LOCUS       sequence-338629-        7316 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 7316)
  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 7316)
  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: pJH574
FEATURES             Location/Qualifiers
     source          1..7316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      31..58
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        77..168
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(169..237,238..1029)
                     /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(387..406)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1200..1788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1689..1708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1942..1959
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1974..2114
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2040..2059)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2200..2222
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2378..3256)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3283..3567
                     /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     3490..3509
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3616..3909
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(6999..7016)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      7169..7255
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
       61 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
      121 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      181 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      241 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      301 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
      361 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
      421 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      481 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      541 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
      601 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
      661 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
      721 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
      781 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
      841 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
      901 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
      961 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     1021 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1081 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1141 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1201 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1261 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1321 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1381 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1441 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1501 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1561 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1621 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1681 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1741 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1801 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1861 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     1921 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     1981 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     2041 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2101 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2161 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2221 ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc gcgaaggcga
     2281 agcggcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa accctatgct
     2341 actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg acggctacat
     2401 cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt
     2461 taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga
     2521 taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc
     2581 agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca
     2641 agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga
     2701 tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg
     2761 cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc
     2821 gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt
     2881 gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt
     2941 catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg
     3001 gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc
     3061 aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa
     3121 tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa
     3181 tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat
     3241 tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat
     3301 attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg
     3361 taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc
     3421 gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca
     3481 cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt
     3541 tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga ggaattaacc
     3601 atgatgggca gcagcatgtc ggactcagaa gtcaatcaag aagctaagcc agaggtcaag
     3661 ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatgggtc ttcagagatc
     3721 ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt cgctaaaaga
     3781 cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat tcaagctgat
     3841 cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca cagagaacag
     3901 attggtggtc atatgattaa gaagatggcg actgaagatc tggatcgttt tggcgaaaaa
     3961 attattgaag gatttcagtt atctgacgac gatttacgcg ctttgcttag cctggaatcg
     4021 gaagaagaat tggaaaagct gtattacgtc gcacgtaaag tacgcaacca ttactttgga
     4081 aatcgcgtat tcctgagctg ttttatctac ttcagcacat actgcaaaaa ccaatgtgcg
     4141 ttttgctatt ataactgcaa aaacaagatt aaccgctatc gtttgtcccg cgaagaaatc
     4201 aaagagactt gcaaggccct taaaggtgcg ggtttccata tgattgatct gactatgggg
     4261 gaggatccct attattacaa ggaacctgat cgcttcgtgg agctggtcca aatggttaag
     4321 aatgagcttg gacttccaat catgatttcg ccaggagtaa tggataatgc taccctttta
     4381 aaagcacgcg aaaagggagc taatttcttc gccttatatc aagaaaccta tgatcaagag
     4441 ttgtacgaga agttgcgcgt aggtcagtcg ttcaaggggc gctacaatac tcgtcgtttt
     4501 gctaaagaac aagggtattg cattgaagac ggtatcttaa cgggagtcgg gaacgatatc
     4561 gagtcaacga tcaaatcttt acgcggcatg aaatcaaatg atcccgacat ggttcgcgtg
     4621 atgactttct taccccagga aggaactccc cttgagcgtt ttaaggaaaa tagtaacctg
     4681 agcgaactga aaatcattgc gatcttgcgt ctgatctttc cgaagtgctt aatccccgcg
     4741 tcgttggacc tggaagggat cgacgggatg gtacaccgtc ttaacgcagg tgctaatatc
     4801 gtgactagca tccttcctag tgattcaaaa cttgagggag ttgctaacta cgatcgtgac
     4861 ctggaagagc gcgaccgcga cattaaaagc gtcatcaagc gccttgaggg aatgggcatg
     4921 gaaccagcgc cacaggcgga attcgagact gtactgcgct gctaataagg aggaaaaatg
     4981 aaaaccatct gcctgattgg tggtaaattg cagggttttg aggccgcgta tctgtctaaa
     5041 aaagccgata tgaaagtagt ggttatcgac aagaaccccc aagctcttat ccgcaattac
     5101 gcagacgagt ttcattgttt tgatgttatc aaggaaccag agaaacttct tgaaatcagc
     5161 aaaaatgtag acgctatctt gcccgtcaac gaaaacttag aatgtattaa attcttaagt
     5221 agtgtaaaag aggaattttc ttgcccagta ctgttcgatt ttgaggctta ccgcatctcg
     5281 cgcgataaga agaaatcgaa agagtatttc cgttccatcg gcattccgac accgcaagac
     5341 aaacctggtc gtcctccata cttcgttaag ccaccgtgcg aaagctcgtc ggttggtgct
     5401 cgcattatct acgatgaaga agaactggga gaattggagc ccggcatgct ggtagaagag
     5461 tatgtggaag gtgaggttat ttcacttgaa gtaatcgggg acggtaccca ctttgcagtt
     5521 gttaaagaga cacttattca catcgaccgc acctatgact gccatatggt aactcccctg
     5581 ccatctgatt tgtctttccg tgagatcagc tattctttag cggccaacct gcctttgaag
     5641 gggatcatgg acgttgaagc aatttcgggc ccgcaaggtt tgaaggtcat tgaaattgat
     5701 tcacgtttcc cctcccagac ccccacagcc gtgtattatt cgtcgggagt gaatttaatt
     5761 gagctgttgt ttcgtgcctt tggagaaggg gtggaggaag ttaaaaccct tccagaggac
     5821 cgctactgta tctacgagca cctgatgctt gcagaaaatg gggccctgat cccagtaggt
     5881 gagcaggttc tgtcgatggg gaatgactat gggaaatact atgaagatcc aggaattgaa
     5941 atctttttat gtcgcggaga ggaccccgtg tttacgcttg tcttttgggg aaaggaccgt
     6001 gaggaagccg aaaacaaaaa gcgcactggt ttgctgattt tgaaggaccg cttcggggca
     6061 gccgtttaaa atggcaaaaa tcggataaga ataaaaattt gcccaaagta aaatttgttt
     6121 aaaacagaaa aaggagataa aaaatggctt tgttgactcc cgaggacctg gagaacatta
     6181 accgtcaact tcacgaagcc gattctacgg tacgtcgtgt gactgggtta gatatcaagg
     6241 gagtttgtaa agatttctat gggactactc catgctgtga gaaggtcggt atcgtcccgg
     6301 taactagtgg caacggcatc attggcaact tttctgaaag tcttcgtgcg atcactgagt
     6361 actttggatt tgactcattc gtgacgacca tgccagacgt tagtggctac tatgaggcgg
     6421 tccgtaatgg agcacaaatt attcttatgg cagatgataa tacgtttctt gcacacaact
     6481 taaagaacgg caagatcgct aataatcaac cgtgtaccgg aattatctac gcggagatcg
     6541 cctcgcgcta tatgaaagcc gattccaaag atgtgcttgc cgtcgggctt ggaaaagtgg
     6601 ggtttccagg cgctgcacat ttggttaata aggggtttcg tgtgtatggg tacgacgcgg
     6661 ataaggcgtt gttagaaaag actgtatcag accttgggat cacacctttc gagcctgaga
     6721 atccccgtcg cttctcaatc atttttgagg cgaccccgtg tgctgataca gtaccagagt
     6781 ctgtcatcag cgacaattgt gtcatctcta cgcccggtat cccttgcgcg atcagccgtg
     6841 aacttcaaga aaaatacggg gtggagttga ttatggaacc tctgggtatt ggaactgctt
     6901 ctatgctgta tagcattttg taaagctcga gatctgcagc tggtaccata tgggaattcg
     6961 aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     7021 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     7081 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     7141 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     7201 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     7261 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcc
//
