LOCUS       sequence-447357-        8120 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses precursor peptide and lanthipeptide synthetase from 
            LanIII-6 (FAST-RiPPs) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8120)
  AUTHORS   Ayikpoe RS, Shi C, Battiste AJ, Eslami SM, Ramesh S, Simon MA, 
            Bothwell IR, Lee H, Rice AJ, Ren H, Tian Q, Harris LA, Sarksian R, 
            Zhu L, Frerk AM, Precord TW, van der Donk WA, Mitchell DA, Zhao H
  TITLE     A scalable platform to discover antimicrobials of ribosomal origin.
  JOURNAL   Nat Commun. 2022 Oct 17;13(1):6135. doi: 10.1038/s41467-022-33890-w.
  PUBMED    36253467
REFERENCE   2  (bases 1 to 8120)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-10-17"; volume: "13(1):6135. doi"; pages: " 
            10.1038/s41467-022-33890-w"
COMMENT     Sequence Label: pET28:LanIII-6
FEATURES             Location/Qualifiers
     source          1..8120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3508..3532
                     /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)."
     CDS             3549..3566
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3838..3885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3890..3908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3909..3933
                     /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)."
     terminator      6549..6596
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6637..7092
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6724..6743)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6934..6955
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7184..7999)
                     /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(7184..7999)
                     /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     7907..7926
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7991..8010
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaatatggta atacgactca ctatagggga attgtgagcg gataacaatt ccaaggagat
     3541 atacaatgca ccaccaccac caccacgcaa tgaaagacgt tctcgaatta cagcagttga
     3601 acgaaggtac tgaagtcgaa gcctcgaaag gaaaaggcgg cttgctgacg ctgatcacca
     3661 ccattgtggg gtcgtctacg atctcaaaca attgttaact agaagcccgg cagagttagt
     3721 tcgaacctaa tgccacaaat gagacaggac gccaatgggt accggacatt aggtcgagct
     3781 cagttcggta acggagagac cctgcggcgt acttaattat gcatatgaaa cgccggtcta
     3841 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttggctct aatacgactc
     3901 actatagggg aattgtgagc ggataacaat tccaaggaga tatacaatgg ataagaagct
     3961 ggtctacttt cagcatatga gcgaggattc taagtactat atcgagaaaa gtccgaacgc
     4021 cgaaaccgat ccgtatgaag tgcacaacat tccaaactat tataccgtga gcacggaata
     4081 taagagccca tggaaattct acaacaatcc taatcatact cttaaagagc aaggttggaa
     4141 aatccatgtg tcagcaacta tgaataacgc ccaggatatt ctcgaggtgg tgagccagat
     4201 tatctttaag caccaaatcc cgttcaagca cattaaggat aagcagagtc tctttaatat
     4261 gaactctaag catggaaatc gtacgtcctc aggcaaattc attgctattt acccttcctc
     4321 taataacatg ttcttccgtt tactgaacga actgtacgaa gaactgaagc attacgagaa
     4381 cgggccgtat atcctgaatg ataaatcatg gaaagacagc aacatttatt atcgttacgg
     4441 cgcctttaag aatatttaca atgagcgcgg agagctgtgt attaaagatg aaaacggaaa
     4501 cctgatccct gataatcgtc agccgtatta taaaattccg gaatttgtaa gcgactttga
     4561 tcagtatctg gacagcaaga ataacctgct ggacactcag gatgtgagca atttcaatga
     4621 ttatgagttt cagggcgtgc tgcgcttcaa taacggcggc ggaatttaca cgggcatccg
     4681 caaaagcgac ggcaagaagg tagttattaa agagggtcgc cccaaagttg gattagatgg
     4741 ccagttcaac gatgcgatcg accgtcttaa tattgaatac gaagcgctga ataaactgac
     4801 cgatgtcgaa ggcgtcgtga acgttctgga ttactttaag gcctggaaac acgtctttct
     4861 tgttgaggag ttcgtcgagg gcgtcacctt acagacctgg tgtgcgatta actatccgtt
     4921 taacgtagag aacgacaaga atgagtatat ttccaaagtt aagaaaatta tcaacagcct
     4981 ggtgaaaacc atcaagcaag tccacagcaa agatgttgga atgggagatc tgcaacctca
     5041 gaacattatg atcacccccg aaagcaacgt gaagattatt gatttcgagg ctgcctcggc
     5101 gttaagcgaa gaaaacggcg cgctgcagac gattgggtat actaacaaga agaataagaa
     5161 ccataaggaa cgcgattggt acgcgctcaa gaaggtttta cgcttctgca ttctgccaat
     5221 cgggcccgtg gccgacattg aagagacgat caagaagaaa cacgacatgt ggatcgagga
     5281 cgaatttggt aaggacgtct accagtatgt tgtggatacc gagaatctgt gtgacaaata
     5341 cctgtccgac accaaggaaa agatcttcaa gaacagttat gattctaaaa tccagaagga
     5401 aatggatatg tcgaaagtaa tcaagggact ccgtaactca atcgagagta acattcttcc
     5461 gaatagctcg cttattcacg gtgatgtacg ccaatttgtc atgccgtctg ggaaactgaa
     5521 tgtactgacg ggcggcacgg gcgctgcctt agcactgtac cgttctggga acgtctccaa
     5581 agaggtttgg gactggatcg agaacgttct gctgaagaag ttaaatgtgc tcaacgacga
     5641 tgttggtttg ttcacgggcc gcgccgggat tgccgccatc ctgtacgaat taggctacat
     5701 tgaacaggcg atggacttat ttaacaatct tagctacgaa gatatgaaag acatttcact
     5761 gcgcagtggc ctgtcaggga ttggactgag tttaatcagc ctgtacatgg aagaaaacaa
     5821 tgatgacttc cttgtgactt cggagaaaat tgccgaccaa atcgaaaacc gtatcatcga
     5881 ggacgacaaa ttgagtgtga ccgatttcgc tgcagtgcca attggcttga ttgatggctg
     5941 gagcggtatc agcctgttct atgcggcgtt atattccgta actaagaatg agaagtacta
     6001 tctgaaatca aaggaatata tcgagcgcga cctgaagcac tgcaaagtgg atgaagagac
     6061 gaacaccctt cagacgtatg atgaccagaa tccgcgcctt ctcccgtacc ttagcggtgg
     6121 cagcatcggg atcggtatcg caatctggta cctgaattac gttaagaata ataacctttt
     6181 ccgcgaagaa ctggagcaga tcatcaatct gaacaaggtc cgcattgcct attctggtgg
     6241 ccttttcgat ggtatcggta gcttcctgct gattcctagc atgaccaaag agaagaacga
     6301 gacttctcaa cacatcgaac tggcacttcg caaactggaa ttatttctga ttcgtaaaga
     6361 tgaccaggtc ctgtttccgg gcaacttcag cttccgtctc gccgatgact actactcggg
     6421 ctccgcgggc attattctgg gtatcaactc gattcagttc tctaacccac tgtactggct
     6481 gcccattatc aaaatcgatt cgttcctgcg taagacaaag aaagaactgt tactgaacaa
     6541 ttaacggtct agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgagcg
     6601 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     6661 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     6721 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     6781 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     6841 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     6901 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     6961 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     7021 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     7081 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7141 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     7201 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     7261 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     7321 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     7381 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     7441 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     7501 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     7561 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     7621 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     7681 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     7741 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     7801 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     7861 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     7921 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     7981 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     8041 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     8101 gaaaagatca aaggatcttc
//
