LOCUS       sequence-447354-        8645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses precursor peptide and lanthipeptide synthetase from 
            LanII-23 (FAST-RiPPs) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8645)
  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 8645)
  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-23
FEATURES             Location/Qualifiers
     source          1..8645
                     /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      7074..7121
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      7162..7617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7249..7268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7459..7480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7709..8524)
                     /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(7709..8524)
                     /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     8432..8451
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8516..8535
                     /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 tgtcagagaa aattctgaat atgagcggcg
     3601 actttgattt tgaatttaat gcttaccaag ccccggccaa tactggcgaa atcctggcta
     3661 ccacgatgga gtgcaacacc tttggcacct gtaccaacat tgggtgtagc acaattggtt
     3721 gctgactaga agcccggcag agttagttcg aacctaatgc cacaaatgag acaggacgcc
     3781 aatgggtacc ggacattagg tcgagctcag ttcggtaacg gagagaccct gcggcgtact
     3841 taattatgca tatgaaacgc gcccaagtga cgcggtctag cataacccct tggggcctct
     3901 aaacgggtct tgaggggttt tttgccctta atacgactca ctatagggga attgtgagcg
     3961 gataacaatt ccaaggagat atacaatgct ggtaaacgca ttaaagttag atcaacgcgg
     4021 caagtatggc ttttcatcgg aaggttgcat tgccttattt gaaaacaacc tgagccgtct
     4081 gcatacggac gatcaggcgc tgctggacca tttcggcgtg gatcgtgaac aacttgtatc
     4141 acacattaat agtccccttg acaacgaacc gctgcctatc gatggcgata cggtgctcaa
     4201 tgccctgaag aagaaattgg cccgtctgga tagcattaac atggaaacca atacgagctc
     4261 cagcgacaaa gataacttct actggttcgg gtaccgcttc tttctgtatg caattgaaat
     4321 cttccagacc gatggccgct attcgcatgc cagcacatat atcaatgaac cagctttctt
     4381 cacgagcctt gaaccgtacg tactggcacg cgtaggccgc acaagcgagc agtcgttggt
     4441 tcactatctg aataccaaga tcgcgcatgg agatgatatt gatttatcga cctttaacga
     4501 acagctgcgc actctgcctt tactggaatt ctttgaagcc tacccggtgt tggcgaccct
     4561 gttgtttgat ctgctcgagg acaccctgaa ctatttatat gcagtaatct tagattttgc
     4621 agcggacatc gagagtcttg aagcggagtt ttcagtacca agccgccaaa ttgatgcgat
     4681 cgaactgggc ctgggtgatc ctcatggtcg tggcgaaacc gtgtgtcagg tcaaaatttg
     4741 cgccatctca cttgtataca aaccacgcgc aagccgcgag gcgctgttct tcaaccgcct
     4801 cttaggtcag ctgcatgaat ggacgaaagc ggactgcttt agcatccgcg caccacgcat
     4861 cctcagccgc gaacgtcgct cctgggtgga gaaaattgac aacgaaccat gtgataacgc
     4921 agccgacgtt gagttattct ataagaaaat gggagctcag attgcagtca ttcatgccct
     4981 gaacggcatt gatttccact acgaaaatat cattgcctgc ggcaatagcc ctgtgatgat
     5041 cgatctggaa tgtctcttta cggcgagtac tagcgattta ctgtcagatc tgccggcagg
     5101 ctgtgcgctg agcaattcat tcaagtcggt acagcaaagt gtgtgctcat caggcttctt
     5161 acccttttca cagcacccca ataatgacca aagcggcttg agtcgtcagg aactctttat
     5221 tgcgacccgt catagtctcg tgtttgaaga tggtttctat cacttacgca aaatcagctt
     5281 tgaacatcag cttattcaga aacacttacc gctgttgcaa ggtgaacgca aaggcctgga
     5341 agcttataaa accgaattgt ttcagggctt tgaatatgca tacaaggaac tgatggtgca
     5401 ccgttataaa atcatgcgca tgttggagca gagcgcgggc gaactgacaa ctcgtgtgct
     5461 tttaaagaac agccagcgtt atgcagattt cattggcctg atccgtcacc cacgtttcac
     5521 tcagaacatg ctggaccgcg aacttctttt agcgacactg tggaaagatt tgaaagaacc
     5581 atatcgcaaa atgggcatcc cgcgttatga gattgcggat ttgcagcgct cctcaatccc
     5641 gtgctttacc atgcccctgt ctgccaatca tttcatgtcg gcgcagggtg aaaccatcga
     5701 aatggccgcc gtagaaccgc ctattaagtc ttgcctgcag aaaattgcaa atctgagccg
     5761 tcgcgactgg gcattgcagc gtacgttgtt agagatttgc ctctttccgg aaccaattgg
     5821 ccatccgccg ccccgcgtcg gccaggtgcc gggagatttg aagaaaactc tgcaagccga
     5881 tcgcttagat gcgattagca gcatctccag tcgcctggaa gaactggcaa ttgcgggcac
     5941 gaccactgat gtgagctggc tctacttcca tactcatccc acgacgcgcc gtaagtatcc
     6001 gtcacccatg gatcacggtc tttactccgg cattgcagga atcggcctgt tttatctttc
     6061 actgttcaaa gtatccggac agcccgtttt cctgtcgcgc atggaccagg ttctgtattc
     6121 actggaacag cgctttggat tctttaaaac tgatctgact gttagcgcct accacggcct
     6181 gggcgcgttc atttaccttt tggttaaccg ccgccagatt accggcgacg ccagccatga
     6241 tgaacgtatc gcgggattac ttgcacagct tatcgaggtg ccgcctgaaa actatgaatt
     6301 tgattttctg ggcggctgct gcggtaccgt gaccctgtta gccaatattc acgccttgag
     6361 cgcccagaaa gatgtgctgc ccgccatccg ccgcatggtc gaatacatta aggaccaaat
     6421 tttaattgag gatggtcagt tactgcgtcg ctccgatcgc tcggttattc tgactgggtt
     6481 atctcacggc cttagcggtg tcatccatgc cctgtgcaaa gcctatgatg taactggaga
     6541 ctctgcactg gtgccgctgg ccattcaagt gctggaaggc gagaaccgcc tgtcccgtga
     6601 tggtttctgg ctcgatctgc gcaatctggg tccggcggac catacgagca agtggtgcca
     6661 tggagacggc ggcatcctga ttgcgcgccg ccagttgatg gaaagtatgg gcgatgcgct
     6721 cgatgaagca actcgtcaaa cagtgcacga tgatattcgc cgctgcgagc gcaatttatg
     6781 ggcccacggc tttggagaag gctacaacct ttgccacggc gactttggca accttatctg
     6841 tctttatgat ttgtatcgtc acagcgataa tccggaaggt atgggccgcg ttcgcgaagc
     6901 cctggacgaa gtaagccgtc aattctttga gggtccttat ctggattcag atcgcgtgcc
     6961 cgatgtaagt ctgttgacgg ggattaccgg cgcgggttac gctctcttat atgaaattga
     7021 tcctactatc ccaaatgtcc tctccctgga cttcgccgtt ctgggataac ggtctagcat
     7081 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gagcgctgaa aggaggaact
     7141 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     7201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     7261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     7321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     7381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     7441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     7501 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     7561 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     7621 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     7681 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     7741 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     7801 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     7861 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     7921 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     7981 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     8041 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     8101 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     8161 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     8221 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     8281 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     8341 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     8401 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     8461 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     8521 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     8581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     8641 tcttc
//
