LOCUS       sequence-447348-        8504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide and lanthipeptide synthetase from LanII 
            (FAST-RiPPs) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8504)
  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 8504)
  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
FEATURES             Location/Qualifiers
     source          1..8504
                     /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      6933..6980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7021..7476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7108..7127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7318..7339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7568..8383)
                     /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(7568..8383)
                     /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     8291..8310
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8375..8394
                     /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 tgaataatga catgaacttt gcgagcaact
     3601 cagttagtcg cgcggcgcag tggcgtaccc aggcagataa gaacaaccca gccggtgcct
     3661 tattcgcaaa tgagtttgcc gaagcggata tcattggcgg gaccgatatg ctggttacgg
     3721 actgtgcaat gtgtaccggc agcattaaga ataccatcca tatttactgc gcttaaggcc
     3781 tttctgcccg aagtttcgat ggactggtgc caacgcgcag gcatagtttt aggagaatta
     3841 ttcgggggca gtgacaacca acatctcggg tccggtctag cataacccct tggggcctct
     3901 aaacgggtct tgaggggttt tttgccctta atacgactca ctatagggga attgtgagcg
     3961 gataacaatt ccaaggagat atacaatgac ctcgtcaact gatgcggatg attttagtga
     4021 aagcctgggt tgtttgattg ccgatgcaat ggttgatttg gctgctcgct tggcaggtgt
     4081 ggcgggactc acctgcgccg aacgtggtgt agttgccgat gcgacgcgtg cgtccctgac
     4141 agaggctttg catggtcgtc tcggacgtgt cttactgctt gaactgcacg cggcagttga
     4201 agatggcctg ctcgtggcag ctgattcggc aggccgttgg catgaattta ttgaacgtag
     4261 ttcgagtcca gccttttggg aagaattgac tacacattat cccactctga tggatcgtgt
     4321 gaatcgttta gttgaacaac gttgtgcgtc cgctctggca tttgctactc attttgcaac
     4381 cgatcgtgaa gccttggacg tctttcgtgg aagtcccttg ggagaactgg tagccctggc
     4441 tatcggtgcg ggagattctc accgcggtgg ccgcaccgtg gcactggtga catgtgctcg
     4501 tgggcgcatc gtgtacaaac cacgtagcgt gggaatcgat gccgcgcttt cccgttttat
     4561 tgaaccatta acagcacaag gtgctagttt gcgtgtcccg ccagttgtgg aacgcgatgg
     4621 ttatggctgg gctgcattcg ttgaacatcg ttatgctgcg gatgatggcg agctttcgcg
     4681 cttttaccgc ggattgggtg aatggttagc cgttatgcgt ttgcttggcg gttctgatct
     4741 gcatgcggaa aacttgatcg cacatggcgc acatccgttc gtcattgatt gtgaaacttt
     4801 atttaccccg catccgcctg tgaccccgtc tggtatgggt ttagcccatg atcgtgcagt
     4861 agggatggtc gaaggctcgg ttctgtcaat gggtttctta cctggacgtg gtgcggctct
     4921 ggcctggcgt ggcgttgatt cgagcggcat gggtgcgctg ccaggccaac aaccttcagt
     4981 ggaagtcccg gccattgttg atgcagggac ggatcgtgca cgtctgggga ccgtcatggt
     5041 tgaacctggc ggtgcagcca accatccatc acgcgcacct gcactggccg ctcattggcc
     5101 gaaggttgtt gaaggcttcg atcaggtgac agcgtggtta cgtagtgagg acgcagcagg
     5161 aaaacttgct gatcgcttac gtgcatttgc aggttgtgaa gttcgtgctg ttgtccgcag
     5221 caccgaggtt tatgcggaac tggcccgtat gttatggcat ccggttagct tacacgcacg
     5281 cgatgaagcg acgcatcgcg cagctgcatt gttgcgccgc atgggattaa atcgtgcgat
     5341 ttcaccggat cgcgacgacg ttattgcggc cgaaattgcg gacctgttgg atggtgacat
     5401 tcctttattt tcgacaatcg tgtcagaagg tcgtcttagt ggcccgcgtg gcacgtcttg
     5461 gctggatccg cgtgatttag ctgaagatgc actgcgtcgt tggcgcgaag cagatttagc
     5521 gctggaacgt caagctattc gtgcgagctt agccagtgct tatgcggacg gggatcgtga
     5581 tgaggcccac gtttcattgc gtccagtgcg tttggcactt catgatctgg atcgtcgccg
     5641 tcgtttgctg gccgcgggcg taatgaaaga tattgtacgt cgtgcgatcc gtggagatga
     5701 cggttccgtg gcttggattg cgtcagtgtt aggtccaaat ggtcgttcag ttcagccgat
     5761 tgcgcaggat gtctatggcg gtattgcggg aattgcgtta ttagcccatg gatatacacg
     5821 tgaacgcgat gcagaacgcg cagatgatgt tgcagaaatt gatgcaattg cagcagccgc
     5881 ggtggatact atgcgctggg ccgaggctca actttgggaa cgtcgtacgc acgttgcccg
     5941 tccacgtccc gttccacctg gtggatacat tggcttgggg agccaaattg cgagctggct
     6001 tttattagag gcttggggcg caacagcagc gggtgaaggt gtggaacgtg cacaacgtct
     6061 ggccggtttg attgcagaga gtgtggcagc ggatgataca cgtgatttat tgactggttc
     6121 gagcggtgct attgccccat tgttaacttt acatgaacgt actaaagacg cacgtcacct
     6181 gacacaggcc gttacagtag gcgacgcatt aattgaagca gcccgtcgtc aggatggacg
     6241 cgcctggtgg cctcatgagc gttggccgga cggtttgggc ggttttgccc acggtagcac
     6301 tggtattgga tgggcactgg cgaaattggc tcaagccagc ggccaagatc gttttctggc
     6361 catgtcgcgt gcagctcttg cgtttgatgc atcactgttt gatccagtta ccggctggcg
     6421 tgatcttcgt ggcggtgatc ctgcagcggc ttggtgtcat ggtgcagtcg gcattggact
     6481 tgcattggca gatctcgatc ctgcgttgga caatgcagat cgtcgtgccc agtttcgtgt
     6541 tgcgtcaact ttagcgtggc agaatgggat cggcggtgga catggtttat gtcatggcga
     6601 tatgggcgca tgggaattgt tagacattgc ggtgcgtact ggtttcgccc cggccggatg
     6661 tgacagtgac acccttaagg cgaccattat tggctcaatc gaaagccacg gtgcacgttg
     6721 tggtcgtcaa agcgaggatc ttagtccggg tctgattgcc ggcttaggtg gcgtcgcatg
     6781 gcaactgctt cgcatgcacc ctgagagtgc actcccgagc gtattgttac catcttttgc
     6841 taatcaaaca tcaacccgtt ctgcgcatag tgataatgtt cctagtccgc cgcccatgac
     6901 tgtggccgca tcaacttctt taggataacg gtctagcata accccttggg gcctctaaac
     6961 gggtcttgag gggttttttg agcgctgaaa ggaggaacta tatccggatt ggcgaatggg
     7021 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     7081 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     7141 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     7201 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     7261 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     7321 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     7381 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     7441 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     7501 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     7561 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     7621 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     7681 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     7741 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     7801 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     7861 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     7921 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     7981 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     8041 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     8101 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     8161 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     8221 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     8281 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     8341 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     8401 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     8461 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
