LOCUS       sequence-339055-        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: pJH858
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..37
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        18..36
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    37..61
                     /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             76..98
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             117..134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             144..164
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             180..473
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(1568..1586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1582..1629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1666..2121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1753..1772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1963..1984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2213..3028)
                     /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(2213..3028)
                     /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     2936..2955
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3020..3039
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3150..3738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3639..3658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3892..3909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3924..4063
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3989..4008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4149..4171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4165..4356)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5165..6247)
                     /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"
     primer_bind     6209..6228
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6248..6325)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6531..6550
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
       61 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
      121 atcatcatca tcacagcagc ggcgaaaacc tgtattttca gggcatgatg ggcagcagca
      181 tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg
      241 agactcacat caatttaaag gtgtccgatg ggtcttcaga gatcttcttc aagatcaaaa
      301 agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg
      361 actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc cctgaagatt
      421 tggacatgga ggataacgat attattgagg ctcacagaga acagattggt ggtcatatga
      481 ttaagaagat ggcgactgaa gatctggatc gttttggcga aaaaattatt gaaggatttc
      541 agttatctga cgacgattta cgcgctttgc ttagcctgga atcggaagaa gaattggaaa
      601 agctgtatta cgtcgcacgt aaagtacgca accattactt tggaaatcgc gtattcctgt
      661 catgttttat ctacttcagc acatactgca aaaaccaatg tgcgttttgc tattataact
      721 gcaaaaacaa gattaaccgc tatcgtttgt cccgcgaaga aatcaaagag acttgcaagg
      781 cccttaaagg tgcgggtttc catatgattg atctgactat gggggaggat ccctattatt
      841 acaaggaacc tgatcgcttc gtggagctgg tccaaatggt taagaatgag cttggacttc
      901 caatcatgat ttcgccagga gtaatggata atgctaccct tttaaaagca cgcgaaaagg
      961 gagctaattt cttcgcctta tatcaagaaa cctatgatca agagttgtac aagaagttgc
     1021 gcgtaggtca gtcgttcaag aggcgctaca atactcgtcg ttttgctaaa gaacaagggt
     1081 attgcattga agacggtatc ttaacgggag tcgggaacga tatcgagtca acgatcaaat
     1141 ctttacgcgg catgaaatca aatgatcccg acatggttcg cgtgatgact ttcttacccc
     1201 aggaaggaac tccccttgag cgttttaagg acaatagtaa cctgagcgaa ctgaaaatca
     1261 ttgcgatctt gcgtctgatc tttccgaagt gcttaatccc cgcgtcgttg gacctggaag
     1321 ggatcgacgg gatggtacac cgtcttaacg caggtgctaa tatcgtgact agcatccttc
     1381 ctagtgattc aaaacttgag ggagttgcta actacgatcg tgacctggaa gagcgcgacc
     1441 gcgacattaa aagcgtcatc aagcgccttg agggaatggg catggaacca gcgccacagg
     1501 cggaattcga gactgtactg cgctgctaat aacaaagccc gaaaggaagc tgagttggct
     1561 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1621 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1681 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1741 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1801 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1861 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1921 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1981 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2041 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2101 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2161 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2221 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2281 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2341 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2401 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2461 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2521 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2581 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2641 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2701 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2761 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2821 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2881 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2941 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     3001 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     3061 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3121 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3181 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3241 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3301 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3361 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3421 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3481 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3541 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3601 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3661 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3721 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3781 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3841 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3901 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3961 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4021 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4081 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4141 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4201 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4261 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4321 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4381 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4441 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4501 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4561 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4621 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4681 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4741 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4801 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4861 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4921 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4981 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5041 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5101 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5161 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5221 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5281 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5341 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5401 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5461 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5521 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5581 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5641 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5701 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5761 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5821 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5881 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5941 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6001 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6061 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6121 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6181 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6241 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6301 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6361 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6421 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6481 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6541 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6601 gcgtagagga tcgaga
//
