LOCUS       sequence-338837-        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: pJH577
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(65..151)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     304..321
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(3411..3704)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     promoter        complement(3753..4037)
                     /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(3811..3830)
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4064..4942
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     primer_bind     complement(5098..5120)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5206..5346
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5261..5280
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5361..5378)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5543..6131)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5623..5642)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6302..7093,7094..7162))
                     /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     6925..6944
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7163..7254)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(7273..7300)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg
       61 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
      121 agtctttcga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg
      181 ggagacccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca
      241 ggtgggacca ccgcgctact gccgccaggc aaattctgtt ttatcagacc gcttctgcgt
      301 tctgatttaa tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca gccaagcttc
      361 gaattcccat atggtaccag ctgcagatct cgagctttac aaaatgctat acagcataga
      421 aacagttcca atacccagag gttccataat caactccacc ccgtattttt cttgaagttc
      481 acggctgatc gcgcaaggga taccgggcgt agagatgaca caattgtcgc tgatgacaga
      541 ctctggtact gtatcagcac acggggtcgc ctcaaaaatg attgagaagc gacggggatt
      601 ctcaggctcg aaaggtgtga tcccaaggtc tgatacagtc ttttctaaca acgccttatc
      661 cgcgtcgtac ccatacacac gaaacccctt attaaccaaa tgtgcagcgc ctggaaaccc
      721 cacttttcca agcccgacgg caagcacatc tttggaatcg gctttcatat agcgcgaggc
      781 gatctccgcg tagataattc cggtacacgg ttgattatta gcgatcttgc cgttctttaa
      841 attgtgtgca agaaacgtat tatcatctgc cataagaata atttgtgctc cattacggac
      901 cgcctcatag tagccactaa cgtctggcat ggtcgtcacg aatgagtcaa atccaaagta
      961 ctcagtgatc gcacgaagac tttcagaaaa gttgccaatg atgccgttgc cactagttac
     1021 cgggacgata ccgaccttct cacagcatgg agtagtccca tagaaatctt tacaaactcc
     1081 cttgatatct aacccagtca cacgacgtac cgtagaatcg gcttcgtgaa gttgacggtt
     1141 aatgttctcc aggtcctcgg gagtcaacaa agccattttt tatctccttt ttctgtttta
     1201 aacaaatttt actttgggca aatttttatt cttatccgat ttttgccatt ttaaacggct
     1261 gccccgaagc ggtccttcaa aatcagcaaa ccagtgcgct ttttgttttc ggcttcctca
     1321 cggtcctttc cccaaaagac aagcgtaaac acggggtcct ctccgcgaca taaaaagatt
     1381 tcaattcctg gttcttcata gtatttccca tagtcattcc ccatcgacag aacctgctca
     1441 cctactggga tcagggcccc attttctgca agcatcaggt gctcgtagat acagtagcgg
     1501 tcctctggaa gggttttaac ttcctccacc ccttctccaa aggcacgaaa caacagctca
     1561 attaaattca ctcccgacga ataatacacg gctgtggggg tctgggaggg gaaacgtgaa
     1621 tcaatttcaa tgaccttcaa accttgcggg cccgaaattg cttcaacgtc catgatcccc
     1681 ttcaaaggca ggttggccgc taaagaatag ctgatctcac ggaaagacaa atcagatggc
     1741 aggggagtta ccatatggca gtcataggtg cggtcgatgt gaataagtgt ctctttaaca
     1801 actgcaaagt gggtaccgtc cccgattact tcaagtgaaa taacctcacc ttccacatac
     1861 tcttctacca gcatgccggg ctccaattct cccagttctt cttcatcgta gataatgcga
     1921 gcaccaaccg acgagctttc gcacggtggc ttaacgaagt atggaggacg accaggtttg
     1981 tcttgcggtg tcggaatgcc gatggaacgg aaatactctt tcgatttctt cttatcgcgc
     2041 gagatgcggt aagcctcaaa atcgaacagt actgggcaag aaaattcctc ttttacacta
     2101 cttaagaatt taatacattc taagttttcg ttgacgggca agatagcgtc tacatttttg
     2161 ctgatttcaa gaagtttctc tggttccttg ataacatcaa aacaatgaaa ctcgtctgcg
     2221 taattgcgga taagagcttg ggggttcttg tcgataacca ctactttcat atcggctttt
     2281 ttagacagat acgcggcctc aaaaccctgc aatttaccac caatcaggca gatggttttc
     2341 atttttcctc cttattagca gcgcagtaca gtctcgaatt ccgcctgtgg cgctggttcc
     2401 atgcccattc cctcaaggcg cttgatgacg cttttaatgt cgcggtcgcg ctcttccagg
     2461 tcacgatcgt agttagcaac tccctcaagt tttgaatcac taggaaggat gctagtcacg
     2521 atattagcac ctgcgttaag acggtgtacc atcccgtcga tcccttccag gtccaacgac
     2581 gcggggatta agcacttcgg aaagatcaga cgcaagatcg caatgatttt cagttcgctc
     2641 aggttactat tgtccttaaa acgctcaagg ggagttcctt cctggggtaa gaaagtcatc
     2701 acgcgaacca tgtcgggatc atttgatttc atgccgcgta aagatttgat cgttgactcg
     2761 atatcgttcc cgactcccgt taagataccg tcttcaatgc aatacccttg ttctttagca
     2821 aaacgacgag tattgtagcg cctcttgaac gactgaccta cgcgcaactt cttgtacaac
     2881 tcttgatcat aggtttcttg atataaggcg aagaaattag ctcccttttc gcgtgctttt
     2941 aaaagggtag cattatccat tactcctggc gaaatcatga ttggaagtcc aagctcattc
     3001 ttaaccattt ggaccagctc cacgaagcga tcaggttcct tgtaataata gggatcctcc
     3061 cccatagtca gatcaatcat atggaaaccc gcacctttaa gggccttgca agtctctttg
     3121 atttcttcgc gggacaaacg atagcggtta atcttgtttt tgcagttata atagcaaaac
     3181 gcacattggt ttttgcagta tgtgctgaag tagataaaac agctcaggaa tacgcgattt
     3241 ccaaagtaat ggttgcgtac tttacgtgcg acgtaataca gcttttccaa ttcttcttcc
     3301 gattccaggc taagcaaagc gcgtaaatcg tcgtcagata actgaaatcc ttcaataatt
     3361 ttttcgccaa aacgatccag atcttcagtc gccatcttct taatcatatg accaccaatc
     3421 tgttctctgt gagcctcaat aatatcgtta tcctccatgt ccaaatcttc aggggtctga
     3481 tcagcttgaa ttctaatacc gtcgtacaag aatcttaagg agtccatttc cttaccctgt
     3541 cttttagcga acgcttccat cagccttctt aaaggagtgg tctttttgat cttgaagaag
     3601 atctctgaag acccatcgga cacctttaaa ttgatgtgag tctcaggctt gacttctggc
     3661 ttgacctctg gcttagcttc ttgattgact tctgagtccg acatgctgct gcccatcatg
     3721 gttaattcct cctgttagcc caaaaaacgg gtatggagaa acagtagaga gttgcgataa
     3781 aaagcgtcag gtaggatccg ctaatcttat ggataaaaat gctatggcat agcaaagtgt
     3841 gacgccgtgc aaataatcaa tgtggacttt tctgccgtga ttatagacac ttttgttacg
     3901 cgtttttgtc atggctttgg tcccgctttg ttacagaatg cttttaataa gcggggttac
     3961 cggtttggtt agcgagaaga gccagtaaaa gacgcagtga cggcaatgtc tgatgcaata
     4021 tggacaattg gtttcttctc tgaatggcgg gagtatgaaa agtatggctg aagcgcaaaa
     4081 tgatcccctg ctgccgggat actcgtttaa tgcccatctg gtggcgggtt taacgccgat
     4141 tgaggccaac ggttatctcg atttttttat cgaccgaccg ctgggaatga aaggttatat
     4201 tctcaatctc accattcgcg gtcagggggt ggtgaaaaat cagggacgag aatttgtttg
     4261 ccgaccgggt gatattttgc tgttcccgcc aggagagatt catcactacg gtcgtcatcc
     4321 ggaggctcgc gaatggtatc accagtgggt ttactttcgt ccgcgcgcct actggcatga
     4381 atggcttaac tggccgtcaa tatttgccaa tacggggttc tttcgcccgg atgaagcgca
     4441 ccagccgcat ttcagcgacc tgtttgggca aatcattaac gccgggcaag gggaagggcg
     4501 ctattcggag ctgctggcga taaatctgct tgagcaattg ttactgcggc gcatggaagc
     4561 gattaacgag tcgctccatc caccgatgga taatcgggta cgcgaggctt gtcagtacat
     4621 cagcgatcac ctggcagaca gcaattttga tatcgccagc gtcgcacagc atgtttgctt
     4681 gtcgccgtcg cgtctgtcac atcttttccg ccagcagtta gggattagcg tcttaagctg
     4741 gcgcgaggac caacgtatca gccaggcgaa gctgcttttg agcaccaccc ggatgcctat
     4801 cgccaccgtc ggtcgcaatg ttggttttga cgatcaactc tatttctcgc gggtatttaa
     4861 aaaatgcacc ggggccagcc cgagcgagtt ccgtgccggt tgtgaagaaa aagtgaatga
     4921 tgtagccgtc aagttgtcat aattggtaac gaatcagaca attgacggct tgacggagta
     4981 gcatagggtt tgcagaatcc ctgcttcgtc catttgacag gcacattatg catgccgctt
     5041 cgccttcgcg cgcgaattga tctgctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     5101 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     5161 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     5221 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     5281 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     5341 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5401 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5461 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca gcaaaaggcc aggaaccgta
     5521 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5581 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5641 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5701 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5761 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5821 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5881 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5941 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6001 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6061 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6121 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6181 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6241 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6301 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6361 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6421 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6481 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6541 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6601 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6661 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6721 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6781 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6841 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6901 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6961 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7021 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7081 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7141 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7201 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaaagagtt
     7261 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg atgcctggca
     7321 gtttatg
//
