LOCUS       sequence-338630-        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: pJH768v2
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     948..967
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(987..1064)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1270..1289
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1373..1392
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1373..1391
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1392..1416
                     /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             1431..1453
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1472..1489
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1499..1519
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1535..1828
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2923..2941)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2937..2984
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3021..3476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3108..3127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3318..3339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3568..4383)
                     /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(3568..4383)
                     /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     4291..4310
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4375..4394
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4505..5093
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4994..5013
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5247..5264
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5279..5418
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5344..5363)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5504..5526
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5520..5711)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6520..6616,1..986))
                     /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 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
       61 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
      121 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
      181 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
      241 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
      301 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
      361 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
      421 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
      481 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
      541 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
      601 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
      661 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
      721 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
      781 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
      841 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
      901 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
      961 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     1021 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     1081 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     1141 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     1201 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     1261 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     1321 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     1381 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     1441 tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcga
     1501 aaacctgtat tttcagggca tgatgggcag cagcatgtcg gactcagaag tcaatcaaga
     1561 agctaagcca gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc
     1621 cgatgggtct tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat
     1681 ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg
     1741 tattagaatt caagctgatc agacccctga agatttggac atggaggata acgatattat
     1801 tgaggctcac agagaacaga ttggtggtca tatgattaag aagatggcga ctgaagatct
     1861 ggatcgtttt ggcgaaaaaa ttattgaagg atttcagtta tctgacgacg atttacgcgc
     1921 tttgcttagc ctggaatcgg aagaagaatt ggaaaagctg tattacgtcg cacgtaaagt
     1981 acgcaaccat tactttggaa atcgcgtatt cctgagctgt tttatctact tcagcacata
     2041 ctgcaaaaac caatgtgcgt tttgctatta taactgcaaa aacaagatta accgctatcg
     2101 tttgtcccgc gaagaaatca aagagacttg caaggccctt aaaggtgcgg gtttccatat
     2161 gattgatctg actatggggg aggatcccta ttattacaag gaacctgatc gcttcgtgga
     2221 gctggtccaa atggttaaga atgagcttgg acttccaatc atgatttcgc caggagtaat
     2281 ggataatgct acccttttaa aagcacgcga aaagggagct aatttcttcg ccttatatca
     2341 agaaacctat gatcaagagt tgtacaagaa gttgcgcgta ggtcagtcgt tcaagaggcg
     2401 ctacaatact cgtcgttttg ctaaagaaca agggtattgc attgaagacg gtatcttaac
     2461 gggagtcggg aacgatatcg agtcaacgat caaatcttta cgcggcatga aatcaaatga
     2521 tcccgacatg gttcgcgtga tgactttctt accccaggaa ggaactcccc ttgagcgttt
     2581 taaggacaat agtaacctga gcgaactgaa aatcattgcg atcttgcgtc tgatctttcc
     2641 gaagtgctta atccccgcgt cgttggacct ggaagggatc gacgggatgg tacaccgtct
     2701 taacgcaggt gctaatatcg tgactagcat ccttcctagt gattcaaaac ttgagggagt
     2761 tgctaactac gatcgtgacc tggaagagcg cgaccgcgac attaaaagcg tcatcaagcg
     2821 ccttgaggga atgggcatgg aaccagcgcc acaggcggaa ttcgagactg tactgcgctg
     2881 ctaataacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     2941 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     3001 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3061 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3121 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3181 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3241 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3301 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3361 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3421 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     3481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3541 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     3601 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     3661 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     3721 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     3781 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     3841 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     3901 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     3961 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     4021 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     4081 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     4141 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     4201 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     4261 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     4321 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     4381 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     4441 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4501 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4561 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4621 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4681 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4741 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4801 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4861 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4921 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4981 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5041 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5101 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5161 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5221 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5281 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     5341 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     5401 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5461 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5521 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     5581 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     5641 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     5701 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     5761 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     5821 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     5881 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     5941 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     6001 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     6061 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     6121 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     6181 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     6241 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     6301 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     6361 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     6421 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6481 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6541 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6601 cgtattgggc gccagg
//
