LOCUS       sequence-338849-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pylBCD variant in pBAD backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7327)
  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 7327)
  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: pJH578
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..285
                     /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     208..227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             334..627
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3717..3734)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3887..3973
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4065..4092
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4111..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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     complement(4421..4440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5234..5822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5723..5742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5987..6004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6019..6159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6085..6104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6245..6267
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6423..7301)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct
       61 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca
      121 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg
      181 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg
      241 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc
      301 taacaggagg aattaaccat gatgggcagc agcatgtcgg actcagaagt caatcaagaa
      361 gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt aaaggtgtcc
      421 gatgggtctt cagagatctt cttcaagatc aaaaagacca ctcctttaag aaggctgatg
      481 gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt gtacgacggt
      541 attagaattc aagctgatca gacccctgaa gatttggaca tggaggataa cgatattatt
      601 gaggctcaca gagaacagat tggtggtcat atgattaaga agatggcgac tgaagatctg
      661 gatcgttttg gcgaaaaaat tattgaagga tttcagttat ctgacgacga tttacgcgct
      721 ttgcttagcc tggaatcgga agaagaattg gaaaagctgt attacgtcgc acgtaaagta
      781 cgcaaccatt actttggaaa tcgcgtattc ctgagctgtt ttatctactt cagcacatac
      841 tgcaaaaacc aatgtgcgtt ttgctattat aactgcaaaa acaagattaa ccgctatcgt
      901 ttgtcccgcg aagaaatcaa agagacttgc aaggccctta aaggtgcggg tttccatatg
      961 attgatctga ctatggggga ggatccctat tattacaagg aacctgatcg cttcgtggag
     1021 ctggtccaaa tggttaagaa tgagcttgga cttccaatca tgatttcgcc aggagtaatg
     1081 gataatgcta cccttttaaa agcacgcgaa aagggagcta atttcttcgc cttatatcaa
     1141 gaaacctatg atcaagagtt gtacaagaag ttgcgcgtag gtcagtcgtt caagaggcgc
     1201 tacaatactc gtcgttttgc taaagaacaa gggtattgca ttgaagacgg tatcttaacg
     1261 ggagtcggga acgatatcga gtcaacgatc aaatctttac gcggcatgaa atcaaatgat
     1321 cccgacatgg ttcgcgtgat gactttctta ccccaggaag gaactcccct tgagcgtttt
     1381 aaggacaata gtaacctgag cgaactgaaa atcattgcga tcttgcgtct gatctttccg
     1441 aagtgcttaa tccccgcgtc gttggacctg gaagggatcg acgggatggt acaccgtctt
     1501 aacgcaggtg ctaatatcgt gactagcatc cttcctagtg attcaaaact tgagggagtt
     1561 gctaactacg atcgtgacct ggaagagcgc gaccgcgaca ttaaaagcgt catcaagcgc
     1621 cttgagggaa tgggcatgga accagcgcca caggcggaat tcgagactgt actgcgctgc
     1681 taataaggag gaaaaatgaa aaccatctgc ctgattggtg gtaaattgca gggttttgag
     1741 gccgcgtatc tgtctaaaaa agccgatatg aaagtagtgg ttatcgacaa gaacccccaa
     1801 gctcttatcc gcaattacgc agacgagttt cattgttttg atgttatcaa ggaaccagag
     1861 aaacttcttg aaatcagcaa aaatgtagac gctatcttgc ccgtcaacga aaacttagaa
     1921 tgtattaaat tcttaagtag tgtaaaagag gaattttctt gcccagtact gttcgatttt
     1981 gaggcttacc gcatctcgcg cgataagaag aaatcgaaag agtatttccg ttccatcggc
     2041 attccgacac cgcaagacaa acctggtcgt cctccatact tcgttaagcc accgtgcgaa
     2101 agctcgtcgg ttggtgctcg cattatctac gatgaagaag aactgggaga attggagccc
     2161 ggcatgctgg tagaagagta tgtggaaggt gaggttattt cacttgaagt aatcggggac
     2221 ggtacccact ttgcagttgt taaagagaca cttattcaca tcgaccgcac ctatgactgc
     2281 catatggtaa ctcccctgcc atctgatttg tctttccgtg agatcagcta ttctttagcg
     2341 gccaacctgc ctttgaaggg gatcatggac gttgaagcaa tttcgggccc gcaaggtttg
     2401 aaggtcattg aaattgattc acgtttcccc tcccagaccc ccacagccgt gtattattcg
     2461 tcgggagtga atttaattga gctgttgttt cgtgcctttg gagaaggggt ggaggaagtt
     2521 aaaacccttc cagaggaccg ctactgtatc tacgagcacc tgatgcttgc agaaaatggg
     2581 gccctgatcc cagtaggtga gcaggttctg tcgatgggga atgactatgg gaaatactat
     2641 gaagatccag gaattgaaat ctttttatgt cgcggagagg accccgtgtt tacgcttgtc
     2701 ttttggggaa aggaccgtga ggaagccgaa aacaaaaagc gcactggttt gctgattttg
     2761 aaggaccgct tcggggcagc cgtttaaaat ggcaaaaatc ggataagaat aaaaatttgc
     2821 ccaaagtaaa atttgtttaa aacagaaaaa ggagataaaa aatggctttg ttgactcccg
     2881 aggacctgga gaacattaac cgtcaacttc acgaagccga ttctacggta cgtcgtgtga
     2941 ctgggttaga tatcaaggga gtttgtaaag atttctatgg gactactcca tgctgtgaga
     3001 aggtcggtat cgtcccggta actagtggca acggcatcat tggcaacttt tctgaaagtc
     3061 ttcgtgcgat cactgagtac tttggatttg actcattcgt gacgaccatg ccagacgtta
     3121 gtggctacta tgaggcggtc cgtaatggag cacaaattat tcttatggca gatgataata
     3181 cgtttcttgc acacaattta aagaacggca agatcgctaa taatcaaccg tgtaccggaa
     3241 ttatctacgc ggagatcgcc tcgcgctata tgaaagccga ttccaaagat gtgcttgccg
     3301 tcgggcttgg aaaagtgggg tttccaggcg ctgcacattt ggttaataag gggtttcgtg
     3361 tgtatgggta cgacgcggat aaggcgttgt tagaaaagac tgtatcagac cttgggatca
     3421 cacctttcga gcctgagaat ccccgtcgct tctcaatcat ttttgaggcg accccgtgtg
     3481 ctgatacagt accagagtct gtcatcagcg acaattgtgt catctctacg cccggtatcc
     3541 cttgcgcgat cagccgtgaa cttcaagaaa aatacggggt ggagttgatt atggaacctc
     3601 tgggtattgg aactgtttct atgctgtata gcattttgta aagctcgaga tctgcagctg
     3661 gtaccatatg ggaattcgaa gcttggctgt tttggcggat gagagaagat tttcagcctg
     3721 atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt
     3781 agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat
     3841 ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa
     3901 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     3961 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     4021 gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac
     4081 ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5341 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5701 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5761 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5821 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctggcc
     5881 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5941 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6001 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     6061 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     6121 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     6181 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     6241 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     6301 cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca aatggacgaa
     6361 gcagggattc tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc
     6421 aattatgaca acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg
     6481 ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa
     6541 ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc
     6601 tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa
     6661 agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa
     6721 ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc
     6781 ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga
     6841 tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca
     6901 aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca
     6961 aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac
     7021 tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg
     7081 aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc
     7141 tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa
     7201 aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac
     7261 gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgcca
     7321 ttcagag
//
