LOCUS       sequence-447356-        8768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide and lanthipeptide synthetase from 
            LanII-2E (FAST-RiPPs) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8768)
  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 8768)
  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: "2022-10-17"; volume: "13(1):6135. doi"; pages: " 
            10.1038/s41467-022-33890-w"
COMMENT     Sequence Label: pET28:LanII-2E
FEATURES             Location/Qualifiers
     source          1..8768
                     /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      3877..3924
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3929..3947
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3948..3972
                     /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      7197..7244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7285..7740
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7372..7391)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7582..7603
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7832..8647)
                     /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(7832..8647)
                     /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     8555..8574
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8639..8658
                     /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 tgtttaacct ggatattatc cgcgcctgga
     3601 aagatgagaa ctaccgcgat agtctcaccg aggcgcagcg ttctcaatta ccggaaaacc
     3661 cggcaggatt gattgagttg accgacgaag atatgagctc ggtatcgggc ggttgctgtt
     3721 gcggaagcag cgcgtgctcg agcttagtac atcatatggg agtggaagag agttccacac
     3781 tggcctaaag ctcttctccc ctgcggtcac gtctatagaa attacaccat taaccctcct
     3841 gagaaccggg aggcgggaat ccgtcacgta tgcggtctag cataacccct tggggcctct
     3901 aaacgggtct tgaggggttt tttgccctta atacgactca ctatagggga attgtgagcg
     3961 gataacaatt ccaaggagat atacaatgga tctgtctgtt ttccagggtt tcaagggtga
     4021 gcgcgcactg accctgtccg aacgtagcct ggccttatcc caggaaaagg ggtcgagcgc
     4081 actgccaatg aacgctgaat tttccgaccg ccgccgccag cgttggcaaa ccgaatttcg
     4141 cgggaacacg gcattctttc aacagcacct ggcccagatt ggaattaccg aaagccagtt
     4201 tcgctatctg ctgggtgagt ttccggaaaa tcttcgcaag cgccatccgg aactgttagc
     4261 atgggagact caaattcaaa cagcctttgc gcagtttcca cctcagtccc aagagagtgg
     4321 cttctttagc gcgatcgcgc cactcattga acaaggtaaa acccgtttat cccagcagct
     4381 gtcagccctc gcagcccagc atagcaccgc ccttccattc gaaatcgaaa gcttagaacg
     4441 tctgcttctg ccggccttgt cagagcgttt acaagaaatt ttcaatccta cgttcgttct
     4501 ggagctgaac gttgcgcgcc tgcaggaccg tctttccggc gacaccccga gcgatcgctt
     4561 tcagtccttt gttcaacact tgcaagaccc agaattttgt ctgagcctgc tccaggaata
     4621 tccggtgctg gtacgtcagt gcgcgattgc cattgattgt tgggtcgagg gttcccagga
     4681 acttctgcaa cgcctgtgta cggactggga cctgatttgt gcacagctgt cgccgcagac
     4741 cgaaccgggc attctggttc acattcatcc gggagccggc gatcgtcatc atcgcggccg
     4801 ttcggttacg ctgctccagt ttagctcagg ttttcgcctc gtatacaaac ctaaaccact
     4861 ggctgttgat ctgcattttc aagaactgct ggcctggatt aatcagaact ctaactttct
     4921 gccgttttca tgctggcagg gcattgatcg tggggaatac ggctggatgg aatttatcgc
     4981 cccgcagact tgccagaccc cggcagaaat tgaacgcttt tatcagcgtc tgggcgggta
     5041 cctggcgctg ctgtatttgc tggccggcac cgactttcat gccgagaatc ttattgcagt
     5101 tggcgagcaa ccgattttga tcgacctgga atccttattt cagccatacc ctgacgcatc
     5161 ggaagatctg aacacagtac tgccggcacg tcgccagatt gcacaaagcg tattgcgcat
     5221 tggtctgtta ccgcgccgtc tccttttgaa ccgtcagcac gaggctgtcg acgctagcgg
     5281 aatgggtggg gacagtcaac aactggcagc tgaccgcatc ccgcatcttg cagccattgg
     5341 caccgatact atgcgcgttg ttcgccagcc cggccgtctg cgtgaaacgc ataatcagcc
     5401 gtcattaaat gaccgtccca tccaaattct tgattatcgc gaagccatcg catgtggttt
     5461 ctcagcgctg tatcgcttca ttctcaaatc tcgcgaccgc ctggaaccgt tacttgagtc
     5521 gttcgcctca gacccgattc gcatcttctt gcgtgaaacc cgcacctaca gcttactgtt
     5581 acgcgaaagt tttcacccgg acgtgctgcg tgatgcactg gaacgtgatc gcttattcaa
     5641 caaattatgg gtggatgtaa agaaccaccc ataccttgcg cagctgatcc cagcggaacg
     5701 tgaggcactg tggctgggcg atattcctaa attcactacc cgtccgaatt cgcgtcatct
     5761 ggcgctgaat aatgccgagt ctctgacgca tttcttctcg caaacgggat tagaattagt
     5821 gcaacaccgc ctgcaatgtt tctcagagga cgacctgcaa cagcagttgc agtttattga
     5881 ctcggcgctg accacactgg caatggttgc cgaggtgaaa actgcccctg catacgctat
     5941 tccgaaattt agcaccgtta gtgacgaagc aagcgccatt gcgtcgtacc tgcaggccgc
     6001 atgtgagatt ggtgatcgct tggaaggctt agcagcacgt gatcgtcagc tggctggatg
     6061 ggttgcaccc gttttaaccg atgcccgtca ctgggccgtg tccccgttgg gttgggacct
     6121 gtttgagggc cttcctggta ttgctttgtt tctggcgtat ttgggcgaaa tcacccaaga
     6181 acaacgctat accaccttgg cacaggccgc cattgcaagc ctgctgtatc agattcgcag
     6241 cgaggattat cttattgcgt cgatcggagc gtttagtggc tggggcggct tgatttactc
     6301 tctgactcac ctgggcattt tatggcagca accgcacctc ttggaacagg ccttggcctg
     6361 ggtggaattg ctgccggagc gcatcgcgca ggatcgccag ttcgatatta tgggcggtgc
     6421 tgcaggttgc attggcgccc tggtagcact tggggagtgc gtcggagcaa gcaacgttcg
     6481 cgcggctgca attcagtgtg gcgaccgtct cttacagaac gcgcagacca tgccacaggg
     6541 catgggatgg attacgttgc cacagtgtcc gccgcttagc ggttttagcc atggtgccgc
     6601 tggcatcgcc tgggcactgc tgaaacttgc ccaccttagc ggagaagccc gcttccaaga
     6661 agctgccggg gcagctattg cgtatgaacg tagcctcttt tcgatccgtg cacaaaattg
     6721 gctggatctc cgccaccgtg gcgatagcgg caccgatcgc tatgcaaatc gtgcgttcgg
     6781 catgggctgg tcccatggag cgccgggcat tggcttagga cgcttacata gcctgaaact
     6841 gatggatgat ccggatctgc gcgtcgaact ggatctggca ctggaaacga cgacccgtca
     6901 tggctttggc tacaaccact cgcttggcaa tggtgatctt ggaaatctgg aatttctgga
     6961 ttccgcccag cagcagctga gtctgccgca gggtcgttca caagtgcatc aaatggccat
     7021 gggcatctta gcgtcaatcc agcagcaggg atgggtatgc gatttgccgc tcggcgtgga
     7081 atctttaggc cttatgaccg gtcttgcagg tattgggtat ggtttcctgc gcttagccaa
     7141 accggatcgc gtaccgtcgc tgttggtatt agcaccgccg cccctgcgtt aacggtctag
     7201 cataacccct tggggcctct aaacgggtct tgaggggttt tttgagcgct gaaaggagga
     7261 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     7321 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     7381 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     7441 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     7501 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     7561 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     7621 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     7681 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     7741 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     7801 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     7861 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     7921 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     7981 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     8041 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     8101 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     8161 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     8221 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     8281 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     8341 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     8401 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     8461 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     8521 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     8581 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     8641 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     8701 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     8761 ggatcttc
//
