LOCUS       sequence-338632-        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: pJH769v2
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1463..1480
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1490..1510
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1526..1819
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2914..2932)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2928..2975
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3012..3467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3099..3118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3309..3330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3559..4374)
                     /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(3559..4374)
                     /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     4282..4301
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4366..4385
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4496..5084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4985..5004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5238..5255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5270..5409
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5335..5354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5495..5517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5511..5702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6511..6616,1..977))
                     /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 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcg aaaacctgta
     1501 ttttcagggc atgatgggca gcagcatgtc ggactcagaa gtcaatcaag aagctaagcc
     1561 agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatgggtc
     1621 ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt
     1681 cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat
     1741 tcaagctgat cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca
     1801 cagagaacag attggtggtc atatgattaa gaagatggcg actgaagatc tggatcgttt
     1861 tggcgaaaaa attattgaag gatttcagtt atctgacgac gatttacgcg ctttgcttag
     1921 cctggaatcg gaagaagaat tggaaaagct gtattacgtc gcacgtaaag tacgcaacca
     1981 ttactttgga aatcgcgtat tcctgagctg ttttatctac ttcagcacat actgcaaaaa
     2041 ccaatgtgcg ttttgctatt ataactgcaa aaacaagatt aaccgctatc gtttgtcccg
     2101 cgaagaaatc aaagagactt gcaaggccct taaaggtgcg ggtttccata tgattgatct
     2161 gactatgggg gaggatccct attattacaa ggaacctgat cgcttcgtgg agctggtcca
     2221 aatggttaag aatgagcttg gacttccaat catgatttcg ccaggagtaa tggataatgc
     2281 taccctttta aaagcacgcg aaaagggagc taatttcttc gccttatatc aagaaaccta
     2341 tgatcaaaag ttgtacaaga agttgcgcgt aggtcagtcg ttcaagaggc gctacaatac
     2401 tcgtcgtttt gctaaagaac aagggtattg cattgaagac ggtatcttaa cgggagtcgg
     2461 gaacgatatc gagtcaacga tcaaatcttt acgcggcatg aaatcaaatg atcccgacat
     2521 ggttcgcgtg atgactttct taccccagga aggaactccc cttgagcgtt ttaaggacaa
     2581 tagtaacctg agcgaactga aaatcattgc gatcttgcgt ctgatctttc cgaagtgctt
     2641 aatccccgcg tcgttggacc tggaagggat cgacgggatg gtacaccgtc ttaacgcagg
     2701 tgctaatatc gtgactagca tccttcctag tgattcaaaa cttgagggag ttgctaacta
     2761 cgatcgtgac ctggaagagc gcgaccgcga cattaaaagc gtcatcaagc gccttgaggg
     2821 aatgggcatg gaaccagcgc cacaggcgga attcgagact gtactgcgct gctaataaca
     2881 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     2941 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     3001 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3061 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     3601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     3661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     3721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     3781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     3841 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     3901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     3961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     4381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     4441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     5341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     6001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     6061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     6121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     6181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     6241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     6301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     6361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     6421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     6481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6601 cgccagggtg gttttt
//
