LOCUS       sequence-338578-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PylB overexpression and purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6616)
  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 6616)
  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: pJH859
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     973..992
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1012..1089)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1295..1314
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1497..1514
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1524..1544
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1560..1853
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MLDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2948..2966)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2962..3009
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3046..3501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3133..3152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3343..3364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3593..4408)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3593..4408)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4316..4335
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4400..4419
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4530..5118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5019..5038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5272..5289
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5304..5443
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5369..5388)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5529..5551
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5545..5736)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6545..6616,1..1011))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
       61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
      241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      301 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
      901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
      961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     1501 atcatcatca tcacagcagc ggcgaaaacc tgtattttca gggcatgatg ggcagcagca
     1561 tgctagactc agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg
     1621 agactcacat caatttaaag gtgtccgatg ggtcttcaga gatcttcttc aagatcaaaa
     1681 agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg
     1741 actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc cctgaagatt
     1801 tggacatgga ggataacgat attattgagg ctcacagaga acagattggt ggtcatatga
     1861 ttaagaagat ggcgactgaa gatctggatc gttttggcga aaaaattatt gaaggatttc
     1921 agttatctga cgacgattta cgcgctttgc ttagcctgga atcggaagaa gaattggaaa
     1981 agctgtatta cgtcgcacgt aaagtacgca accattactt tggaaatcgc gtattcctgt
     2041 catgttttat ctacttcagc acatactgca aaaaccaatg tgcgttttgc tattataact
     2101 gcaaaaacaa gattaaccgc tatcgtttgt cccgcgaaga aatcaaagag acttgcaagg
     2161 cccttaaagg tgcgggtttc catatgattg atctgactat gggggaggat ccctattatt
     2221 acaaggaacc tgatcgcttc gtggagctgg tccaaatggt taagaatgag cttggacttc
     2281 caatcatgat ttcgccagga gtaatggata atgctaccct tttaaaagca cgcgaaaagg
     2341 gagctaattt cttcgcctta tatcaagaaa cctatgatca aaagttgtac aagaagttgc
     2401 gcgtaggtca gtcgttcaag aggcgctaca atactcgtcg ttttgctaaa gaacaagggt
     2461 attgcattga agacggtatc ttaacgggag tcgggaacga tatcgagtca acgatcaaat
     2521 ctttacgcgg catgaaatca aatgatcccg acatggttcg cgtgatgact ttcttacccc
     2581 aggaaggaac tccccttgag cgttttaagg acaatagtaa cctgagcgaa ctgaaaatca
     2641 ttgcgatctt gcgtctgatc tttccgaagt gcttaatccc cgcgtcgttg gacctggaag
     2701 ggatcgacgg gatggtacac cgtcttaacg caggtgctaa tatcgtgact agcatccttc
     2761 ctagtgattc aaaacttgag ggagttgcta actacgatcg tgacctggaa gagcgcgacc
     2821 gcgacattaa aagcgtcatc aagcgccttg agggaatggg catggaacca gcgccacagg
     2881 cggaattcga gactgtactg cgctgctaat aacaaagccc gaaaggaagc tgagttggct
     2941 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     3001 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     3061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3241 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3361 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3481 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3541 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     3601 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     3661 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     3721 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     3781 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     3841 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     3901 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     3961 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     4021 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     4081 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     4141 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     4201 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     4261 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     4321 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     4381 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     4441 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4501 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4561 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4621 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     4681 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4741 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4801 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4861 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4921 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4981 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5041 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5101 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5161 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5221 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     5281 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5341 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5401 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     5461 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5521 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5581 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     5641 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     5701 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     5761 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     5821 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     5881 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     5941 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     6001 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     6061 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     6121 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     6181 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     6241 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     6301 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     6361 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     6421 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     6481 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     6541 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6601 ccaacgcgcg gggaga
//
