LOCUS       sequence-447346-        8876 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide and lanthipeptide synthetase from 
            LanII-2D (FAST-RiPPs) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8876)
  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 8876)
  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-2D
FEATURES             Location/Qualifiers
     source          1..8876
                     /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"
     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      7305..7352
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7393..7848
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7480..7499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7690..7711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7940..8755)
                     /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(7940..8755)
                     /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     8663..8682
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8747..8766
                     /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 tgaatctggt acgcgcatgg aaagaccctg
     3601 aatatcgtgc gacactgtct gaggccccag ctaatccggc gggccttgtt gaattggcag
     3661 atgatcaact ggacggtgtc gcaggtggca ctacttgggc atgcgcaacc gtaaccttga
     3721 cggttactgt ttgttccccg acgggtacac tgtgtggtag ctgttcaatg ggtacccgcg
     3781 gatgttgtta atagatagac tagccgctgg taaacacacc atgaccccgg ctctccattg
     3841 atgccacggc gattgttgga gagccagcag cgcggtctag cataacccct tggggcctct
     3901 aaacgggtct tgaggggttt tttgccctta atacgactca ctatagggga attgtgagcg
     3961 gataacaatt ccaaggagat atacaatgcc agatgatgct tccgaacatc ctactgtacg
     4021 tcctgatggt gtgcatctgc catggcaccg tgcagcaacg ctgtcagaac gtttgggtgg
     4081 aacaccatcg cgttcggatc cagttcgtgg tgagaaacgc ttggcgatgt ggcgtgagct
     4141 tgctccgctc acgggtgaac gtgccacctt agatgcacgt ttagctccgc ttggtgttga
     4201 tgcggcggaa ctgaccgcgt tacttggcga gtcagatgat agtcttgcag cccgtaccgc
     4261 cgacgaaccg gattggcatc gtacctttcg tcgctggtgg gcagaaggga tggccactac
     4321 ggatgcagcc cgtcgtttgg atccggatat gggcttgctt gaagtgattc gtccgctggt
     4381 cgaaggagcc gtacatgaat tggatgctct ggtggaagaa cgcgttcgtg cgtccgatga
     4441 tcctgtaatg gctgatcccg ggcatattct ggacgtcgtc cgtgcgactg cccccgcagc
     4501 ggatctcttg ggtcatgttc aacgtacgtt agttcttgaa cttaacattg cccgtgttga
     4561 ggaacgttta ggtggtgaga ctcctgaaga acgttttagt tcctatgtcg agttgctgcg
     4621 cagccagggt aacgcattag cattgtggga tgaatatccc gtgttagctc gtttggtggt
     4681 taatcagctt cgcttttgga ttgatacccg tgttgaactg atcgatgccc tggtagccga
     4741 tttgccagcc ttgcgcggtg ggcttctccc gagcccaggg ccacgtcgtc tggaacgttt
     4801 agaatttggg gcgggtgata aacatcgtcg tggccgtagc gtggctatgg tagaatttga
     4861 taccggaacc ctggtgttta aaccacgtag tttagcgatg gatacggcgt ttgatggttt
     4921 gctggcatgg gtaaaccgtc aacaaccccc acatgatctg gcacgcattg gtgtgttaga
     4981 acgcggcggc cacggctggg ttgaacaagt agataccacg gccaccaccg atgctgccgg
     5041 tggtgatcgt tatgcatggc gtctcggcgc actgacctca ctgttatact tacttcacgc
     5101 gaccgatttt cattttgaaa atgttttggc ggcaggtgaa catcccgtct tggttgatct
     5161 ggaagcactt ttacataacg ataaaactgc cgcagttgtt aagattgatg gagagactga
     5221 tattgcagcc gttaccctgg cggatagtgt acaaagtgta ggaatcttac cgaatcactt
     5281 gttagtacgt ggcgaagatg gcacgtttgg tctggatgtt tcaggattgg caggccacgg
     5341 tggccaatta acgcctatgc ccgtgccgac ctgggaagat tctggcactg atcgtatgcg
     5401 tctcgtgaaa cgccgtatgg aaatggatgc agaacgtaac cacccgatga ccgccgatgg
     5461 cgaacccatt gacctcgttg gtcgtgtgga cgcatttgtc gatgggttta cacacgttta
     5521 ccgtctcctg gaagccggcc gtgatgagct tctggatccg gctggtcctt tggcagcctt
     5581 tggctcggtg cgcacccgtt taattgctcg cccaacccat atttatggtc gtttattact
     5641 cgagtcaacg catcccgatt tcttgcgtga tgggttagat catgcacgta gtcttgcgcg
     5701 cctggcgggc ggacatgtag aaatgccgga atctcgtgaa gccatggttc aggatgaggt
     5761 agcagaattg acactcggag atatcccgat gtttaccgtg gacgcagaaa gtggcacctt
     5821 gcgtggcggt tttgatggcc gtccaattgg aactcgctat ccaccgttag cggcagtacg
     5881 cgaacgttta gccgccttgg gcgatgctga tctggcattt caggaacgcg ttattcgttc
     5941 atgtgtcgca gcgaccgcca tgggtgattc ggacgcgcgt tggcctaatt ggcatcgtcc
     6001 tcgcctggat ggtgcagcag acccgggtga ctttgcagaa gaagcgctgc gcttagcacg
     6061 ccgtttaggc gacttatgtg tacgtgatga acatggtctt ggctggattg gattggatct
     6121 ggttgatgaa aagtattggc aatacgcgcc ggcccctatt gggctttata ccggtacggc
     6181 aggaattgcg cacgcgttag atgcggttgc agccgtaacg ggagatgaag ctaccgggga
     6241 actggcacgc gcggcattcg atcaagtagc caaacgctca gtattaattg cggaaaccct
     6301 cgctagctta gcaaagaaac ctggcgctgc ggaattcggt attggtgcgt ttggcccgtt
     6361 tggtggtgca gtgtatgcct tggctcatgc cgcggtgcgt catgatcgtc ctgattatgc
     6421 agaggctgca gcagcattac tccctgcaat tgatgaatta gtagaagagg accccctgct
     6481 tgacgttgtg tcgggttcag ccggtgctat tctggctctg ctggctcttg agaaagcacg
     6541 tcctgcagcg ggtgctgctc gcattgcggc tcgttgtgcg gagcgtctgc tggctacacg
     6601 tcaagaatgt gcagagggct ggggttgggc aacgcctatt aaccctgagg ctcccttggc
     6661 aggcttcagc catggtgcgt cagggatcgc atatgccctt gcgcgtttag atgcggtggc
     6721 gccgcgccca gaatatggcg aagcggtgcg taacgccctg cgttacgaac gtacggtatt
     6781 tgatcctgaa ttacgtactt ggcgtgatct tcgtccggca aatctgggtg gacgcactgt
     6841 aatgaacgcc tggtgtcacg gggcgcccgg cattgcgctg gcccgcgatg cttttcaggg
     6901 tttaggtacg gcggccgatc tggcagattt ggttgaaagc gatcgtcata ctgccgtttt
     6961 ggcagctgtt agcacgggtt tagatctgga tccagtgagc gggctgggta atcatagctt
     7021 atgtcacggg gatgttggta atttattaat tatcgaagca agcgcgcgcg cagatcgcga
     7081 accggaagtt gcgggtttgt tgccacgtgt gtggaatacc ctgctgcatg agggccgtga
     7141 aaacggctgg ctctgtggtg tccctaaagg agtggaaact ccagggttaa tgaccggctt
     7201 agctggcatc gcttggggac tggcacgtaa ggcggcacct gaacgtgttc cggatctgtt
     7261 gaccttggcg gctccagcag gacctggcgg gaccccctaa cggtctagca taaccccttg
     7321 gggcctctaa acgggtcttg aggggttttt tgagcgctga aaggaggaac tatatccgga
     7381 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     7441 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     7501 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     7561 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     7621 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     7681 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     7741 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     7801 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     7861 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     7921 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     7981 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     8041 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     8101 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     8161 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     8221 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     8281 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     8341 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     8401 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     8461 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     8521 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     8581 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     8641 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     8701 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     8761 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     8821 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
