532)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(join(5848..6639,6640..6708))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6471..6490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6709..6800)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(6819..6846)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcccgccacc ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc
       61 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
      121 tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac
      181 actaccatcg gcgctacggc gtttcacttc tgagttcggc atggggtcag gtgggaccac
      241 cgcgctactg ccgccaggca aattctgttt tatcagaccg cttctgcgtt ctgatttaat
      301 ctgtatcagg ctgaaaatct tctctcatcc gccaaaacag ccaagcttcg aattcccata
      361 tggtaccagc tgcagatctc gagtcaatga tgatgatgat gatgtgagcc tttatacagt
      421 tcgtccatac cgtgcgtgat gccggccgcg gtcacgaatt ccagcaggac catgtggtca
      481 cgcttttcgt tcggatcttt actcagcaca gactgcgtgc tcaggtaatg attgtccggc
      541 agcaggaccg ggccatcacc aatcggggtg ttttgctgat agtggtcggc cagttgaacg
      601 ctgccatctt ccacattatg gcggatcttg aagttagcct tgataccgtt cttctgctta
      661 tctgcggtga tctacacgtt gtgcgaattg aagttatatt ccagtttatg gcccagaata
      721 ttaccgtctt ccttaaaatc gatgcctttc agttcaatac ggttaaccag cgtatcacct
      781 tcgaatttca cttcggcgcg cgttttgtag gtgccgtcat ctttaaatga gatggtacgt
      841 tcttggacat aaccttccgg catcgcggac ttgaaaaagt cgtggcgttt catgtgatcc
      901 gggtaacgac tgaaacactg aacaccatac gtcagcgtgg tgaccagggt cggccacgga
      961 accggcagtt tacccgtggt gcaaataaac ttcagcgtca gtttacccta ggttgcatcg
     1021 ccttcacctt cgccgcgcac gctgaatttg tgaccattga catcaccatc cagttccacc
     1081 agaatcggca ccacgcccgt aaacagttct tcgcccttgc tcatcctagg ttaattcctc
     1141 ctgttagccc aaaaaacggg tatggagaaa cagtagagag ttgcgataaa aagcgtcagg
     1201 taggatccgc taatcttatg gataaaaatg ctatggcata gcaaagtgtg acgccgtgca
     1261 aataatcaat gtggactttt ctgccgtgat tatagacact tttgttacgc gtttttgtca
     1321 tggctttggt cccgctttgt tacagaatgc ttttaataag cggggttacc ggtttggtta
     1381 gcgagaagag ccagtaaaag acgcagtgac ggcaatgtct gatgcaatat ggacaattgg
     1441 tttcttctct gaatggcggg agtatgaaaa gtatggctga agcgcaaaat gatcccctgc
     1501 tgccgggata ctcgtttaat gcccatctgg tggcgggttt aacgccgatt gaggccaacg
     1561 gttatctcga tttttttatc gaccgaccgc tgggaatgaa aggttatatt ctcaatctca
     1621 ccattcgcgg tcagggggtg gtgaaaaatc agggacgaga atttgtttgc cgaccgggtg
     1681 atattttgct gttcccgcca ggagagattc atcactacgg tcgtcatccg gaggctcgcg
     1741 aatggtatca ccagtgggtt tactttcgtc cgcgcgccta ctggcatgaa tggcttaact
     1801 ggccgtcaat atttgccaat acggggttct ttcgcccgga tgaagcgcac cagccgcatt
     1861 tcagcgacct gtttgggcaa atcattaacg ccgggcaagg ggaagggcgc tattcggagc
     1921 tgctggcgat aaatctgctt gagcaattgt tactgcggcg catggaagcg attaacgagt
     1981 cgctccatcc accgatggat aatcgggtac gcgaggcttg tcagtacatc agcgatcacc
     2041 tggcagacag caattttgat atcgccagcg tcgcacagca tgtttgcttg tcgccgtcgc
     2101 gtctgtcaca tcttttccgc cagcagttag ggattagcgt cttaagctgg cgcgaggacc
     2161 aacgtatcag ccaggcgaag ctgcttttga gcaccacccg gatgcctatc gccaccgtcg
     2221 gtcgcaatgt tggttttgac gatcaactct atttctcgcg ggtatttaaa aaatgcaccg
     2281 gggccagccc gagcgagttc cgtgccggtt gtgaagaaaa agtgaatgat gtagccgtca
     2341 agttgtcata attggtaacg aatcagacaa ttgacggctt gacggagtag catagggttt
     2401 gcagaatccc tgcttcgtcc atttgacagg cacattatgc atgccgcttc gccttcgcgc
     2461 gcgaattgat ctgctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2521 agctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag
     2581 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc
     2641 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat
     2701 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     2761 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     2821 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     2881 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     2941 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     3001 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     3061 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     3121 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     3181 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     3241 ctgaaaggac aagttttggt ggctgcgctc ctccaagcca gttacctcgg ttcaaagagt
     3301 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     3361 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     3421 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     3481 gatataagtt gtaattctca tgtttgacag cttatcatcg ataagcttta atgcggtagt
     3541 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
     3601 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
     3661 ctgccgggcc tcttgcggga tctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     3721 gtaacgccat gcaaaaaagc ctgctcgttg agcaggcttt tcgaatttgg cggaaacccc
     3781 gggaatctaa cccggctgaa cggatttaga gtccattcga tctacatgat caggtttcca
     3841 atgcggggcg catcttactg cgcagatacg ccctcgtcaa tcccttaata gcaaaatgcc
     3901 ttttgatcgg cgagaaagtc agcaggccgc ttagttagcc agggtggttt ttcttttcac
     3961 cagcgagacg ggcaacagaa cgccatgagc ggcctcattt cttattatga gttacaacag
     4021 tccgcaccgc tgccggtagc tccttcctgt gggcgcgggc catagatctc agcggtggca
     4081 gcagcctagg ttacaggttg gtagaaatcc cgttatagta agactcggac cttgcagctc
     4141 tcttgatatt tttaaagtcg tgtttaacct ttagaaggcg ttcgagcccg aaacctgccc
     4201 ctatccaggg tttatcaata ccccattccc ggtcaagcgg tatgggtccg actactgcag
     4261 aggaaagttc caggtctccg tgcattacat caagggtatc cccatagacc atgcaggaat
     4321 cgcctacgat cttgaaatca attcccaggt ggttcaggaa gtccgtaatt atgctttcaa
     4381 gattttcccg tgtgcatccc gatcccatct ggcagaagtt cagcatggta aactcttcga
     4441 ggtgttcttt gccgtcggac tcttttctgt agcatgggcc tatttcaaaa atttttattg
     4501 gatcaggcag ggccctgtca agcttgcgca ggtagttgta aaggtttgga gcaagcatgg
     4561 gtctcaggca gaagttcttg tcaaccctga agatctgttt tgaaagttcg gtatcattat
     4621 caatgcccat cctttcgata tactcaagag ggatcaggat cggggatttt atttccagaa
     4681 aacccctgtc cacaaagaac ctggtaattt cacgctcgag tttccccaga taattctccc
     4741 tttcttccgc gtagatctgc tgcaggtctt tttttctgcg agagagcaat tcggactcaa
     4801 gctccctgaa aggcttgccg gaattcaggg aaatctcatc ttttgggttt aacaggactt
     4861 caagcctgtc agtctggctc ttcgtaagtg cgggggcact tgcagaggtc ggaaccggag
     4921 agttcggggt agatttcgcc ggagacggaa cagaacgaga ggtgtcggta gacgctttcg
     4981 cagaaaccgg attttccagc ggtttcggcg cacgagaaac agatttcggc atcgcttttt
     5041 tcactttcgg cgcagaaaca actttaactt taacagaggt tttgccttcg gtagaacggg
     5101 tcaggaagtt gttgatatct tcgtcagaaa cacgacaacg tttgcaggtt ttacggtatt
     5161 tatgatgacg gaatgcacgt gcggtacgac aagaacgaga gttgttcaca accagatggt
     5221 caccacacgc catttcgatg tagattttag aacgagaaac ctcatagtgc ttgatcttgt
     5281 gcagcgtgcc ggtacgggac atccacagac cggtcgcaga gatcagaaca tccagcggct
     5341 tcttatccat actagttacc ctctagattg agttaatctc catgtagcgt tacaagtatt
     5401 acacaaagtt ttttatgttg agaatatttt tttgatggga aggcacttat ttttgatcgt
     5461 tcgctcaaag aagcatcgaa cgctgggttg cggccgcccg ggtcgaattt gctttcgaat
     5521 ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
     5581 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta
     5641 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     5701 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtc tgagattatc
     5761 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5821 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5881 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5941 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6001 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6061 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6121 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6181 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6241 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6301 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6361 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6421 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     6481 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6541 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6601 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6661 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6721 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6781 tatttagaaa aataaacaaa agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg
     6841 ccttctgctt aatttgatgc ctggcagttt atggcgggcg tcct
//
